WO2018194015A1 - Method for obtaining information of antigen-specific antibody - Google Patents

Abstract

Provided is a method whereby a high-affinity antibody can be efficiently produced with the use of a natural type protein as an antigen. Also provided is a method for obtaining information of an antigen-specific antibody derived exclusively from a blistered fish without being affected by microorganisms existing in the rearing environment. Information of a high-affinity antibody, i.e., an antigen-specific antibody is obtained with the use of a natural type protein as an antigen through the following steps, etc.: (A) a step for administering an antigen to a blistered fish and allowing the fish to produce an antibody; (B) a step for collecting the blister fluid containing the antibody from the fish treated in (A); and (C) a step for constructing an antigen-specific polyclonal antibody gene library from the blister fluid collected in (B). Thus, information of the antigen-specific antibody is obtained using primers, said primers being capable of amplifying only the gene of the antigen-specific antibody derived exclusively from the blistered fish but not amplifying genes of microorganisms existing in the rearing environment such as Propionibacterium acnes.

Description

Method for obtaining antigen-specific antibody information

The present invention relates to a method for acquiring antigen-specific antibody information. More specifically, the present invention relates to a method for obtaining antigen-specific antibody information including a step of producing an antibody by administering an antigen to fish having water bubbles. Further, the present invention relates to base sequence information, amino acid sequence information and the like obtained by the method.

Conventionally, various antibody acquisition methods such as a mammalian immunization method using various animals such as mice and a phage display method have been developed. However, in order to obtain an antibody having a high affinity for a natural target protein by these methods, conditions such as the target protein having high purity and maintaining a three-dimensional structure are necessary. It was.
In addition, a method for obtaining an antibody by a DNA immunization method is also known, but this method is effective when targeting a part of a protein that can be expressed as a natural form in a dendritic cell. However, it has been extremely difficult to obtain a high-affinity antibody by targeting a protein that cannot be expressed in dendritic cells or a protein with a low expression level.
Therefore, depending on these conventionally known antibody production methods, a high-affinity antibody that can be used for drug discovery, etc., can be efficiently used with a natural protein modified with a sugar chain, fatty acid, etc. as an antigen after protein translation. It could not be said that it could be produced.

Accordingly, the present inventor has set an object of the present invention to provide a method capable of efficiently producing a high affinity antibody using these natural proteins as antigens. In providing this method, attention was focused on obtaining antigen-specific antibody information from fish having water bubbles. Since fish with water bubbles are bred in water, information on antigen-specific antibodies derived only from fish with water bubbles is obtained without being affected by microorganisms such as acne bacteria present in the breeding environment. It is also a subject of the present invention.
In addition, in the patent document 1, the present inventors have disclosed an antibody that can repeatedly obtain an antibody produced in a fish while keeping the fish alive by subjecting a fish having water bubbles to an antigen administration target. A manufacturing method is being developed.

Japanese Patent Application No. 2012-058566

An object of the present invention is to provide a method capable of efficiently producing a high affinity antibody using a natural protein as an antigen. Another object of the present invention is to provide a method for obtaining information on antigen-specific antibodies derived only from fish having water bubbles without being affected by microorganisms present in the breeding environment.

The present inventor has conducted intensive research to solve the above problems, and (A) a step of administering an antigen to fish having water bubbles to produce antibodies, (B) a water bubble containing antibodies from the fish of (A) above. And (C) a step of constructing an antigen-specific polyclonal antibody gene library from the foam solution collected in (B) above, and the like. It has been found that information on affinity antibodies, that is, antigen-specific antibodies can be obtained, and the present invention has been completed.

In the method of the present invention, a fish library having a blister is immunized with a target protein serving as an antigen, and a cDNA library is prepared using the IgM gene of B cells contained in a blister liquid that has been confirmed to have sufficiently increased antibody titer as a template. Based on the obtained fragments, antigen-specific antibody gene and / or amino acid sequence information is obtained from the clones to which the antigen protein is bound by phage display.
In such a method of the present invention, a specific primer for amplification of an antibody gene is used in obtaining the base sequence and / or amino acid sequence of a clone in which binding is found by direct protein-protein interaction with an antigen protein. By combining these, it is possible to obtain the base sequence of the antigen-specific VH region and / or VL region and the amino acid sequence information of the VH region and / or VL region.
The specific primer designed for amplification of this antibody gene does not amplify the genes of microorganisms such as acne bacteria present in the breeding environment, but only antigen-specific antibody genes derived from fish with water bubbles. It is important that the primer be a primer that can amplify the gene in the leader peptide region.

