CN115197968B - Construction, screening method and application of chimeric antigen receptor modified cell library with automatic optimization of antigen binding domain - Google Patents

Abstract

The present invention discloses a construction and screening method of a chimeric antigen receptor modified cell library with automatic optimization of antigen binding domain and its application. The method inserts a chimeric antigen receptor sequence into a cell and continuously induces mutations in the extracellular antigen binding domain through cis-acting elements and trans-acting factors to establish a dynamic library of antibody sequences for subsequent screening; the dynamic library of antibody sequences is displayed on the cell surface by receptor 2 to form a chimeric receptor library cell library; receptor 1 can recognize the label on the target antigen and form a dimer with receptor 2 that can recognize the target antigen, thereby starting the expression of the screening gene and simultaneously starting the inhibition system to maintain the stability of the antigen binding domain. The chimeric antigen receptor library is screened, and the antigen binding domain of the screened positive cell chimeric receptor is amplified and sequenced to obtain the nucleic acid sequence of the antigen recognition region. This method can be used for the preparation of a variety of antibodies, which is simpler and more efficient.

Description

Construction, screening method and application of chimeric antigen receptor modified cell library with automatic optimization of antigen binding domain

Technical Field

The present invention relates to the construction and screening methods of a chimeric antigen receptor modified cell library with automatic optimization of antigen binding domain and its application, and belongs to the technical field of genetic engineering.

Background technique

At present, antibodies play an irreplaceable role in the fields of scientific research, detection, diagnosis, and medical treatment. With the rapid development of chimeric antigen receptor T cell immunotherapy (CAR-T) and immune checkpoint therapy, there is a greater demand and higher requirements for the preparation of antibodies. For example, the preparation of single-chain antibodies (scFv) of the extracellular antigen binding domain of chimeric antigen receptor (CAR). The extracellular antigen binding domain of CAR also mostly uses single-domain antibodies (sdAb) that naturally lack light chains produced by camelids and sharks, which are called heavy chain antibodies (HcAb). The antigen binding fragment in each arm of HcAb has a single heavy chain variable domain (VHH), which can have high affinity for antigens without the help of light chains. VHH is called the smallest functional antigen binding fragment with a molecular weight of about 15Kd and is widely used in CAR receptors. In addition, humanized antibodies are often required in the treatment of humans.

At present, scFv and VHH are mostly obtained through traditional antibody preparation methods, and the main steps include: 1. Preparation of antigens and adjuvants; 2. Animal immunization; 3. Antiserum collection; 4. B lymphocyte separation; 5. Hybridoma preparation/antibody sequence amplification and library construction; 6. Antibody screening; 7. Obtaining antibody sequences; 8. Further preparation of scFv and VHH sequences. In addition, the prepared antibodies need to be humanized, involving the use of humanized mice or splicing between antibodies. The entire process is cumbersome, time-consuming, labor-intensive, and costly.

Summary of the invention

The purpose of the present invention is to provide a method for constructing a chimeric antigen receptor modified cell library with automatic optimization of antigen binding domain.

The second object of the present invention is to provide a chimeric antigen receptor modified cell library prepared by the above construction method.

The third object of the present invention is to provide a method for screening a chimeric antigen receptor modified cell library with automatic optimization of the antigen binding domain.

The fourth object of the present invention is to provide the application of the above screening method in preparing antibodies.

The fifth object of the present invention is to provide antibodies screened by the above method.

In order to achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a method for constructing a chimeric antigen receptor modified cell library with automatic optimization of antigen binding domains, wherein one or more vectors comprising a first gene element, a second gene element, a third gene element, a fourth gene element, and a fifth gene element are transfected into cells to obtain a chimeric antigen receptor modified cell library;

The first gene element is a nucleotide sequence encoding receptor 1, and the receptor 1 includes an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain can recognize biotin or a 6×His tag marked on the target antigen, and the intracellular domain includes tobacco etch virus protease TEVP;

The second gene element is a nucleotide sequence encoding receptor 2, and the receptor 2 includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the antigen binding domain is a dynamic library of antibody sequences, and the dynamic library is obtained by inducing a series of mutations under the action of the fourth gene element and introducing sequence diversity; the intracellular signaling domain includes a tTA transcription factor domain and a connecting sequence, and the connecting sequence connects the tTA transcription factor domain and the transmembrane domain, and includes a sequence that can be recognized and cut by TEVP;

The third gene element is a nucleotide sequence encoding a screening gene, and the tTA transcription factor can activate the expression of the screening gene, so that the host produces fluorescence, acquires certain resistance, or obtains a growth advantage under certain conditions;

The fourth gene element is a nucleotide sequence encoding one or more cis-regulatory elements and one or more trans-acting factors; the cis-regulatory elements can cause DNA to produce a DNA sequence with a special structure, wherein the special structure refers to a DNA single-stranded structure in which no base complementary pairing occurs in the DNA structure, and the special structure includes R-loop, G-triplet and G-quadruplex; the trans-acting factor is a combination of one or more of the Cas protein family and variants of the Cas protein family, gRNA, uracil glycosylase inhibitor, adenine deaminase, cytidine deaminase, RNA-DNA hybrid chain binding protein, photosensitive protein, Nucleolin, DNA helicase, error-prone DNA polymerase, recombinase, endonuclease, exonuclease, and alkyl adenine glycosylase;

The fifth gene element is a nucleotide sequence encoding an inhibition system, and the tTA transcription factor can activate the expression of the inhibition system, thereby knocking down or knocking out the trans-acting factor, so that the dynamic library of antibody sequences remains stable; or the fifth gene element is a nucleotide sequence encoding a light control system, and the light control system can regulate the expression of the fourth gene element: the expression of the fourth gene element is activated under light conditions to start building the antibody dynamic sequence library; after stopping the light, the function of the fourth gene element is inhibited, so that the dynamic library of antibody sequences remains stable.

Preferably, the cell is a eukaryotic cell or a prokaryotic cell. More preferably, the cell is a human or mouse cell. Even more preferably, the cell is 293T.

Preferably, the dynamic library of antibody sequences includes a traditional antibody library, a single-chain antibody library and a nanobody library.

Preferably, the nucleotide sequence of the receptor 1 is as shown in SEQ ID NO.1.

Preferably, the receptor 2 is a synNotch receptor transformed from the Notch receptor, and its nucleotide sequence is shown as SEQ ID NO.2 or SEQ ID NO.3.

Preferably, the trans-acting factors include the following Cas protein family members and partial domains thereof: Cas3-8s, Cas10s, Cas11s, Cas9s, xCas9, Cas12s, Cas13s and Cas14s.

Preferably, the trans-acting factors include the following adenine deaminases and partial domains thereof: ecTadA, mADA, hADAR2 and hADAT2.

Preferably, the trans-acting factors include the following cytidine deaminases and partial domains thereof: AID (activation-induced cytidine deaminase), APOBEC3G, APOBEC1 and CDA1.

Preferably, the nucleotide sequence encoding the light control system includes the following photosensitive proteins and their partial domains: BphS, Cph1, PCB, PhyB-PIFs, PΦB, BphP1-PpsR2, BphP1-Q-PAS1, LOV2-Jα, PhyB, PIF6, Epdz and Photoreceptor Vivid (VVD).

Preferably, the Cas protein family is a family of proteins that recognize and direct specific genomic sites through gRNA.

Preferably, the light control system needs the cooperation of light to exert its regulatory effect on the fourth gene element, and the light includes visible light, invisible light, blue light, red light, far-infrared light and ultraviolet light.

Preferably, the screening genes include GFP, mCherry, Luciferase, puromycin resistance gene, bleomycin and neomycin resistance gene.

Preferably, the inhibition system is to initiate the expression of negative regulatory proteins, repressor proteins, siRNA, CAS9/gRNA through tTA transcription factors or knock down or knock out trans-acting factors through reverse transcription, so as to maintain the stability of the dynamic library of antibody sequences.

In a second aspect, the present invention provides a chimeric antigen receptor modified cell library with automatically optimized antigen binding domain obtained by the above construction method.

In a third aspect, the present invention provides a method for screening a chimeric antigen receptor modified cell library for the above-mentioned antigen binding domain automatic optimization, comprising the following steps:

1) preparing a target antigen, wherein the target antigen carries a tag that can be recognized by the chimeric receptor 2, wherein the tag includes biotin or 6×His;

2) Co-incubating the target antigen with the chimeric antigen receptor modified cell library, and screening out positive chimeric antigen receptor expressing cells based on the expression of the screening gene;

3) Amplifying and sequencing the extracellular antigen binding domain of the chimeric antigen receptor of positive cells to obtain the antibody nucleotide sequence that can specifically bind to the target antigen;

Preferably, after obtaining an antibody nucleotide sequence that can specifically bind to a target antigen, a genetic engineering method is used to obtain an antibody against the target antigen.

Preferably, the target antigen is expressed on the cell surface, coated on the carrier surface or is a soluble protein.

Preferably, the carrier includes magnetic microspheres, non-magnetic microspheres, graphene, agarose microspheres, nanoparticles, silica-based magnetic beads, GMA magnetic beads and polystyrene magnetic microspheres.

In a fourth aspect, the present invention provides the use of the above screening method in preparing antibodies against a specific target antigen.

In a fifth aspect, the present invention provides antibodies screened by the above method.

The present invention uses GFP-biotin as the target antigen, constructs a chimeric receptor modified cell library in different ways and screens it to obtain a GFP nanobody, wherein the amino acid sequence of the anti-GFP nanobody comprises any one of SEQ ID NO.4-13, or any combination of CDR1, CDR2, and CDR3 regions in SEQ ID NO.4-13.

The beneficial effects of the present invention are:

The library obtained by the construction method of the chimeric antigen receptor modified cell library provided by the present invention is a dynamic library, which can automatically optimize the antigen binding domain while screening, overcoming the defect of limited sequence diversity of the static library in the prior art; and the whole process can use human antibodies as a framework for optimization, directly realizing the humanization of antibodies. The dynamic library constructed by this method can be reused. According to the screening method of the chimeric antigen receptor modified cell library provided by the present invention, the corresponding antigen binding domain sequences for multiple antigens can be obtained. Compared with the traditional antigen binding domain sequence/antibody sequence acquisition method, the efficiency is greatly improved and the cost is greatly reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of the technical solution of the present invention;

Figure 2 is a schematic diagram of a gRNA-guided base editor;

Figure 3 is a schematic diagram of a G-quadruplex-guided base editor;

Figure 4 is a schematic diagram of an optimized gRNA-guided base editor;

FIG5 is a schematic diagram of the structure of a carrier 1;

FIG6 is a schematic diagram of the structure of a carrier 2;

FIG7 is a schematic diagram of the structure of a carrier 3;

FIG8 is a schematic diagram of the structure of a carrier 4;

FIG9 is a schematic diagram of the structure of a carrier 5;

FIG10 is a schematic diagram of the structure of adenosine deaminase fused to dCas9;

FIG11 is a schematic diagram of the structure of dCas9-fused cytidine deaminase;

Figure 12 is a schematic diagram of the dCas9-Vivid-Mcm7 structure;

FIG13 is a schematic diagram of the structure of Vivid fused cytidine deaminase and adenosine deaminase;

FIG14 is a graph showing the results of VHH sequence recognition of eGFP obtained by ELISA;

In the figure, a, based on the DNA G-quadruplex-guided base editor positioning library construction and identification of the screened nanoantibodies; b, based on the gRNA-guided base editor positioning library construction and identification of the screened nanoantibodies; c, based on the gRNA-guided improved base editor positioning library construction and identification of the screened nanoantibodies.

FIG15 is a graph showing the results of PCR amplification of all antibody heavy chains;

FIG16 is a diagram showing the results of a large-scale PCR amplification of all antibody heavy chains;

Figure 17 is a graph showing the results of PCR amplification of nanobodies;

FIG18 is flow cytometry verification of the expression of receptor 1 and receptor 2;

FIG19 is a cell ELISA and immunofluorescence validation of the expression of receptor 1 and receptor 2;

In the figure, a, schematic diagram of the expression structure pattern and detection method of the dimerization-activated dual receptor system in cells; b, cell ELISA to detect the expression of streptavidin (receptor 1); c, GFP was co-incubated with cells expressing the dimerization-activated dual receptor system for 15 minutes and the expression of receptor 2 was observed under a fluorescence microscope.

Figure 20 is a diagram of screening nanobodies using biotin-labeled GFP protein;

Figure 21 is the result of specific identification of nanobodies.

In the figure, a, unlabeled GFP protein was used for flow cytometry to detect the 5 positive cell clones screened; b, ELISA was used to detect the specificity of 5 nanoantibodies to GFP protein after prokaryotic expression and purification, and anti-CD19 nanoantibody was used as negative control; c, positive cells carrying VHH5 gene were incubated with unlabeled GFP protein for 30 minutes, and the binding status of GFP protein and cells was observed under fluorescence microscope.

Detailed ways

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; unless otherwise specified, the equipment and reagents used in the examples are conventionally commercially available.