That is, the present invention relates to the following methods (1) to (10) for obtaining information on antigen-specific antibodies.
(1) A method for obtaining information on an antigen-specific antibody comprising the following steps (A) to (D).
(A) A step of administering an antigen to a fish having blisters and producing an antibody (B) A step of collecting a blister fluid containing an antibody from the fish of (A) (C) A blister fluid sampled in (B) above Step of constructing a more antigen-specific polyclonal antibody gene library (D) Step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in the above (C) (2) Antigen-specific antibody The method according to (1) above, wherein the information is antigen-specific VH region and / or VL region base sequence information and / or amino acid sequence information.
(3) In the construction of an antigen-specific polyclonal antibody gene library, the primer containing the base sequence shown in SEQ ID NO: 1 and / or the primer containing the base sequence shown in SEQ ID NO: 2 is used (1 ) Or the method according to (2).
(4) The method according to any one of the above (1) to (3), wherein the fish having a water bubble is a water bubble eye or ranchu.
(5) Information on the base sequence of an antigen-specific antibody obtained by the method according to any one of (1) to (4) above.
(6) Amino acid sequence information of the antigen-specific antibody obtained by the method according to any one of (1) to (4) above.
(7) A phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody obtained through the following steps (A) to (E).
(A) A step of administering an antigen to a fish having blisters and producing an antibody (B) A step of collecting a blister fluid containing an antibody from the fish of (A) (C) A blister fluid sampled in (B) above Step of constructing a more antigen-specific polyclonal antibody gene library (D) Step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in (C) (E) Step of (D) (8) screening a phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody that binds to an antigen from a phage library constructed by (8) any one of SEQ ID NOS: 31 to 34 in the sequence listing Described in (7) above, which is encoded by the nucleotide sequence of or shown by the amino acid sequence of any one of SEQ ID NOs: 35 to 38 in the sequence listing Phage display recombinant protein.
(9) A primer comprising the base sequence set forth in SEQ ID NO: 1 and / or a primer comprising the base sequence set forth in SEQ ID NO: 2
(10) The primer according to (9) above for the construction of an antigen-specific polyclonal antibody gene library.

According to the present invention, it is possible to obtain base sequence information of the VH region and / or VL region and amino acid sequence information of the VH region and / or VL region as information on the antibody specific to the target protein antigen. It is also possible to create an antibody based on the acquired information and obtain a test / diagnostic drug or antibody drug.

It is the figure which showed the schematic diagram of the fragment | piece amplified by cDNA synthesis and PCR reaction (Example). It is the figure which showed the schematic diagram of scFv recombinant amino acid sequence and recombinant protein (Example). It is the figure which showed the detection result of scFv-His by CBB dyeing | staining and Western * Blotting (Example). It is the figure which showed the detection result of the anti-EGFP activity of scFv-His by ELISA (Example). FIG. 10 is a diagram showing scFv9 sequence results (Example). It is the figure which showed the sequence result of scFv10 (Example). It is the figure which showed the sequence result of scFv12 (Example). It is the figure which showed the sequence result of scFv13 (Example).

The “method for obtaining information on an antigen-specific antibody” of the present invention refers to the nucleotide sequence information and / or amino acid sequence information of an antibody capable of specifically recognizing a target protein, that is, the VH region of the antibody and / or Alternatively, the present invention relates to a method for obtaining nucleotide sequence information of a VL region and amino acid sequence information of a VH region and / or a VL region. This method of the present invention includes the following steps (A) to (D) as essential steps.

(A) A step of administering an antigen to a fish having blisters and producing an antibody (B) A step of collecting a blister fluid containing an antibody from the fish of (A) (C) A blister fluid sampled in (B) above Step of constructing a more antigen-specific polyclonal antibody gene library (D) Step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in the above (C) In performing each of these steps, Any known reagent, kit, instrument, etc. may be used.

“(A) The step of producing an antibody by administering an antigen to a fish having water bubbles” is a step of administering the antigen to the fish having a water bubble by a method such as injection, oral, or transdermal to produce an antibody in the fish. I mean. In this step, it is preferable to administer the antigen to the water bubbles of the fish by injection. In the method of the present invention, the antigen to be administered is particularly preferably a natural protein in which sugar chains, fatty acids and the like are modified after protein translation. In addition, in this process, the frequency | count of administering an antigen to the fish which has a water bubble may be 1 time, and may be multiple times.
The “fish having water bubbles” may be any conventionally known fish having water bubbles, and examples thereof include water bubbles and ranchu.

“(B) The step of collecting the bubbly liquid containing the antibody from the fish of (A)” as described above, the bubbling liquid containing the antibody produced in the fish body by administering the antigen is aspirated and collected by injection or the like. It means the process to do. It is preferable to collect the amount of the foam solution to be collected so that the fish having the foam does not die.

“(C) the step of constructing an antigen-specific polyclonal antibody gene library from the blister liquid collected in (B)” is a cDNA library using the IgM gene of B cells contained in the collected blister liquid as a template. Is the process of building This cDNA library is preferably a phage library.
In this step, the designed primer does not amplify genes of microorganisms such as acne bacteria present in the breeding environment, but can amplify only antigen-specific antibody genes derived from fish having water bubbles. Acquisition of antigen-specific VH and / or VL region base sequence information and VH and / or VL region amino acid sequence information by using primers, ie, primers that target the gene in the leader peptide region It becomes possible to do.
Examples of such a primer include a primer containing the base sequence shown in SEQ ID NO: 1 and / or a primer containing the base sequence shown in SEQ ID NO: 2. These primers are particularly preferably combined as a primer set.
These primers can be used as primers for the construction of an antigen-specific polyclonal antibody gene library.