The present invention constructs a vector expressing receptor 1 and receptor 2, receptor 1 can recognize a biotin tag, and the extracellular antigen binding domain of receptor 2 can display a nano antibody library. When a target antigen with a biotin tag is co-incubated with a chimeric antigen receptor modified cell library, the target antigen can induce receptor 1 and receptor 2 to produce dimers. After dimerization, tobacco etch virus protease (TEV Proteinase, TEVP) in the intracellular domain of receptor 1 recognizes and cuts and releases the tTA transcription factor domain of the intracellular signal transduction domain of receptor 2, and the tTA transcription factor can start the expression of the screening gene and the inhibition system (as shown in Figure 1).

Then, camel peripheral blood mononuclear cells were taken to obtain natural nanoantibody sequences, and the natural nanoantibody sequence library was used to replace the nanoantibody sequence region outside the receptor 2 cell. The vector was used to package the lentivirus and transfected into 293T cells, and the transfection-positive cells were screened with puromycin, thereby displaying the nanoantibody library on 293T cells through receptor 2 to obtain a chimeric antigen receptor modified cell library.

GFP protein was obtained by prokaryotic expression and purification, and biotin-labeled GFP protein (GFP-biotin) was prepared as the target antigen. The above-mentioned modified cell library was screened, which proved that the chimeric receptor modified cell library activated by dimerization of receptor 1 and receptor 2 was successfully constructed and nanoantibodies to GFP protein could be screened.

Furthermore, the present invention introduces a fourth gene element into the chassis cells, that is, by introducing one or more cis-regulatory elements and one or more trans-acting factors, mutations are continuously induced in the antigen binding domain, sequence diversity is introduced, and a dynamic library of nanoantibody sequences is established, a dynamic chimeric antigen receptor modified cell library is constructed and screened, and nanoantibodies to GFP protein are obtained, which proves the feasibility of the construction and screening methods of the chimeric antigen receptor modified cell library provided by the present invention.

Example 1: Vector construction

The vector inserted with a series of gene elements can express receptor 1 and receptor 2 in 293T cells. Receptor 1 can recognize the biotin tag, and the extracellular antigen binding domain of receptor 2 can display the nanoantibody library. When the target antigen with the biotin tag is co-incubated with the chimeric antigen receptor modified cell library, the target antigen can induce receptor 1 and receptor 2 to dimerize. After dimerization, the tobacco etch virus protease (TEV Proteinase, TEVP) in the intracellular domain of receptor 1 recognizes and cuts the tTA transcription factor domain that releases the intracellular signal transduction domain of receptor 2. The tTA transcription factor can initiate the expression of the screening gene and the inhibition system (as shown in Figure 1).

Preferably, the key DNA sequence of the vector 1 (as shown in FIG. 5 ) is as shown in SEQ ID NO.14 (SEQ ID NO.14 is the sequence between the cPPT/CTS element and the EF-1αcore promoter element in FIG. 5 , and this sequence can be connected to any lentiviral vector with a similar structure). The key DNA sequence of the vector 2 (as shown in FIG. 6 ) is as shown in SEQ ID NO.15 (SEQ ID NO.15 is the sequence between the cPPT/CTS element and the EF-1αcore promoter element in FIG. 6 , and this sequence can be connected to any lentiviral vector with a similar structure). The vector is delivered to a gene synthesis company for full gene synthesis.

Example 2: Construction of Nanobody Library

200 ml of camel blood was purchased from Tianjin Mo Shang Ke for camel blood monocyte separation experiment, and finally 2.03×10 ⁹ monocytes were separated and obtained. After resuspending with Trizol, they were divided into 10 centrifuge tubes (2 ml Trizol/centrifuge tube) and frozen in a -80°C refrigerator.

RNA was extracted from the cells in one of the centrifuge tubes, and a total of 95.8 μg (A260/A280=2.0) was extracted. All of it was reverse transcribed into cDNA using 5×All In One RT MasterMix (Cat#G490, abm) and frozen in a -20°C refrigerator.

1) First PCR amplification of all antibody heavy chains

Experimental Materials:

Hieff Canace ^R Gold High-Fidelity DNA Polymerase, product number 10148ES10;

Amplification Primers:

CALL001:5′-GTCCTGGCTGCTCTTCTACAAGG-3′;

CALL002:5′-GGTACGTGCTGTTGAACTGTTCC-3′;

reaction system:

Reaction procedure:

Reaction results:

The PCR amplification results are shown in Figure 15. It can be seen from the figure that the best effect is achieved when cDNA is added to 5 μl, a total of 0.5 μg.

Re-PCR amplification: add 5 μl of cDNA and perform PCR amplification again, for a total of 8 tubes.

PCR amplification product recovery:

The 700 bp target band was cut and the target band DNA was recovered using the SanPrep column DNA gel recovery kit (REF B518131-0100, OLD#SK8132, LOT E710KA8969) (as shown in Figure 16). The total volume of 8 tubes of PCR product recovery was 240 μl, the product concentration was 42 ng/μl, and the purity was (OD260/280=1.81).

2) Second PCR amplification of nanobodies (using the recovered 8 tubes of PCR products to amplify again with nested primers)

Experimental Materials:

Hieff Canace ^R Gold High-Fidelity DNA Polymerase, product number 10148ES10.

The primers are as follows: Xba: ⅠTCTAGA, EcoR: ⅠGAATTC

reaction system:

Reaction procedure: Same as above

Amplification results: as shown in Figure 17

reaction system:

Reaction procedure: Same as above

3) PCR product purification

Experimental materials: DNA product purification kit (D1300);

Experimental results: The concentration of the purified product was 150 ng/μl, the total volume was 240 μl; the purity A260/A280 = 1.84

4) Enzyme digestion of vector 1 or vector 2, enzyme digestion of PCR products and enzyme ligation. Experimental materials:

XmaI (NEB, R0180S), (NEB, R3136S), T4 DNA ligase (Takara, Cat#2011A, Lot#AIF2071A), SanPrep column PCR product purification kit (REF#B518141-0100, LOT#B206KA5188).

Experimental steps:

a) Enzyme digestion of plasmid vector

b) Enzyme digestion of PCR products

c) Purify the cleavage product after the cleavage is completed

The digested products were purified using the SanPrep column-based PCR product purification kit.

Vector 1 obtained purified product 60 μL 150 ng/μL A260/A280=1.86

VHH PCR product 40 μL 100 ng/μL A260/A280＝1.85

d) Enzyme ligation

Ligation was allowed to proceed overnight at 16°C.

e) Purification of ligation product

The ligation product was purified using the SanPrep column-based PCR product purification kit and eluted with 100 μL of ddH ₂ O buffer.

Example 3: Preparation of E. coli competent cells

This study used electroporation to transform the recombinant plasmid into the E. coli strain Stbl3. The preparation method of competent cells of E. coli for electroporation is as follows (Note: it needs to be done in front of an alcohol lamp):

1) Use an inoculation loop to streak the E. coli Stbl3 bacterial solution on an LB solid medium plate without antibiotics (operate in a clean bench), invert the plate and incubate at 37°C for 24 hours to obtain a single colony with a diameter of about 1 mm;

2) Pick a single Stbl3 colony and place it in 3 ml of LB liquid culture medium without antibiotics, place it in a 37°C constant temperature shaker, and culture it overnight at 220 rpm;

3) Transfer 3 ml of overnight culture solution to 250 ml of LB liquid medium (without antibiotics), place in a 37°C constant temperature shaker, and culture at 220 rpm for about 3-5 hours. Stop the culture when the OD value of the culture solution reaches 0.4-0.5;

4) Divide 250 ml of bacterial solution into two 85 ml sterile centrifuge tubes (twice), place in a low-temperature centrifuge, centrifuge at 4°C, 4000 rpm for 10 min, pour off the supernatant, and place the centrifuge tubes in an ice box;

Note: The following operations need to be performed at low temperature (ice bath operation).

5) Use 30 ml sterile ddH ₂ O (pre-cooled in a 4°C refrigerator) to fully resuspend the bacterial precipitate, place it in a low-temperature centrifuge, centrifuge it at 4°C, 4000 rpm for 10 min, pour off the supernatant, and place the centrifuge tube in an ice box;

6) Repeat the previous step once;

7) Use 30 ml of 10% glycerol (pre-cooled in a 4°C refrigerator) to fully resuspend the bacterial pellet, place it in a low-temperature centrifuge, centrifuge at 4°C, 4000 rpm for 10 min, pour off the supernatant, and place the centrifuge tube in an ice box;

8) Repeat the previous step once;

9) Use 0.5 ml 10% glycerol (pre-cooled in a 4°C refrigerator) to resuspend the precipitate, and use a pipette to gently blow to break up small bacterial clumps. Dispense the bacterial solution into 1.5 ml sterile centrifuge tubes, 45 μl per tube, and place the divided solutions in an ice box.

10) Place the packed centrifuge tubes in a foam box, pour in an appropriate amount of liquid nitrogen, freeze for 1 minute, and store at -20°C for later use.

Example 4: Electroporation and plating of recombinant vectors

Experimental materials: American Bio-Rad MicroPulser electroporator, Bio-Rad electroporation cup (Cat. No.: 165-2089).

Experimental method: 100 μL of purified ligation product + 1 mL of electrocompetent cells were electrotransferred 20 times (50 μL each time). The specific operation was as follows:

1) Pre-cool the electric transfer cup (new, sterile) in a -20℃ refrigerator;

2) Set the dot punch to "Bacteria" and "Time ms" and run the experiment in automatic mode;

3) Transfer the mixed competent cells to a pre-cooled sterile electroporation cup, cover it with the lid, and tap it a few times to allow the bacterial solution to fully enter the electroporation groove;

4) Place the electroporator cup on the dot perforator and click. The normal display time should be between 3-5ms.

5) After electroporation, add 0.5 mL of preheated SOC recovery medium to each electroporation cup, then transfer 20 cells to the same 50 mL centrifuge tube and incubate at 37°C, 170 rpm for 1 hour.

6) Take 100 μL of the electroporated cells and dilute them 10× with LB medium (1:10,000, 1:1,000, 1:100), spread 100 μL of the diluted cells on a 9 cm LB agar plate (100 μg mL ^-1 ampicillin, 2% (wt/vol) glucose) at 37°C overnight.

7) The remaining cells were divided into four portions, plated in 245 mm square LB culture dishes (100 μg ml ^-1 ampicillin and 2% (wt/vol) glucose) and incubated at 37° C. overnight.

Example 5: Calculate electroporation efficiency and collect libraries

1) Count the number of colonies on a 9 cm LB agar plate, calculate the library size by the number of colonies × dilution factor, pick 20 single colonies and perform colony PCR using vF and vR as primers, analyze the size of the PCR product to ensure that > 75% of the colonies contain DNA fragments of the correct size.

2) 100 single colonies were selected and sequenced using vF and vR as primers to obtain the library size of the 100 single colonies, and then the size of the entire nanobody library was calculated to be 2.15×10 ⁷ .

3) Add 4 mL of LB medium to each dish, collect cells with a sterile cell scraper, and transfer to a 50 mL centrifuge tube. Rinse each dish once with 2 mL of LB medium, collect cells, and add 20% volume of glycerol. Measure _OD600 and divide the cell suspension into 20 portions of 150 μL. Keep the rest in a large centrifuge tube and freeze at -80°C.

4) Take out one of the bacterial solutions to amplify and extract the plasmid, and the extraction steps are carried out according to the instructions of the kit.

Example 6: Preparation of lentivirus and screening of stable transfected strains

According to the ratio of 4:3:1, add 23.3μg of the connected vector 1 or vector 2 plasmid and two lentiviral packaging plasmids PSPAX2 17.5μg, PMD2.G 5.8μg to 1.8mL calcium chloride. Then add the mixture to 1.8mL BBS, let it stand for 20min, and then add it to a ^175cm2 293T cell culture flask. Collect the 48h and 72h virus supernatants respectively, and concentrate them with 5×PEG. Add the obtained virus and polybrene to the culture dish with 293T for transfection. After 2 days, screen with 6μg/mL puromycin, and change the solution every day to keep the puromycin concentration unchanged for 5 consecutive days.

Example 7: Stable Transformation Strain Detection

Since the 293T stable strain of transfection vector 1 screened in Example 6 expresses receptor 1 and receptor 2, and the extracellular domains of these two receptors carry Flag and HA tags, respectively, we used flow cytometry to verify the expression of receptor 1 and receptor 2 by using flow antibodies that recognize these two tags. As shown in Figure 18, the two receptors were successfully expressed.

Since the receptor 1 extracellular domain expressed by the 293T stable strain of transfection vector 2 screened in Example 6 is a single-domain streptavidin that can recognize biotin, we used biotin-labeled antibodies and HRP-labeled streptavidin to perform cell ELISA to detect the expression of receptor 1 (as shown in Figure 19b). The receptor 2 extracellular domain has a nanobody domain, and we also detected the expression of receptor 2 using FITC-labeled goat antibodies (Goat Anti-Llama IgG H&L, Abcam, ab112785) that can recognize nanobodies (as shown in Figure 19c).

Example 8: GFP protein purification and GFP-biotin ligand preparation

We expressed and purified the GFP protein in prokaryotes as follows:

1) The eGFP protein expression vector is pET-28a(+), and the host bacteria is E. coli BL21(DE3);

2) Use an inoculation loop to streak the E. coli BL21 (DE3) bacterial solution on a LB solid medium plate containing kanamycin (operate in a clean bench), invert the plate and incubate at 37°C for 24 hours to obtain a single colony with a diameter of about 1 mm;

3) Pick a single colony and place it in 3 ml of LB liquid medium without antibiotics, place it in a 37°C constant temperature shaker, and culture it overnight at 220 rpm;

4) Transfer 3 ml of overnight culture solution to 200 ml of LB liquid medium (without antibiotics), place in a 37°C constant temperature shaker, and culture at 220 rpm for about 3-5 hours. Stop the culture when the OD value of the culture solution reaches 0.8;

5) Add 0.5 mM IPTG to induce protein expression (IPTG stock solution concentration is 1 M, add 100 μl to 200 ml LB liquid medium), place in a 16°C constant temperature shaker, and shake and culture at 170 rpm for 16 h;

6) Centrifuge at 4000g for 5 min, discard the supernatant, and wash once with 30 ml Buffer A;

7) Add 25 ml of Buffer A and mix well. Place on ice and perform ultrasonic disruption with a working time of 2 s, a rest time of 5 s, and a working number of 40 times.

8) Centrifuge at 10,000 rpm, 4°C for 30 min, and filter the supernatant with a 0.45 μm filter;

9) Rinse the nickel column with 10 times the volume of Buffer A, and then add clear solution;

10) After the supernatant has flowed through, add 5 volumes of Buffer A containing 10mM imidazole to wash the protein once, then add 5 volumes of Buffer A containing 20mM imidazole to wash the protein once, then add 5 volumes of Buffer A containing 40mM imidazole to wash the protein once, then add 5 volumes of Buffer A containing 250mM imidazole, and collect the eluate into different 1.5ml centrifuge tubes. Note: When washing the protein with low concentration imidazole, use Quick Start ^TM Bradford Protein Assay protein detection solution to observe whether the protein is eluted.

11) Rinse the nickel column with 10 volumes of Buffer A, then rinse the nickel column with 10 volumes of ddH2O, and finally keep the nickel column in 20% ethanol for later use.

12) SDS-PAGE gel was used to detect the protein content and purity in each tube.

13) Combine the protein solutions in the target tubes and concentrate them using a suitable ultrafiltration tube. During this period, add protein preservation solution three times to replace the elution solution.

14) Use a thin pipette tip to transfer the protein solution.

15) Biotin Super Biotin Rapid Labeling Kit (ARL0020S-30K-0.5 mL, purchased from Fuinde Technology (Wuhan) Co., Ltd.) was used to prepare biotin-labeled GFP protein (GFP-biotin), and the Biotin labeling step was carried out according to the instruction manual.

Example 9: Nanobody screening and specificity identification

Experimental materials: sequencing primer 5’-GAGGGCAGAGGAAGTCTGCT-3’; Blasticidin (Solebio);

The biotin-labeled GFP protein (GFP-biotin, 0.1 μg/ml) ligand was co-incubated with the transfected 293T cells for 24 hours, and the positive cells were screened with blastcidin (5 μg/ml). After the positive cells were selected for monoclonal amplification, the nanobody region was sequenced to obtain the GFP-specific nanobody. The left side of Figure 20 shows 293T cells that were not screened with blastcidin, and one of the monoclonal cells after screening with blastcidin (the right side of Figure 20).

A total of 5 positive cell clones were obtained by screening, namely VHH1, VHH2, VHH3, VHH4 and VHH5. The results of flow cytometry detection using unlabeled GFP protein are shown in Figure 21a. The staining results of the two cell clones, VHH3 and VHH5, were positive.

After sequencing, 5 nanobody sequences were obtained. After prokaryotic expression and purification (prokaryotic expression purification step, expression vector, and induced expression conditions are as described in Example 8), the specificity of the 5 nanobodies screened for GFP protein was verified by ELISA. The results are shown in Figure 21b, which is consistent with the flow cytometry results. The nanobody ELISA results corresponding to the two cell lines VHH3 and VHH5 with positive flow cytometry results were also positive.

The ELISA experimental steps are as follows:

1) Add 100 μL of PBS containing GFP protein (1 μg/mL) to each well of the ELISA plate, shake gently for 1 min to mix evenly, seal the plate, and coat at room temperature for 2 h.

2) Pour off the solution and add 400 μL Wash Solution to each well for 4 washes, 1 min each wash. After the last wash, pour off the liquid and absorb the remaining liquid with clean absorbent paper.

3) Add 100 μL of PBS containing VHH1-5 protein (1 μg/mL) and PBS containing anti-CD19 VHH protein (1 μg/mL) (control group) to each well, seal the plate, and coat at room temperature for 2 h.

4) Add 100 μL of HRP-labeled anti-His tag antibody working solution to each well, seal the plate, and coat at room temperature for 2 h.

5) Pour off the solution and wash with 400μL Wash Solution; repeat three times; after the last wash, pour off the liquid and absorb the residual liquid with clean absorbent paper.

6) Add 200 μL of Substrate Solution to each well and coat for 20-30 minutes at room temperature in the dark.

Add 50 μL Stop Solution to each well and shake well. Detect the absorbance within 30 minutes, set the detection wavelength of the microplate reader to 450 nm, and the reference wavelength to 540 or 570 nm.

Finally, positive cells carrying the VHH5 gene were co-incubated with unlabeled GFP protein for 30 minutes. The results are shown in FIG21c . Observation under a fluorescence microscope revealed that the GFP protein could effectively bind to the cells.

Afterwards, in order to increase the affinity of VHH5 nanobody to GFP protein, we performed a series of artificial mutations on the three CDR regions of the VHH5 gene, and finally obtained a series of nanobodies with different affinities to GFP including the VHH5 gene. The VHH5 nanobody comprises any one of SEQ ID NO.16-25, or any combination of CDR1, CDR2, and CDR3 regions in SEQ ID NO.16-25.

Example 10: Guided base editor positioning based on DNA G-quadruplexes and library construction and nanobody screening and identification

In this example, a base editor is used to construct a base editing library in vitro in a nanobody CDRs region of known sequence based on the G-quadruplex high-level structure of DNA.

The principle is shown in Figure 3. Since all base editors can only work on single-stranded DNA, in addition to using dCas9 and gRNA to unwind a single DNA strand, it can also induce the generation of higher-order structures on a single DNA strand, such as G-quadruplexes, thereby leading to the generation of another single DNA strand. A minimum of ~25bp of G-base-rich DNA sequence can induce the formation of G-quadruplexes. At the same time, nucleolin-linked cytidine deaminase (promoting mutation of G or C bases to any base) and nucleolin-linked adenosine deaminase (promoting mutation of A base to any base) are designed. Since nucleolin can recognize and bind to G-quadruplexes, the two base editors are located in the CDRs region. Nucleolin-Vivid and the two base editor genes linked to Vivid were transferred into 293T cells together through the Sleeping Beauty transposon system SB100X to construct a stable transgenic strain. By optimizing the length of the linker sequence between Vivid and deaminase and the blue light irradiation time, extensive mutations were induced in the CDRs region of VHH.

Vector 2 (see Example 1) was packaged into lentivirus and transformed into 293T cells, and stable transfectants were obtained by puromycin selection.

Construct vector 3 (as shown in FIG. 7 ), wherein the key DNA sequence of vector 3 is shown in SEQ ID NO. 26 (SEQ ID NO. 26 is the sequence between the cPPT/CTS element and the EF-1α core promoter element in FIG. 7 , and this sequence can be connected to any lentiviral vector with a similar structure);

Construct vector 4 (as shown in FIG8 ), the key DNA sequence of vector 4 is shown in SEQ ID NO.27 (SEQ ID NO.27 is the sequence between the cPPT/CTS element and the EF-1α core promoter element in FIG8 , and this sequence can be connected to any lentiviral vector with a similar structure); (the vector is delivered to a gene synthesis company for full gene synthesis)

By packaging lentivirus and constructing a stable 293T cell line, vector 3 and vector 4 were sequentially transferred into 293T cells.

After the vector transfection was completed, the cells were cultured for 5 days, and then the cell genomic DNA was extracted. The nanobody fragments after library construction were amplified by nested PCR. The sequences of the two pairs of nested PCR primers were as follows: F1: 5'-CTAGAGCCACCATGGCCC-3'; R1: 5'-CCAGGATGTGGCACAGCA-3'; F2: 5'-TCTAGATGGCCCTGCTGCTGCACG-3'; R2: 5'-GAATTCAGGTGCCCTGGTTGTAGC-3'. After the ends of the fragments were amplified, the fragments were ligated to the pET-28a-c(+) vector (Novagen, Cat. No. 69864-3) by enzyme digestion (XbaⅠ/EcoRI) and enzyme ligation (T4 ligase). The size of the cell-displayed nanobody library was calculated to be approximately 2.67×10 ⁶ through steps such as electroporation, plating, picking single clones for sequencing, etc. (see Reference Example 2-5 for specific steps).

The GFP-biotin ligand was co-incubated with the transfected 293T cells for 24 hours, and the positive cells were screened with blastcidin. After the positive cells were selected for monoclonal amplification, the nanobody region was sequenced to obtain the GFP-specific nanobody.

Three nanobody sequences were selected from the obtained positive cells and purified by prokaryotic expression (see Example 8 for protein expression and purification method) to obtain VHH antibody protein.

ELISA was used to detect whether the obtained VHH sequence could recognize eGFP. The specific steps were as follows: 1. The 96-well plate was coated with eGFP protein and washed three times after coating; 2. Three purified VHH antibody proteins were added, and anti-CD19 VHH antibody protein was used as a negative control, and washed three times after coating; 3. HRP-labeled Anti-llama IgG (H+L) secondary antibody was added, and washed three times after coating. 4. TMB colorimetric solution was added to identify whether the obtained VHH antibody protein recognized eGFP. The results are shown in Figure 14a. One of the three antibodies has a good recognition of eGFP.

Afterwards, in order to increase the affinity of the nanobody to the GFP protein, we performed a series of artificial mutations on the three CDR regions of the screened VHH3 gene, and finally obtained a series of nanobodies with different affinities to GFP including the VHH3 gene. Among them, the VHH5 nanobody contains any one of SEQ ID NO.4-13, or any combination of the CDR1, CDR2, and CDR3 regions in SEQ ID NO.4-13.

Example 11: Construction of library based on gRNA-guided base editor positioning and nanobody screening and identification

This example is to construct a library for in vitro base editing based on gRNA-guided base editors in the CDRs region of a nanobody with a known sequence.

The principle is shown in Figure 2. First, a VHH sequence is obtained, and three gRNAs targeting the CDR1, CDR2 and CDR3 regions of VHH are designed. At the same time, the Vivid photosensitive protein (dCas9-Vivid) connected to the inactive Cas9 (dCas9) is designed. Then, two highly active base editors are designed, namely, Vivid-fused cytidine deaminase (promoting the mutation of G or C base to any base) and Vivid-fused adenosine deaminase (promoting the mutation of A base to any base). Under the condition of blue light irradiation, the Vivid photosensitive protein will form a homodimer, so the protein containing the Vivid domain will randomly form a dimer, resulting in the two base editors randomly binding to the dCas9-Vivid protein, thereby localizing in the CDRs region. The gRNAs, dCas9-Vivid and two base editor genes were transferred into 293T cells through the Sleeping Beauty transposon system SB100X to construct a stable transgenic strain. By optimizing the length of the Linker sequence between the Vivid photosensitive protein and the deaminase and the blue light irradiation time, we can induce extensive mutations in the CDRs region of VHH, thereby diversifying the single VHH sequence and building a nanoantibody library. By detecting the diversity of the nanoantibody library, we can determine the length of the Linker sequence between Vivid and the deaminase and the blue light irradiation time.

Vector 2 (see Example 1) was packaged into lentivirus and transformed into 293T cells, and stable transfectants were obtained by puromycin selection.

Construct vector 5 (as shown in FIG. 9 ), wherein the key DNA sequence of vector 5 is shown in SEQ ID NO. 28 (SEQ ID NO. 28 is the sequence between the U6 promoter element and the EF-1α core promoter element in FIG. 9 , and this sequence can be connected to any pLL7 series lentiviral vector having a similar structure);

A lentiviral expression vector 6 is constructed (as shown in FIG. 10 ). The key DNA sequence of vector 6 is shown in SEQ ID NO. 29 (the sequence can be connected to a suitable position of any lentiviral expression vector). In another preferred embodiment, the amino acid sequence expressed by vector 6 has a homology of ≥ 85% with the sequence of SEQ ID NO. 29.

Constructing a lentiviral expression vector 7 (as shown in FIG. 11 ), wherein the key DNA sequence of vector 7 is shown in SEQ ID NO. 30 (the sequence can be connected to a suitable position of any lentiviral expression vector), (in another preferred embodiment, the amino acid sequence expressed by vector 7 has a homology of ≥ 85% with the sequence of SEQ ID NO. 30);

Vectors 5, 6, and 7 were delivered to a gene synthesis company for full gene synthesis.

By packaging lentivirus and constructing a stable 293T cell line, vector 5, vector 6 and vector 7 were sequentially transferred into 293T cells.

The specific steps for detecting the size of the nanobody library are shown in Example 2-5. The size of the detected nanobody library is about 2.75×10 ⁷ .

The GFP-biotin ligand was co-incubated with the transfected 293T cells for 24 hours, and the positive cells were screened with blastcidin. After the positive cells were selected for monoclonal amplification, the nanobody region was sequenced to obtain the GFP-specific nanobody.

Five nanobody sequences were selected from the obtained positive cells and purified by prokaryotic expression (see Example 8 for protein expression and purification method) to obtain VHH antibody protein.

ELISA was used to detect whether the obtained VHH sequences could recognize eGFP. The results are shown in Figure 14b, and 2 of the 5 antibodies had good recognition of eGFP.

Afterwards, in order to increase the affinity of the nanobody to the GFP protein, we performed a series of artificial mutations on the three CDR regions of the two screened VHH genes, and finally obtained a series of nanobodies with different affinities to GFP including these two VHH genes. The VHH nanobodies contain any two of SEQ ID NO.31-40, or any combination of CDR1, CDR2, and CDR3 regions in SEQ ID NO.31-40.

Example 12: Library construction based on gRNA-guided base editor positioning and nanobody screening and identification

This embodiment can construct a library by performing in vitro base editing based on gRNA guidance in the CDRs region of a nanobody with unknown sequence.

The principle is shown in Figure 4. Since the mutation library is constructed in the CDRs region of the nanobody with a known sequence, after several rounds of mutation, the base editor will not be able to recognize the CDRs region due to sequence changes in the gRNA recognition region or the G-quadruplex region, thereby failing to achieve continuous library construction. In addition, affinity maturation mutations cannot be performed after obtaining specific nanobodies. Therefore, we have made improvements based on the scheme in Figure 1, designing three gRNAs targeting the CDR1, CDR2 and right adjacent regions of CDR3 of VHH, respectively, and designing a guide domain containing an inactivated Cas9 (dCas9) and a flexible Linker at the N-terminus of the dCas9 protein to sequentially connect Vivid light-sensitive protein and helicase Mcm7 (the N-terminus is closer to the CDR region). The helicase Mcm7 will form a complete hexamer with helicase activity with Mcm2-6 expressed in 293T cells, and open the double-stranded CDRs region (3'-5' helicase activity) driven by the energy provided by ATP hydrolysis and guide the Vivid light-sensitive protein to one side of the CDRs region. Two highly active base editors are expressed in addition, namely, Vivid-fused cytidine deaminase (promoting mutation of G or C base to any base) and Vivid-fused adenosine deaminase (promoting mutation of A base to any base). Under the condition of blue light irradiation, Vivid photosensitive protein will form a homodimer, causing the two base editors to randomly bind to the Vivid protein of dCas9-Vivid-Mcm7, thereby localizing in the CDRs region. The (dCas9-Vivid-Mcm7) and two base editor genes were transferred into 293T cells through the Sleeping Beauty transposon system SB100X to construct a stable transgenic strain. By optimizing the length of the Linker sequence between dCas9-Vivid-Mcm7, the length of the Linker sequence between Vivid and deaminase, and the blue light irradiation time, extensive mutations were induced in the CDRs region of VHH, and the optimization scheme was determined by detecting the diversity of the nanoantibody library.

Vector 2 was packaged into lentivirus and transformed into 293T cells, and stable transfected cells were obtained by puromycin selection.

Vector 8 was constructed (as shown in FIG. 12 ). The same lentiviral vector as that of vector 1 was used for vector 8. Vector 8 can express a fusion protein comprising three domains: Mcm7 (SEQ ID NO. 41), Vivid (SEQ ID NO. 42), and dCAS9 (the sequence is consistent with or has a homology ≥ 85% with the inactivated CAS9, i.e., dCAS9, in the Streptococcus pyogenes species commonly used in the literature) (in another preferred embodiment, the amino acid sequences of the two domains of Mcm7 and Vivid expressed by vector 8 have a homology ≥ 85% with the sequences of SEQ ID NO. 41 and SEQ ID NO. 42), and the three domains are sequentially connected by a 5×GGGGS Linker (SEQ ID NO. 43) (in another preferred embodiment, the length of the Linker can be optimized between 2×GGGGS and 10×GGGGS).

A lentiviral expression vector 9 (as shown in FIG. 13 ) was constructed. Vector 9 can express a fusion protein comprising three domains of Vivid, rAPOBEC1 and ecTadA*. The amino acid sequence of Vivid is shown in SEQ ID NO.42, the amino acid sequence of ecTadA* is shown in SEQ ID NO.44 (in another preferred example, the amino acid sequence expressed by ecTadA* has a homology of ≥85% with that of SEQ ID NO.44), and the amino acid sequence of rAPOBEC1 is shown in SEQ ID NO.45 (in another preferred example, the amino acid sequence expressed by rAPOBEC1 has a homology of ≥85% with that of SEQ ID NO.45; in another preferred example, rAPOBEC1 can be replaced with cytidine deaminase AID and its mutants). By packaging the lentivirus and constructing a stable 293T cell strain, vectors 8 and 9 are sequentially transferred into 293T cells.

Finally, vector 10 (the vector structure is shown in Figure 9) was transferred into 293T cells. Vector 10 expressed three gRNAs to localize the protein containing the dCAS9 domain to three different positions. The key DNA sequence of vector 10 is shown in SEQ ID NO.46 (SEQ ID NO.46 is the sequence between the U6 promoter element and the EF-1αcore promoter element in Figure 9. This sequence can be connected to any pLL7 series lentiviral vector with a similar structure).

After 2 days of blue light irradiation of 293T cells (on for 5s and off for 10s, blue light intensity 0.84W/m ² ), the size of the nanobody library was calculated. The specific steps for detecting the size of the nanobody library are shown in Example 2-5. The size of the detected nanobody library was about 6.75×10 ⁷ . The GFP-biotin ligand was co-incubated with the transfected 293T cells for 24h, and positive cells were screened with blastcidin. After the positive cells were selected for monoclonal amplification, the nanobody region was sequenced to obtain the GFP-specific nanobody.

Five nanobody sequences were selected from the obtained positive cells and purified by prokaryotic expression (see Example 8 for protein expression and purification method) to obtain VHH antibody protein.

ELISA was used to detect whether the obtained VHH sequences could recognize eGFP. The results are shown in Figure 14c, and one of the five antibodies had good recognition of eGFP.

Afterwards, in order to increase the affinity of the nanobody to the GFP protein, we performed a series of artificial mutations on the three CDR regions of the screened VHH gene, and finally obtained a series of nanobodies with different affinities to GFP including the VHH gene. The VHH nanobody comprises any one of SEQ ID NO.31-40, or any combination of the CDR1, CDR2, and CDR3 regions in SEQ ID NO.31-40.

<110> Xinxiang Medical College

<120> Construction and screening methods of chimeric antigen receptor modified cell libraries with automatic optimization of antigen binding domains and their applications

<160> 46

<170> PatentIn version 3.5

<210> 1

<211> 1416

<212> DNA

<213> Artificial sequence

<221> Receptor 1 sequence

<400> 1

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgattaccc atacgatgtt ccagattacg ctgcggaagc gggtatcacc 120

ggcacgtggt acaaccagca tggttctacc ttcaccgtta ccgcgggtgc ggacggtaac 180

ctgaccggtc agtacgaaaa ccgtgcgcag ggcactggtt gccagaactc tccgtacacc 240

ctgaccggtc gttacaacgg taccaaactg gaatggcgtg ttgaatggaa caactctacc 300

gaaaactgcc actctcgtac cgaatggcgt ggtcagtacc agggtggtgc ggaagcgcgt 360

atcaacaccc agtggaacct gacctacgaa ggtggttctg gtccggcgac cgaacagggt 420

caggacacct tcaccaaagt taaaatgtac ttcagccact tcgtgccggt cttcctgcca 480

gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 540

cagcccctgt ccctgcgccc agaggcgagc cggccagcgg cggggggcgc agtgcacacg 600

aggggggctgg acttcgccag cgatatctac atctgggcgc ccttggccgg gacttgtggg 660

gtccttctcc tgtcactggt tatcaccctt tactgcggag gcggggagag tttgtttaag 720

gggccaaggg actataaccc aatatcatcc actatttgcc acctcactaa cgagagcgat 780

ggacatacaa cctctctcta cgggataggc ttcggtcctt tcatcattac caataagcat 840

ctgtttcgcc gaaacaacgg tactcttctg gttcaatctc ttcatggtgt cttcaaggtg 900

aaaaacacca ctacgcttca gcaacacctg attgatggta gggatatgat aattatcaga 960

atgccgaaag atttcccacc ttttccacag aagctgaaat tcagggaacc gcagagag 1020

gagaggattt gtttggtaac gaccaacttc cagacgaaga gtatgagttc tatggtgtcc 1080

gacactagct gcacgttccc ctcaagtgat gggatattct ggaaacactg gatacagact 1140

aaagacggac agtgtggaag cccattggtt tccacccgag atggttttat cgtgggtatc 1200

catagcgcct ctaatttcac aaacacgaac aactatttca cttcagtgcc caaaaacttt 1260

atggagctgc tcacaaacca agaagcgcag cagtgggtaa gcggttggag acttaacgct 1320

gactcagttc tctggggggg gcacaaagta ttcatggtaa agccagagga gccattccaa 1380

ccagtcaaag aagccacaca acttatgaac agctaa 1416

<210> 2

<211> 2394

<212> DNA

<213> Artificial sequence

<221> Receptor 2 sequence 1

<400> 2

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgactaca aggacgacga cgacaagccc gggatggccc aggtgcagct ggtggagagc 120

ggcggcggcc tggtgcaggc cggcggcagc ctgaggctga gctgcgccgc cccggggcgg 180

gccgggggcg gggtcccggc ggggtggttc aggcaggccc ccggcaagga gagggagttc 240

gtggccgcgg ggcgcttatg gggagggtgg ggagggtggg gaaggtgggg aggaggcagg 300

ttcaccatca gcagggacaa cgacaagaac accgtgtacg tgcagatgaa cagcctgatc 360

cccgaggaca ccgccatcta ctactgcgcc gcccgcggag gggcgggcgc gggaggaagg 420

gggcgggagc ggggctgtgg cgactactgg ggccagggca cccaggtgac cgtggggatcc 480

ccctgcgtgg gcagcaaccc ctgctacaac cagggcacct gcgagcccac cagcgagaac 540

cccttctaca ggtgcctgtg ccccgccaag ttcaacggcc tgctgtgcca catcctggac 600

tacagcttca ccggcggcgc cggcagggac atcccccccc cccagatcga ggaggcctgc 660

gagctgcccg agtgccaggt ggacgccggc aacaaggtgt gcaacctgca gtgcaacaac 720

cacgcctgcg gctgggacgg cggcgactgc agcctgaact tcaacgaccc ctggaagaac 780

tgcacccaga gcctgcagtg ctggaagtac ttcagcgacg gccactgcga cagccagtgc 840

aacagcgccg gctgcctgtt cgacggcttc gactgccagc tgaccgaggg ccagtgcaac 900

cccctgtacg accagtactg caaggaccac ttcagcgacg gccactgcga ccagggctgc 960

aacagcgccg agtgcgagtg ggacggcctg gactgcgccg agcacgtgcc cgagaggctg 1020

gccgccggca ccctggtgct ggtggtgctg ctgccccccg accagctgag gaacaacagc 1080

ttccacttcc tgagggagct gagccacgtg ctgcacacca acgtggtgtt caagagggac 1140

gcccagggcc agcagatgat cttcccctac tacggccacg aggaggagct gaggaagcac 1200

cccatcaaga ggagcaccgt gggctgggcc accagcagcc tgctgcccgg caccagcggc 1260

ggcaggcaga ggagggagct ggaccccatg gacatcaggg gcagcatcgt gtacctggag 1320

atcgacaaca ggcagtgcgt gcagagcagc agccagtgct tccagagcgc caccgacgtg 1380

gccgccttcc tgggcgccct ggccagcctg ggcagcctga acatccccta caagatcgag 1440

gcccataaga gcgagcccgt ggagcccccc ctgcccagcc agctgcacct gatgtacgtg 1500

gccgccgccg ccttcgtgct gctgttcttc gtgctccttt tctttctgct gagcaggaag 1560

aggaggaggc agctgtgcat ccagaagctg ctcggggatcg agggaagggg aggaggcgag 1620

ttcgctagcg agaacctgta tttccagggc atgtctagac tggacaagag caaagtcata 1680

aactctgctc tggaattact caatgaagtc ggtatcgaag gcctgacgac aaggaaactc 1740

gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc 1800

ctgctcgatg ccctggcaat cgagatgctg gacaggcatc atacccactt ctgccccctg 1860

gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc 1920

ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac 1980

gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca 2040

ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggatcaggag 2100

catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg 2160

agacaagcaa ttgagctgtt cgaccatcag ggagccgaac ctgccttcct tttcggcctg 2220

gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac 2280

gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt 2340

gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc aggg 2394

<210> 3

<211> 1584

<212> DNA

<213> Artificial sequence

<221> Receptor 2 sequence 2

<400> 3

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

ccctacccat acgatgttcc agattacgct cccggggatgg cccaggtgca gctggtggag 120

agcggcggcg gcctggtgca ggccggcggc agcctgaggc tgagctgcgc cgccagcggc 180

aggaccttca gcaactacgc catgggctgg ttcaggcagg cccccggcaa ggagaggggag 240

ttcgtggccg ccatcagctg gaccggcgtg agcacctact acgccgacag cgtgaagggc 300

aggttcacca tcagcaggga caacgacaag aacaccgtgt acgtgcagat gaacagcctg 360

atccccgagg acaccgccat ctactactgc gccgccgtga gggccaggag cttcagcgac 420

acctacagca gggtgaacga gtacgactac tggggccagg gcacccaggt gaccgtggga 480

tccatgtact tcagccactt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 540

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 600

gaggcgagcc ggccagcggc gggggcgca gtgcacacga gggggctgga cttcgccagc 660

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 720

atcacccttt actgcaattc gagctcgaac aacaacaaca ataacaataa caacaacctc 780

gggatcgagg gaaggggagg aggcgagttc gctagcgaga acctgtattt ccagggcatg 840

tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tgaagtcggt 900

atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg 960

tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tggcaatcga gatgctggac 1020

aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac 1080

aacgccaagt cattccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc 1140

ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt 1200

cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca 1260

ctgggctgcg tattggagga tcaggagcat caagtagcaa aagaggaaag agagacacct 1320

accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccatcaggga 1380

gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta 1440

aagtgcgaaa gcggcgggcc ggccgacgcc cttgacgatt ttgacttaga catgctccca 1500

gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt 1560

gaccttgaca tgctcccagg gtaa 1584

<210> 4

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 1

<400> 4

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Arg Gly Trp Gly Gly Trp Arg Arg

50 55 60

Trp Gly Gly Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Thr Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu

100 105 110

Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 5

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 2

<400> 5

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 6

<211> 124

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 3

<400> 6

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val

65 70 75 80

Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys

100 105 110

Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 7

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 4

<400> 7

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg

100 105 110

Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 8

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 5

<400> 8

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg

100 105 110

Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 9

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 6

<400> 9

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg Ala Lys Asn Thr

65 70 75 80

Val Asp Leu Gln Met Asp Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ser Ala Arg Ser Arg Gly Tyr Val Leu Ser Val Leu Arg Ser

100 105 110

Val Asp Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 10

<211> 124

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 7

<400> 10

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala

1 5 10 15

Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val

65 70 75 80

Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys

100 105 110

Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 11

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 8

<400> 11

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 12

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 9

<400> 12

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala

1 5 10 15

Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu

100 105 110

Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 13

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 10

<400> 13

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Tyr Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 14

<211> 4946

<212> DNA

<213> Artificial sequence

<221> Vector 1 key sequence

<400> 14

tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60

aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120

taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180

atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240

cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360

ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420

tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480

gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540

gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600

ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660

cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720

ttggggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780

cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840

cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900

cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960

ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020

ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080

acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140

gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200

gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260

ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320

cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380

cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440

caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500

gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560

gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680

aagtctccac cccattgacg tcaatggggag tttgttttgg caccaaaatc aacgggactt 1740

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800

ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860

acgctgtttt gacctccata gaagattcta gagccaccat ggccctgcta ctggccctca 1920

gcctgctggt tctctggact tccccagccc caactctgag tggcaccaat gatgactaca 1980

aagacgatga cgacaaggcg gaagcgggta tcaccggcac gtggtacaac cagcatggtt 2040

ctaccttcac cgttaccgcg ggtgcggacg gtaacctgac cggtcagtac gaaaaccgtg 2100

cgcagggcac tggttgccag aactctccgt acaccctgac cggtcgttac aacggtacca 2160

aactggaatg gcgtgttgaa tggaacaact ctaccgaaaa ctgccactct cgtaccgaat 2220

ggcgtggtca gtaccagggt ggtgcggaag cgcgtatcaa cacccagtgg aacctgacct 2280

acgaaggtgg ttctggtccg gcgaccgaac agggtcagga caccttcacc aaagttaaaa 2340

tgtacttcag ccacttcgtg ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc 2400

cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg 2460

cgagccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gccagcgata 2520

tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 2580

ccctttactg cggaggcggg gagagtttgt ttaaggggcc aagggactat aacccaatat 2640

catccactat ttgccacctc actaacgaga gcgatggaca tacaacctct ctctacggga 2700

taggcttcgg tcctttcatc attaccaata agcatctgtt tcgccgaaac aacggtactc 2760

ttctggttca atctcttcat ggtgtcttca aggtgaaaaa caccactacg cttcagcaac 2820

acctgattga tggtagggat atgataatta tcagaatgcc gaaagatttc ccaccttttc 2880

cacagaagct gaaattcagg gaaccgcaga gagaggagag gatttgtttg gtaacgacca 2940

acttccagac gaagagtatg agttctatgg tgtccgacac tagctgcacg ttcccctcaa 3000

gtgatggggat attctggaaa cactggatac agactaaaga cggacagtgt ggaagcccat 3060

tggtttccac ccgagatggt tttatcgtgg gtatccatag cgcctctaat ttcacaaaca 3120

cgaacaacta tttcacttca gtgcccaaaa actttatgga gctgctcaca aaccaagaag 3180

cgcagcagtg ggtaagcggt tggagactta acgctgactc agttctctgg ggggggcaca 3240

aagtattcat ggtaaagcca gaggagccat tccaaccagt caaagaagcc acacaactta 3300

tgaacagcga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc 3360

caatggccct gcccgtgacc gccctgctgc tgcccctggc cctgctgctg cacgccgcca 3420

ggccctaccc atacgatgtt ccagattacg ctcccgggat ggcccaggtg cagctggtgg 3480

agagcggcgg cggcctggtg caggccggcg gcagcctgag gctgagctgc gccgccagcg 3540

gcaggacctt cagcaactac gccatgggct ggttcaggca ggcccccggc aaggagaggg 3600

agttcgtggc cgccatcagc tggaccggcg tgagcaccta ctacgccgac agcgtgaagg 3660

gcaggttcac catcagcagg gacaacgaca agaacaccgt gtacgtgcag atgaacagcc 3720

tgatccccga ggacaccgcc atctactact gcgccgccgt gagggccagg agcttcagcg 3780

acacctacag cagggtgaac gagtacgact actggggcca gggcacccag gtgaccgtgg 3840

gatccatgta cttcagccac ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc 3900

cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc 3960

cagaggcgag ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcca 4020

gcgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc ctgtcactgg 4080

ttatcaccct ttactgcaat tcgagctcga acaacaacaa caataacaat aacaacaacc 4140

tcgggatcga gggaagggga ggaggcgagt tcgctagcga gaacctgtat ttccagggca 4200

tgtctagact ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg 4260

gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc 4320

tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg 4380

acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga 4440

acaacgccaa gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc 4500

tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt 4560

gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta 4620

cactgggctg cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac 4680

ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg 4740

gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc 4800

taaagtgcga aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc 4860

cagccgatgc ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt 4920

ttgaccttga catgctccca gggtaa 4946

<210> 15

<211> 5762

<212> DNA

<213> Artificial sequence

<221> Vector 2 key sequence

<400> 15

tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60

aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120

taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180

atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240

cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360

ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420

tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480

gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540

gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600

ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660

cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720

ttggggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780

cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840

cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900

cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960

ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020

ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080

acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140

gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200

gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260

ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320

cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380

cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440

caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500

gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560

gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680

aagtctccac cccattgacg tcaatggggag tttgttttgg caccaaaatc aacgggactt 1740

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800

ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860

acgctgtttt gacctccata gaagattcta gagccaccat ggccctgccc gtgaccgccc 1920

tgctgctgcc cctggccctg ctgctgcacg ccgccaggcc cgactacaag gacgacgacg 1980

acaagcccgg gatggcccag gtgcagctgg tggagagcgg cggcggcctg gtgcaggccg 2040

gcggcagcct gaggctgagc tgcgccgccc cggggcgggc cgggggcggg gtcccggcgg 2100

ggtggttcag gcaggccccc ggcaaggaga gggagttcgt ggccgcgggg cgcttatggg 2160

gagggtgggg agggtgggga aggtggggag gaggcaggtt caccatcagc agggacaacg 2220

acaagaacac cgtgtacgtg cagatgaaca gcctgatccc cgaggacacc gccatctact 2280

actgcgccgc ccgcggaggg gcgggcgcgg gaggaagggg gcggggagcgg ggctgtggcg 2340

actactgggg ccagggcacc caggtgaccg tgggatcccc ctgcgtgggc agcaacccct 2400

gctacaacca gggcacctgc gagcccacca gcgagaaccc cttctacagg tgcctgtgcc 2460

ccgccaagtt caacggcctg ctgtgccaca tcctggacta cagcttcacc ggcggcgccg 2520

gcagggacat cccccccccc cagatcgagg aggcctgcga gctgcccgag tgccaggtgg 2580

acgccggcaa caaggtgtgc aacctgcagt gcaacaacca cgcctgcggc tgggacggcg 2640

gcgactgcag cctgaacttc aacgacccct ggaagaactg cacccagc ctgcagtgct 2700

ggaagtactt cagcgacggc cactgcgaca gccagtgcaa cagcgccggc tgcctgttcg 2760

acggcttcga ctgccagctg accgagggcc agtgcaaccc cctgtacgac cagtactgca 2820

aggaccactt cagcgacggc cactgcgacc agggctgcaa cagcgccgag tgcgagtggg 2880

acggcctgga ctgcgccgag cacgtgcccg agaggctggc cgccggcacc ctggtgctgg 2940

tggtgctgct gccccccgac cagctgagga acaacagctt ccacttcctg agggagctga 3000

gccacgtgct gcacaccaac gtggtgttca agagggacgc ccagggccag cagatgatct 3060

tcccctacta cggccacgag gaggagctga ggaagcaccc catcaagagg agcaccgtgg 3120

gctgggccac cagcagcctg ctgcccggca ccagcggcgg caggcagagg agggagctgg 3180

accccatgga catcaggggc agcatcgtgt acctggagat cgacaacagg cagtgcgtgc 3240

agagcagcag ccagtgcttc cagagcgcca ccgacgtggc cgccttcctg ggcgccctgg 3300

ccagcctggg cagcctgaac atcccctaca agatcgaggc ccataagagc gagcccgtgg 3360

agccccccct gcccagccag ctgcacctga tgtacgtggc cgccgccgcc ttcgtgctgc 3420

tgttcttcgt gctccttttc tttctgctga gcaggaagag gaggaggcag ctgtgcatcc 3480

agaagctgct cgggatcgag ggaaggggag gaggcgagtt cgctagcgag aacctgtatt 3540

tccagggcat gtctagactg gacaagagca aagtcataaa ctctgctctg gaattactca 3600

atgaagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc 3660

agcctaccct gtactggcac gtgaagaaca agcgggcct gctcgatgcc ctggcaatcg 3720

agatgctgga caggcatcat accccacttct gccccctgga aggcgagtca tggcaagact 3780

ttctgcggaa caacgccaag tcattccgct gtgctctcct ctcacatcgc gacggggcta 3840

aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg 3900

cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg 3960

gccactttac actgggctgc gtattggagg atcaggagca tcaagtagca aaagaggaaa 4020

gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg 4080

accatcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg 4140

agaaacagct aaagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag 4200

acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc 4260

ttgacgattt tgaccttgac atgctcccag gggagggcag aggaagtctg ctaacatgcg 4320

gtgacgtcga ggagaatcct ggcccaatgg ccctgctact ggccctcagc ctgctggttc 4380

tctggacttc cccagcccca actctgagtg gcaccaatga ttacccatac gatgttccag 4440

attacgctgc ggaagcgggt atcaccggca cgtggtacaa ccagcatggt tctaccttca 4500

ccgttaccgc gggtgcggac ggtaacctga ccggtcagta cgaaaaccgt gcgcagggca 4560

ctggttgcca gaactctccg tacaccctga ccggtcgtta caacggtacc aaactggaat 4620

ggcgtgttga atggaacaac tctaccgaaa actgccactc tcgtaccgaa tggcgtggtc 4680

agtaccaggg tggtgcggaa gcgcgtatca acacccagtg gaacctgacc tacgaaggtg 4740

gttctggtcc ggcgaccgaa cagggtcagg acaccttcac caaagttaaa atgtacttca 4800

gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 4860

caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc 4920

cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgccagcgat atctacatct 4980

gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttat 5040

gcggaggcgg ggagagtttg tttaaggggc caagggacta taacccaata tcatccacta 5100

tttgccacct cactaacgag agcgatggac atacaacctc tctctacggg ataggcttcg 5160

gtcctttcat cattaccaat aagcatctgt ttcgccgaaa caacggtact cttctggttc 5220

aatctcttca tggtgtcttc aaggtgaaaa acaccactac gcttcagcaa cacctgattg 5280

atggtaggga tatgataatt atcagaatgc cgaaagattt cccacctttt ccacagaagc 5340

tgaaattcag ggaaccgcag agagaggaga ggatttgttt ggtaacgacc aacttccaga 5400

cgaagagtat gagttctatg gtgtccgaca ctagctgcac gttcccctca agtgatggga 5460

tattctggaa acactggata cagactaaag acggacagtg tggaagccca ttggtttcca 5520

cccgagatgg ttttatcgtg ggtatccata gcgcctctaa tttcacaaac acgaacaact 5580

atttcacttc agtgcccaaa aactttatgg agctgctcac aaaccaagaa gcgcagcagt 5640

gggtaagcgg ttggagactt aacgctgact cagttctctg gggggggcac aaagtattca 5700

tggtaaagcc agaggagcca ttccaaccag tcaaagaagc cacacaactt atgaacagct 5760

aa5762

<210> 16

<211> 124

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 1

<400> 16

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Ala Ile Ser Ile Asp Gly Ser His Thr Thr Tyr Thr Ala Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Leu Arg Leu Gln Tyr Gly Leu Ala Tyr Asp Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 2

<400> 17

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Tyr Phe Val Ile Pro Asp Ala Gln

20 25 30

His Gly Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Asn Lys Asp Thr Gly Cys Tyr His Val Ala Arg Leu Pro Met Glu

50 55 60

Gln Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Thr Arg Glu Ile His Gly Lys Met Gln Trp Leu Phe Ser Val

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 18

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 3

<400> 18

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Pro Phe Ser Leu Gly Cys Gln Glu

20 25 30

Arg Ala Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Thr Glu Asn Lys His Ala Pro Leu Val Gln Tyr Asp Trp Ile Gly

50 55 60

Met Cys Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Met Asn Arg Ala Ile Glu Asp Gly Phe Lys Cys Ser Trp Gln

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 19

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 4

<400> 19

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Trp Thr Gly Pro Asp Arg Glu Val

20 25 30

Met Phe Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Thr Gln Ile Met Asp Arg Leu Phe Lys Val Pro Asn Ala Ser Glu

50 55 60

Gly Trp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Trp Cys Ala Arg Val His Gly Asp Pro Asn Phe Tyr Met Ile Glu

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 20

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 5

<400> 20

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Cys Tyr Gly Ile His Val Trp Asn

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Asn Met Gln Pro Leu Ser Ala Ile Arg Glu Phe Trp Tyr Cys Val

50 55 60

His Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Cys Phe Pro Thr Asn Arg Tyr Glu His Met Asp Leu Gln Ile

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 21

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 6

<400> 21

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Tyr Phe Asn Glu Trp His Gln Pro

20 25 30

Thr Gly Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Cys Glu Ala Trp Pro Ile Ser Asp Thr Asn Met Phe Tyr Gln Leu

50 55 60

His Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Phe Ala Pro Gln Glu Ser Leu Asp Gly Ile Asn Arg Trp Tyr Lys

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 22

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 7

<400> 22

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala His Ile Phe Pro Thr Gly Trp Asn

20 25 30

Cys Lys Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Glu Met Thr Ile Tyr Ala Gly Val Trp His Asp Cys Pro Gln Lys

50 55 60

Asn Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Met Cys Asn Arg Val Pro Phe Glu Ala Thr Gly Asp Leu Trp

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 23

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 8

<400> 23

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Lys Leu Phe Val Asn Gly Cys Tyr

20 25 30

Ser His Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Ala Cys Ser Gly Val Ile Pro Asp Met His Thr Gln Glu Leu Trp

50 55 60

Tyr Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Cys Arg Ser Phe Asn Ile Gln Gly Met Asp Thr Tyr Val Trp His

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 24

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 9

<400> 24

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Met Val Tyr Pro Ala Thr Glu Ile

20 25 30

His Trp Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Tyr Asp Ala Glu Met Trp Lys Gln Thr Asn Arg Val Gly His Cys

50 55 60

Ser Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Tyr Val Ala Gly Gln Leu Asn Asp Cys Pro Glu Met Lys Phe His

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 25

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 10

<400> 25

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Phe Thr Trp Glu Arg Gly Ile Pro

20 25 30

Ser Ala Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Glu Lys Asn Pro Thr Ser Phe Cys Leu Tyr Ile Asp Ala Gly Met

50 55 60

Trp Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Arg Lys Ala Cys Phe Ser Met Gln Val Pro Ile Asp His Glu

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 26

<211> 3378

<212> DNA

<213> Artificial sequence

<221> Carrier 3

<400> 26

gactacaaag acgatgacga caagtccgaa gtcgagtttt cccatgagta ctggatgaga 60

cacgcattga ctctcgcaaa gagggcttgg gatgaacgcg aggtgcccgt gggggcagta 120

ctcgtgcata acaatcgcgt aatcggcgaa ggttggaata ggccgatcgg acgccacgac 180

cccactgcac atgcggaaat catggccctt cgacaggggag ggcttgtgat gcagaattat 240

cgacttatcg atgcgacgct gtacgtcacg cttgaacctt gcgtaatgtg cgcggggagct 300

atgattcact cccgcattgg acgagttgta ttcggtgccc gcgacgccaa gacgggtgcc 360

gcaggttcac tgatggacgt gctgcatcac ccaggcatga accaccgggt agaaatcaca 420

gaaggcatat tggcggacga atgtgcggcg ctgttgtccg acttttttcg catgcggagg 480

caggagatca aggcccagaa aaaagcacaa tcctctactg actctggtgg ttcttctggt 540

ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt 600

ggttcttctg gtggttcttc cgaagtcgag ttttcccatg agtactggat gagacacgca 660

ttgactctcg caaagagggc tcgagatgaa cgcgaggtgc ccgtgggggc agtactcgtg 720

ctcaacaatc gcgtaatcgg cgaaggttgg aatagggcaa tcggactcca cgaccccact 780

gcacatgcgg aaatcatggc ccttcgacag ggagggcttg tgatgcagaa ttatcgactt 840

atcgatgcga cgctgtacgt cacgtttgaa ccttgcgtaa tgtgcgcggg agctatgatt 900

cactcccgca ttggacgagt tgtattcggt gttcgcaacg ccaagacggg tgccgcaggt 960

tcactgatgg acgtgctgca ttacccaggc atgaaccacc gggtagaaat cacagaaggc 1020

atattggcgg acgaatgtgc ggcgctgttg tgttatactttt ttcgcatgcc caggcaggtc 1080

tttaacgccc agaaaaaagc acaatcctct actgactctg gtggttcttc tggtggttct 1140

agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagttc tggtggttct 1200

tctggtggtt ctatggtgaa gctcgcgaag gcaggtaaaa atcaaggtga ccccaagaaa 1260

atggctcctc ctccaaagga ggtagaagaa gatagtgaag atgaggaaat gtcagaagat 1320

gaagaagatg atagcagtgg agaagaggtc gtcatacctc agaagaaagg caagaaggct 1380

gctgcaacct cagcaaagaa ggtggtcgtt tccccaacaa aaaaggttgc agttgccaca 1440

ccagccaaga aagcagctgt cactccaggc aaaaaggcag cagcaacacc tgccaagaag 1500

acagttacac cagccaaagc agttaccaca cctggcaaga agggagccac accaggcaaa 1560

gcattggtag caactcctgg taagaagggt gctgccatcc cagccaaggg ggcaaagaat 1620

ggcaagaatg ccaagaagga agacagtgat gaagaggagg atgatgacag tgaggaggat 1680

gaggaggatg acgaggacga ggatgaggat gaagatgaaa ttgaaccagc agcgatgaaa 1740

gcagcagctg ctgcccctgc ctcagaggat gaggacgatg aggatgacga agatgatgag 1800

gatgacgatg acgatgagga agatgactct gaagaagaag ctatggagac tacaccagcc 1860

aaaggaaaga aagctgcaaa agttgttcct gtgaaagcca agaacgtggc tgaggatgaa 1920

gatgaagaag aggatgatga ggacgaggat gacgacgacg acgaagatga tgaagatgat 1980

gatgatgaag atgatgagga ggaggaagaa gaggaggagg aagagcctgt caaagaagca 2040

cctggaaaac gaaagaagga aatggccaaa cagaaagcag ctcctgaagc caagaaacag 2100

aaagtggaag gcacagaacc gactacggct ttcaatctct ttgttggaaa cctaaacttt 2160

aacaaatctg ctcctgaatt aaaaactggt atcagcgatg tttttgctaa aaatgatctt 2220

gctgttgtgg atgtcagaat tggtatgact aggaaatttg gttatgtgga ttttgaatct 2280

gctgaagacc tggagaaagc gttggaactc actggtttga aagtctttgg caatgaaatt 2340

aaactagaga aaccaaaagg aaaagacagt aagaaagagc gagatgcgag aacacttttg 2400

gctaaaaatc tcccttacaa agtcactcag gatgaattga aagaagtgtt tgaagatgct 2460

gcggagatca gattagtcag caaggatggg aaaagtaaag ggattgctta tattgaattt 2520

aagacagaag ctgatgcaga gaaaaccttt gaagaaaagc agggaacaga gatcgatggg 2580

cgatctattt ccctgtacta tactggagag aaaggtcaaa atcaagacta tagaggtgga 2640

aagaatagca cttggagtgg tgaatcaaaa actctggttt taagcaacct ctcctacagt 2700

gcaacagaag aaactcttca ggaagtattt gagaaagcaa cttttatcaa agtaccccag 2760

aaccaaaatg gcaaatctaa agggtatgca tttatagagt ttgcttcatt cgaagacgct 2820

aaagaagctt taaattcctg taataaaagg gaaattgagg gcagagcaat caggctggag 2880

ttgcaaggac ccaggggatc acctaatgcc agaagccagc catccaaaac tctgtttgtc 2940

aaaggcctgt ctgaggatac cactgaagag acattaaagg agtcatttga cggctccgtt 3000

cgggcaagga tagttactga ccgggaaact gggtcctcca aagggtttgg ttttgtagac 3060

ttcaacagtg aggaggatgc caaagctgcc aaggaggcca tggaagacgg tgaaattgat 3120

ggaaataaag ttaccttgga ctgggccaaa cctaagggtg aaggtggctt cgggggtcgt 3180

ggtggaggca gaggcggctt tggaggacga ggtggtggta gaggaggccg aggaggattt 3240

ggtggcagag gccggggagg ctttggaggg cgaggaggct tccgaggagg cagaggagga 3300

ggaggtgacc acaagccaca aggaaagaag acgaagtttg aatctggtgg ttctcccaag 3360

aagaagagga aagtctag 3378

<210> 27

<211> 3183

<212> DNA

<213> Artificial sequence

<221> Carrier 4

<400> 27

gactacaaag acgatgacga caagagctca gagactggcc cagtggctgt ggaccccaca 60

ttgagacggc ggatcgagcc ccatgagttt gaggtattct tcgatccgag agagctccgc 120

aaggagacct gcctgcttta cgaaattaat tgggggggcc ggcactccat ttggcgacat 180

acatcacaga acactaacaa gcacgtcgaa gtcaacttca tcgagaagtt cacgacagaa 240

agatatttct gtccgaacac aaggtgcagc attacctggt ttctcagctg gagcccatgc 300

ggcgaatgta gtagggccat cactgaattc ctgtcaaggt atccccacgt cactctgttt 360

atttacatcg caaggctgta ccaccacgct gacccccgca atcgacaagg cctgcgggat 420

ttgatctctt caggtgtgac tatccaaatt atgactgagc aggagtcagg atactgctgg 480

agaaactttg tgaattatag cccgagtaat gaagcccact ggcctaggta tccccatctg 540

tgggtacgac tgtacgttct tgaactgtac tgcatcatac tgggcctgcc tccttgtctc 600

aacattctga gaaggaagca gccacagctg acattcttta ccatcgctct tcagtcttgt 660

cattaccagc gactgccccc acacattctc tgggccaccg ggttgaaaag cggcagcgag 720

actcccggga cctcagagtc cgccacaccc gaaagtatgg tgaagctcgc gaaggcaggt 780

aaaaatcaag gtgaccccaa gaaaatggct cctcctccaa aggaggtaga agaagatagt 840

gaagatgagg aaatgtcaga agatgaagaa gatgatagca gtggagaaga ggtcgtcata 900

cctcagaaga aaggcaagaa ggctgctgca acctcagcaa agaaggtggt cgtttcccca 960

acaaaaaagg ttgcagttgc cacaccagcc aagaaagcag ctgtcactcc aggcaaaaag 1020

gcagcagcaa cacctgccaa gaagacagtt acaccagcca aagcagttac cacacctggc 1080

aagaagggag ccacaccagg caaagcattg gtagcaactc ctggtaagaa gggtgctgcc 1140

atcccagcca agggggcaaa gaatggcaag aatgccaaga aggaagacag tgatgaagag 1200

gaggatgatg acagtgagga ggatgaggag gatgacgagg acgaggatga ggatgaagat 1260

gaaattgaac cagcagcgat gaaagcagca gctgctgccc ctgcctcaga ggatgaggac 1320

gatgaggatg acgaagatga tgaggatgac gatgacgatg aggaagatga ctctgaagaa 1380

gaagctatgg agactacacc agccaaagga aagaaagctg caaaagttgt tcctgtgaaa 1440

gccaagaacg tggctgagga tgaagatgaa gaagaggatg atgaggacga ggatgacgac 1500

gacgacgaag atgatgaaga tgatgatgat gaagatgatg aggaggagga agaagaggag 1560

gaggaagagc ctgtcaaaga agcacctgga aaacgaaaga aggaaatggc caaacagaaa 1620

gcagctcctg aagccaagaa acagaaagtg gaaggcacag aaccgactac ggctttcaat 1680

ctctttgttg gaaacctaaa ctttaacaaa tctgctcctg aattaaaaac tggtatcagc 1740

gatgtttttg ctaaaaatga tcttgctgtt gtggatgtca gaattggtat gactaggaaa 1800

tttggttatg tggattttga atctgctgaa gacctggaga aagcgttgga actcactggt 1860

ttgaaagtct ttggcaatga aattaaacta gagaaaccaa aaggaaaaga cagtaagaaa 1920

gagcgagatg cgagaacact tttggctaaa aatctccctt acaaagtcac tcaggatgaa 1980

ttgaaagaag tgtttgaaga tgctgcggag atcagattag tcagcaagga tgggaaaagt 2040

aaagggattg cttatattga atttaagaca gaagctgatg cagagaaaac ctttgaagaa 2100

aagcagggaa cagagatcga tgggcgatct atttccctgt actatactgg agagaaaggt 2160

caaaatcaag actatagagg tggaaagaat agcacttgga gtggtgaatc aaaaactctg 2220

gttttaagca acctctccta cagtgcaaca gaagaaactc ttcaggaagt atttgagaaa 2280

gcaactttta tcaaagtacc ccagaaccaa aatggcaaat ctaaagggta tgcatttata 2340

gagtttgctt cattcgaaga cgctaaagaa gctttaaatt cctgtaataa aagggaaatt 2400

gagggcagag caatcaggct ggagttgcaa ggacccaggg gatcacctaa tgccagaagc 2460

cagccatcca aaactctgtt tgtcaaaggc ctgtctgagg ataccactga agagacatta 2520

aaggagtcat ttgacggctc cgttcgggca aggatagtta ctgaccggga aactgggtcc 2580

tccaaagggt ttggttttgt agacttcaac agtgaggagg atgccaaagc tgccaaggag 2640

gccatggaag acggtgaaat tgatggaaat aaagttacct tggactgggc caaacctaag 2700

ggtgaaggtg gcttcggggg tcgtggtgga ggcagaggcg gctttggagg acgaggtggt 2760

ggtagaggag gccgaggagg atttggtggc agaggccggg gaggctttgg agggcgagga 2820

ggcttccgag gaggcagagg aggaggaggt gaccacaagc cacaaggaaa gaagacgaag 2880

tttgaatctg gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa 2940

ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 3000

aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 3060

atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 3120

aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc 3180

taa 3183

<210> 28

<211> 589

<212> DNA

<213> Artificial sequence

<221> Carrier 5

<400> 28

gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60

cccgggttcg attcccggct ggtgcacccc gccgggaccc cgccccgttt tagagctaga 120

aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180

gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240

tcgattcccg gctggtgcag agggtgggga gggtggggag ttttagagct agaaatagca 300

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360

agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420

ccggctggtg cactcccgcg cccgcccctc cggttttaga gctagaaata gcaagttaaa 480

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcttttttggatcc 540

agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589

<210> 29

<211> 5328

<212> PRT

<213> Artificial sequence

<221> Carrier 6

<400> 29

atgtccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 60

agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 120

atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 180

atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 240

tacgtcacgc ttgaaccttg cgtaatgtgc gcggggagcta tgattcactc ccgcattgga 300

cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 360

ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 420

tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 480

aaagcacaat cctctactga ctctggtggt tcttctggtg gttctagcgg cagcgagact 540

cccgggacct cagagtccgc cacacccgaa agttctggtg gttcttctgg tggttcttcc 600

gaagtcgagt tttcccatga gtactggatg agacacgcat tgactctcgc aaagagggct 660

cgagatgaac gcgaggtgcc cgtgggggca gtactcgtgc tcaacaatcg cgtaatcggc 720

gaaggttgga atagggcaat cggactccac gaccccactg cacatgcgga aatcatggcc 780

cttcgacagg gagggcttgt gatgcagaat tatcgactta tcgatgcgac gctgtacgtc 840

acgtttgaac cttgcgtaat gtgcgcggga gctatgattc actcccgcat tggacgagtt 900

gtattcggtg ttcgcaacgc caagacgggt gccgcaggtt cactgatgga cgtgctgcat 960

tacccaggca tgaaccaccg ggtagaaatc acagaaggca tattggcgga cgaatgtgcg 1020

gcgctgttgt gttacttttt tcgcatgccc aggcaggtct ttaacgccca gaaaaaagca 1080

caatcctcta ctgactctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1140

acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1200

tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1260

tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 1320

aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1380

aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1440

atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1500

ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1560

gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1620

tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1680

cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1740

ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1800

ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1860

ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1920

tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1980

cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 2040

cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 2100

atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 2160

tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 2220

gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2280

ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 2340

gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 2400

ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 2460

aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 2520

acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 2580

atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 2640

ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 2700

gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2760

acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2820

aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2880

aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2940

gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 3000

gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 3060

ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 3120

gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 3180

aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 3240

agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 3300

aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 3360

gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 3420

gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3480

cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 3540

gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3600

caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 3660

gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa 3720

tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3780

gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3840

cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3900

aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3960

cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 4020

gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 4080

gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 4140

cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 4200

gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 4260

agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 4320

ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 4380

accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 4440

aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 4500

ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 4560

gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta 4620

gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 4680

gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 4740

aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4800

gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4860

gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4920

ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4980

tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5040

gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 5100

caggcggaaa atattatcca tttgtttatctttaccaacc tcggcgctcc agccgcattc 5160

aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5220

gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 5280

cttgggggtg actctggtgg ttctcccaag aagaagagga aagtctaa 5328

<210> 30

<211> 5130

<212> DNA

<213> Artificial sequence

<221> Carrier 7

<400> 30

atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60

catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120

gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180

cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gattatttctg tccgaacaca 240

aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300

actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360

caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420

atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480

ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540

gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600

ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660

cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720

gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780

ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840

aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900

gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960

aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020

ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080

cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140

cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200

gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260

gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320

gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380

tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440

ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500

gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560

aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620

agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680

ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740

gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800

aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860

aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920

gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980

ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040

cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100

cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg 2160

aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220

aactttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280

tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340

cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400

cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460

tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520

ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580

gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640

aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700

acgggctggg gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760

aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg 2820

atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880

ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940

atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000

gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060

agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120

aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180

caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240

gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300

cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360

aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420

ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480

attaaacgtc agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat 3540

tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600

actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660

gagataaata actaccacca tgcgcacgac gctttatctta atgccgtcgt agggaccgca 3720

ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780

gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840

ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900

atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960

ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020

aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080

gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140

cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200

ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260

aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320

aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380

gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440

tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500

ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560

agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620

cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680

aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740

acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800

gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860

gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920

ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980

gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040

aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100

ggtggttctc ccaagaagaa gaggaaagtc 5130

<210> 31

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 1

<400> 31

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Leu Thr Ala Trp Phe

20 25 30

Arg Gln Ile His Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ser Trp Val Pro Ala Lys Met Phe Cys Gln Ile Leu Thr Asp

50 55 60

Glu Arg Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Leu Glu Phe His Arg Met Lys Asp Val Trp Ser Gly Pro

100 105 110

Tyr Ala Asn Gln Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 32

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 2

<400> 32

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Asn Leu Gln Gly

20 25 30

Ala Tyr Met Ile Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ile Ala Ser Gly Lys Val Glu Asp Tyr Met His Cys Trp Leu

50 55 60

Pro Asn Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Ile Gln Tyr His Trp Glu Asn Val Phe Thr Cys Pro Ser

100 105 110

Gly Arg Met Ala Leu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 33

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 3

<400> 33

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Ser Val Ile Pro

20 25 30

Glu Ala Asn Cys Met Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Met Ser Asn Gln Tyr Lys Phe Cys Asp His Ile Arg Gly Pro

50 55 60

Val Glu Ala Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Met Ala Glu Ile His Arg Thr Pro Val Leu Ser Tyr Lys

100 105 110

Phe Asn Cys Gln Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 34

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 4

<400> 34

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Pro Thr Cys Ser Gly

20 25 30

Ile Asn Tyr Val Glu Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Arg Gln Tyr Ala Pro Glu Val Asn Ser Gly Thr Phe Met Leu

50 55 60

Trp Ile Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Gln Trp Ser Arg Ile Asn His Gly Phe Asp Leu Tyr Met

100 105 110

Ala Thr Lys Val Cys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 35

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 5

<400> 35

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Pro Ile Met Trp

20 25 30

Asn Gln Ala Tyr Arg Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Val Ser Phe Gln Pro Cys Tyr Leu His Glu Lys Ile Asn Trp

50 55 60

Thr Met Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Gln Met Ser Asp Arg Ala Tyr Pro His Ile Asn Phe Val

100 105 110

Leu Thr Trp Cys Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125 <210> 36

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 6

<400> 36

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Trp Arg Gln Pro Glu

20 25 30

Met Cys Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Gln Arg Phe Ile Gly Pro Asn Leu His Lys Ala Met Trp Glu

50 55 60

Thr Asp Tyr Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Trp Gly Met Pro Arg Phe Tyr Cys Asp Thr Ser Lys His

100 105 110

Val Gln Asn Leu Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 37

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 7

<400> 37

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Ser Thr Cys Tyr

20 25 30

Lys Pro Trp Leu Phe Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Asp Phe His Thr Gln Gly Ala Leu Ser Glu Met Asn Pro Tyr

50 55 60

Cys Lys Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Gln Tyr Met Glu Arg Val His Lys Cys Gly Ala Leu Thr

100 105 110

Ser Phe Trp Asn Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 38

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 8

<400> 38

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Thr His Gly Leu Ser

20 25 30

Ile Asn Phe Met Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Pro Met Tyr Ser Ala Lys Glu Ile Thr Asn Trp Cys Val Leu

50 55 60

Asp Arg Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Gln Glu Phe Thr Asp Arg Trp Ala Leu Cys Ser His Met

100 105 110

Pro Lys Ile Asn Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 39

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 9

<400> 39

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Gln Glu His

20 25 30

Cys Asn Lys Ala Tyr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Gly Glu Cys Ser Lys Asn Val Gln Asp Arg Tyr Pro Ala Leu

50 55 60

His Trp Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Gln Phe Pro Thr Ala Lys Leu Met Gly His Glu Trp Tyr

100 105 110

Cys Arg Asn Ser Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 40

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 10

<400> 40

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Arg Lys Thr Cys Tyr

20 25 30

Asn Pro Asp Phe His Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Pro Val Gln Arg Met Ala Asp Asn Lys Cys Phe Leu Ile Thr

50 55 60

Trp Tyr Gly Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Ile Arg Gln Pro Cys His Leu Tyr Thr Lys Val Phe Asn

100 105 110

Met Ser Glu Ala Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 41

<211> 719

<212> PRT

<213> Homo sapiens

<221> Mcm7

<400> 41

Met Ala Leu Lys Asp Tyr Ala Leu Glu Lys Glu Lys Val Lys Lys Phe

1 5 10 15

Leu Gln Glu Phe Tyr Gln Asp Asp Glu Leu Gly Lys Lys Gln Phe Lys

20 25 30

Tyr Gly Asn Gln Leu Val Arg Leu Ala His Arg Glu Gln Val Ala Leu

35 40 45

Tyr Val Asp Leu Asp Asp Val Ala Glu Asp Asp Pro Glu Leu Val Asp

50 55 60

Ser Ile Cys Glu Asn Ala Arg Arg Tyr Ala Lys Leu Phe Ala Asp Ala

65 70 75 80

Val Gln Glu Leu Leu Pro Gln Tyr Lys Glu Arg Glu Val Val Asn Lys

85 90 95

Asp Val Leu Asp Val Tyr Ile Glu His Arg Leu Met Met Glu Gln Arg

100 105 110

Ser Arg Asp Pro Gly Met Val Arg Ser Pro Gln Asn Gln Tyr Pro Ala

115 120 125

Glu Leu Met Arg Arg Phe Glu Leu Tyr Phe Gln Gly Pro Ser Ser Asn

130 135 140

Lys Pro Arg Val Ile Arg Glu Val Arg Ala Asp Ser Val Gly Lys Leu

145 150 155 160

Val Thr Val Arg Gly Ile Val Thr Arg Val Ser Glu Val Lys Pro Lys

165 170 175

Met Val Val Ala Thr Tyr Thr Cys Asp Gln Cys Gly Ala Glu Thr Tyr

180 185 190

Gln Pro Ile Gln Ser Pro Thr Phe Met Pro Leu Ile Met Cys Pro Ser

195 200 205

Gln Glu Cys Gln Thr Asn Arg Ser Gly Gly Arg Leu Tyr Leu Gln Thr

210 215 220

Arg Gly Ser Arg Phe Ile Lys Phe Gln Glu Met Lys Met Gln Glu His

225 230 235 240

Ser Asp Gln Val Pro Val Gly Asn Ile Pro Arg Ser Ile Thr Val Leu

245 250 255

Val Glu Gly Glu Asn Thr Arg Ile Ala Gln Pro Gly Asp His Val Ser

260 265 270

Val Thr Gly Ile Phe Leu Pro Ile Leu Arg Thr Gly Phe Arg Gln Val

275 280 285

Val Gln Gly Leu Leu Ser Glu Thr Tyr Leu Glu Ala His Arg Ile Val

290 295 300

Lys Met Asn Lys Ser Glu Asp Asp Glu Ser Gly Ala Gly Glu Leu Thr

305 310 315 320

Arg Glu Glu Leu Arg Gln Ile Ala Glu Glu Asp Phe Tyr Glu Lys Leu

325 330 335

Ala Ala Ser Ile Ala Pro Glu Ile Tyr Gly His Glu Asp Val Lys Lys

340 345 350

Ala Leu Leu Leu Leu Leu Val Gly Gly Val Asp Gln Ser Pro Arg Gly

355 360 365

Met Lys Ile Arg Gly Asn Ile Asn Ile Cys Leu Met Gly Asp Pro Gly

370 375 380

Val Ala Lys Ser Gln Leu Leu Ser Tyr Ile Asp Arg Leu Ala Pro Arg

385 390 395 400

Ser Gln Tyr Thr Thr Gly Arg Gly Ser Ser Gly Val Gly Leu Thr Ala

405 410 415

Ala Val Leu Arg Asp Ser Val Ser Gly Glu Leu Thr Leu Glu Gly Gly

420 425 430

Ala Leu Val Leu Ala Asp Gln Gly Val Cys Cys Ile Asp Glu Phe Asp

435 440 445

Lys Met Ala Glu Ala Asp Arg Thr Ala Ile His Glu Val Met Glu Gln

450 455 460

Gln Thr Ile Ser Ile Ala Lys Ala Gly Ile Leu Thr Thr Leu Asn Ala

465 470 475 480

Arg Cys Ser Ile Leu Ala Ala Ala Asn Pro Ala Tyr Gly Arg Tyr Asn

485 490 495

Pro Arg Arg Ser Leu Glu Gln Asn Ile Gln Leu Pro Ala Ala Leu Leu

500 505 510

Ser Arg Phe Asp Leu Leu Trp Leu Ile Gln Asp Arg Pro Asp Arg Asp

515 520 525

Asn Asp Leu Arg Leu Ala Gln His Ile Thr Tyr Val His Gln His Ser

530 535 540

Arg Gln Pro Pro Ser Gln Phe Glu Pro Leu Asp Met Lys Leu Met Arg

545 550 555 560

Arg Tyr Ile Ala Met Cys Arg Glu Lys Gln Pro Met Val Pro Glu Ser

565 570 575

Leu Ala Asp Tyr Ile Thr Ala Ala Tyr Val Glu Met Arg Arg Glu Ala

580 585 590

Trp Ala Ser Lys Asp Ala Thr Tyr Thr Ser Ala Arg Thr Leu Leu Ala

595 600 605

Ile Leu Arg Leu Ser Thr Ala Leu Ala Arg Leu Arg Met Val Asp Val

610 615 620

Val Glu Lys Glu Asp Val Asn Glu Ala Ile Arg Leu Met Glu Met Ser

625 630 635 640

Lys Asp Ser Leu Leu Gly Asp Lys Gly Gln Thr Ala Arg Thr Gln Arg

645 650 655

Pro Ala Asp Val Ile Phe Ala Thr Val Arg Glu Leu Val Ser Gly Gly

660 665 670

Arg Ser Val Arg Phe Ser Glu Ala Glu Gln Arg Cys Val Ser Arg Gly

675 680 685

Phe Thr Pro Ala Gln Phe Gln Ala Ala Leu Asp Glu Tyr Glu Glu Leu

690 695 700

Asn Val Trp Gln Val Asn Ala Ser Arg Thr Arg Ile Thr Phe Val

705 710 715

SEQ ID NO. 76

<210> 42

<211> 150

<212> PRT

<213> Neurospora crassa

<221> Vivid

<400> 42

His Thr Leu Tyr Ala Pro Gly Gly Tyr Asp Ile Met Gly Tyr Leu Ile

1 5 10 15

Gln Ile Met Asn Arg Pro Asn Pro Gln Val Glu Leu Gly Pro Val Asp

20 25 30

Thr Ser Cys Ala Leu Ile Leu Cys Asp Leu Lys Gln Lys Asp Thr Pro

35 40 45

Ile Val Tyr Ala Ser Glu Ala Phe Leu Tyr Met Thr Gly Tyr Ser Asn

50 55 60

Ala Glu Val Leu Gly Arg Asn Cys Arg Phe Leu Gln Ser Pro Asp Gly

65 70 75 80

Met Val Lys Pro Lys Ser Thr Arg Lys Tyr Val Asp Ser Asn Thr Ile

85 90 95

Asn Thr Met Arg Lys Ala Ile Asp Arg Asn Ala Glu Val Gln Val Glu

100 105 110

Val Val Asn Phe Lys Lys Asn Gly Gln Arg Phe Val Asn Phe Leu Thr

115 120 125

Met Ile Pro Val Arg Asp Glu Thr Gly Glu Tyr Arg Tyr Ser Met Gly

130 135 140

Phe Gln Cys Glu Thr Glu

145 150

<210> 43

<211> 25

<212> PRT

<213> Artificial sequence

<221> Linker

<400> 43

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

SEQ ID NO. 79

<210> 44

<211> 166

<212> PRT

<213> Escherichia coli

<221> ecTadA*

<400> 44

Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr

1 5 10 15

Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val

20 25 30

Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile

35 40 45

Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln

50 55 60

Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr

65 70 75 80

Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser

85 90 95

Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala

100 105 110

Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg

115 120 125

Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu

130 135 140

Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys

145 150 155 160

Ala Gln Ser Ser Thr Asp

165

<210> 45

<211> 228

<212> PRT

<213> Rat (rattus norvegicus)

<221> rAPOBEC1

<400> 45

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser

35 40 45

Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn

50 55 60

Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser

85 90 95

Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe

100 105 110

Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln

115 120 125

Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr

130 135 140

Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro

145 150 155 160

Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu

165 170 175

Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 46

<211> 589

<212> DNA

<213> Artificial sequence

<221> Vector 10 key sequence

<400> 46

gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60

cccgggttcg attcccggct ggtgcagttc aggcaggccc ccggcagttt tagagctaga 120

aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180

gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240

tcgattcccg gctggtgcaa ggcaggttca ccatcagcag ttttagagct agaaatagca 300

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360

agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420

ccggctggtg cactactggg gccagggcac ccgttttaga gctagaaata gcaagttaaa 480

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcttttttggatcc 540

agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589

Claims (17)

1. A construction method of an antigen binding domain automatic optimized chimeric antigen receptor modified cell library is characterized in that a vector comprising a first gene element, a second gene element, a third gene element, a fourth gene element and a fifth gene element is transfected into cells, and one or more vectors are used for obtaining the chimeric antigen receptor modified cell library;

The first genetic element is a nucleotide sequence encoding a receptor 1, the receptor 1 comprising an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain can recognize biotin or a 6 XHis tag marked on a target antigen, the intracellular domain comprises tobacco etch virus protease TEVP, and the nucleotide sequence of the receptor 1 is shown as SEQ ID NO. 1;

The second genetic element is a nucleotide sequence encoding receptor 2, the receptor 2 comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the antigen binding domain is an antibody sequence dynamic library, and the dynamic library is obtained by inducing a series of mutations under the action of a fourth gene element and introducing sequence diversity; the intracellular signal domain comprises a tTA transcription factor domain and a connecting sequence, wherein the connecting sequence connects the tTA transcription factor domain and a transmembrane domain and comprises a TEVP recognizable and cleavable sequence, the receptor 2 is a synNotch receptor modified by a Notch receptor, and the nucleotide sequence of the receptor is shown as SEQ ID NO. 2 or SEQ ID NO. 3;

the third gene element is a nucleotide sequence for encoding a screening gene, and the tTA transcription factor can start the expression of the screening gene;

The fourth gene element is a nucleotide sequence encoding a cis-regulatory element or a trans-acting factor; the cis-regulatory element is capable of producing a single-stranded structure of DNA from the DNA; the trans-acting factors are gRNA, dCAS9, a photosensitive protein, deaminase and a Linker sequence between the photosensitive protein and the deaminase;

The fifth gene element is a nucleotide sequence for encoding an inhibition system, and the tTA transcription factor can start the expression of the inhibition system, so that the trans-acting factor is knocked down or knocked out, and the antibody sequence dynamic library is kept stable; or the fifth gene element is a nucleotide sequence encoding a light control system capable of regulating expression of the fourth gene element: starting a fourth gene element to play a role under the illumination condition, and starting to construct an antibody dynamic sequence library; after the light is stopped, the function of the fourth gene element is inhibited, so that the antibody dynamic sequence library is kept stable.

2. The method of constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the cis-regulatory element causes DNA to produce a DNA single-stranded structure of a specific structure; the DNA single-chain structure with the special structure comprises a G-tetrad, and the trans-acting factors are Nucleolin, a photosensitive protein, deaminase and a Linker sequence between the photosensitive protein and the deaminase.

3. The method for constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the cells are of human or murine origin.

4. The method for constructing a chimeric antigen receptor-modified cell library according to claim 1, wherein the antibody sequence dynamic library comprises a double-chain antibody library, a single-chain antibody library and a nanobody library.

5. The method of constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following Cas protein family members and partial domains thereof: cas3-8s, cas10s, cas11s, cas9s, xCas9, cas12s, cas13s, and Cas14s.

6. The method of constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following adenine deaminase and partial domains thereof: ecTadA, mADA, hADAR2 and hADAT.

7. The method of constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following cytidine deaminase and partial domains thereof: activation-reduced CYTIDINE DEAMINASE, apobe 3G, APOBEC1, and CDA1.

8. The method of claim 1, wherein the nucleotide sequence encoding the light control system comprises the following photoproteins and partial domains thereof: bphS, cph1, PCB, phyB-PIFs, PΦ B, bphP1-PpsR2, bphP1-Q-PAS1, LOV2-Jα, phyB, PIF6, epdz and Photoreceptor Vivid.

9. The method of claim 1, wherein the light control system is adapted to regulate the fourth genetic element in response to light comprising visible light, invisible light, blue light, red light, far infrared light, and ultraviolet light.

10. The method of claim 1, wherein the screening genes include GFP, mCherry, luciferase, puromycin resistance genes, bleomycin and neomycin resistance genes.

11. The method for constructing a chimeric antigen receptor-modified cell library according to claim 1, wherein the inhibition system is to stabilize the antibody sequence dynamic library by activating negative regulatory proteins, repressor proteins, siRNA, CAS9/gRNA expression or by knocking down or knocking out trans-acting factors by reverse transcription.

12. A library of chimeric antigen receptor-modified cells with automatically optimized antigen binding domains, wherein the library of chimeric antigen receptor-modified cells is constructed by the method of any one of claims 1-11.

13. A method for screening a library of chimeric antigen receptor-modified cells for autooptimization of antigen binding domain, comprising the steps of:

Preparing a target antigen on which a tag capable of being recognized by the receptor 1 is marked, wherein the tag comprises biotin or 6 xHis;

Incubating the target antigen and the chimeric antigen receptor modified cell library together, and screening positive chimeric antigen receptor expression cells according to the expression condition of the screening genes;

And (3) amplifying and sequencing the chimeric antigen receptor extracellular antigen binding domain of the positive cell to obtain an antibody nucleotide sequence capable of specifically binding with the target antigen.

14. The method of screening a library of chimeric antigen receptor-modified cells for antibodies according to claim 13, further comprising the steps of, after obtaining the nucleotide sequence of the antibody capable of specifically binding to the target antigen: the antibody against the target antigen is obtained by adopting a genetic engineering method.

15. The method of screening a library of chimeric antigen receptor-modified cells for antibodies according to claim 13, wherein the target antigen is expressed on the cell surface, coated on the surface of a carrier, or is a soluble protein.

16. The method of screening a library of chimeric antigen receptor-modified cells for antibodies according to claim 15, wherein the carrier comprises magnetic microspheres, non-magnetic microspheres, graphene, nanoparticles; the magnetic microsphere comprises silicon-based magnetic beads, GMA magnetic beads and polystyrene magnetic microspheres; the non-magnetic microspheres include agarose microspheres.

17. Use of the method of screening a library of chimeric antigen receptor-modified cells according to any one of claims 13-16 for the preparation of antibodies against a specific target antigen.