“(D) The step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in the above (C)” is performed by using the antigen-specific polyclonal antibody gene library from the antigen-specific polyclonal gene library. This refers to the process of displaying protein molecules on the phage surface.
By making these steps (A) to (D) essential steps, it is possible to obtain antigen-specific antibody information. The methods of the present invention may further include other methods useful for obtaining antigen-specific antibody information.

In obtaining antigen-specific antibody information, it is preferable to obtain a phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody. This protein can be obtained through the following step (E) in addition to the steps (A) to (D).
“(E) A step of screening a phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody that binds to an antigen from the phage library constructed by the step (D)”.
By this step, the next phage contained in the phage library constructed by the step (D) can be selected. This phage is a phage on which the surface of all or part of a protein molecule of a polyclonal antibody capable of binding to an antigen as a target protein is displayed.
This screening may be performed by any conventionally known method, for example, biopanning. Biopanning is preferable because a phage containing an antigen-specific high-affinity antibody gene that is a target protein can be identified by infecting and amplifying a phage bound to the target protein in E. coli. When biopanning is performed, the number of times may be one, and it is more preferable that the number of times of biopanning is further increased because phages can be concentrated and the accuracy of selection is increased.

The “phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody” obtained through such a process is a protein displayed on the surface of a phage, and the The antibody contains all or part of an antibody that can specifically bind to. What is necessary is just to include all or a part of this antigen-specific polyclonal antibody, and may include other proteins.
Such a “phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody” is any one as long as it can be obtained through the steps (A) to (E) of the present invention. It may be a protein. Examples of such a protein include a protein encoded by the base sequence described in any one of SEQ ID NOs: 31 to 34 and a protein represented by the amino acid sequence described in any one of SEQ ID NOs: 35 to 38, etc. Can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Cloning of antibody genes>
1. Preparation of bubbling liquid sample Bubbling liquid collected before immunization (pre (hereinafter may be simply referred to as pre)) and bubbling liquid collected after the third day of immunization (3rd (hereinafter may be simply referred to as 3rd)) ) As a sample. Immunization was performed by directly injecting 100 μg EGFP per time into the water bubble of watermelon, a total of 3 times per week.

2. cDNA synthesis A foam solution was collected, immediately centrifuged at 4 ° C, 1000 xg for 10 minutes to separate into supernatant and precipitate, and the precipitate was suspended in 1 ml QIAzol (registered trademark) Lysis Reagent (QIAGEN). Thus, after extracting total RNA, DNase I (TaKaRa) treatment was performed to decompose genomic DNA, and purification was performed with QIAzol (registered trademark) Lysis Reagen. CDNA synthesized by reverse transcription from purified total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche) was used as a template for cloning goldfish scFv. CDNA synthesis was performed with the gIgL CL reverse 1 and gIgH_2D primers listed in Table 1. Tm values are also shown in Table 1.

3. Cloning of scFv Gene fragments of the VL region (hereinafter sometimes simply referred to as VL) and VH region (hereinafter sometimes simply referred to as VH) were amplified with degenerate primers, and scFv was cloned. The PCR reaction was performed in three steps: 1) First PCR, 2) Second PCR, and 3) Overlap PCR. A schematic diagram of the gene fragment amplified by this is shown in FIG. In FIG. 1, the primers at each stage are indicated by red arrows.
Each PCR amplification product was subjected to agarose gel electrophoresis and then purified by Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).

1) First PCR
2. Using the cDNA synthesized in step 1 as a template, the gene fragment containing the VL region was amplified with the primers and Tm values shown in Table 2 under the reaction conditions shown in Tables 3 and 4. Further, the gene fragment containing the VH region was amplified with the primers and Tm values shown in Table 5 under the reaction conditions shown in Tables 6 and 7.

2) Second PCR
Tables 9 and 10 show the restriction enzyme sites obtained by the first PCR of 1) above and a gene fragment containing a partially added VL region as a template and the primers and Tm values shown in Table 8 as shown in Tables 9 and 10. Amplified under reaction conditions.
Similarly, a gene fragment containing a VH region to which a restriction enzyme site and a part of a linker were added was used as a template, and the primers and Tm values shown in Table 11 were used for amplification under the reaction conditions shown in Tables 12 and 13.

3) Overlap PCR
(1) First step
Using the VH secondPCR product obtained in the second PCR of 2) above as a template, the primers described in Table 14 (PrimerF is a primer of a linker sequence, PrimerR is a primer corresponding to VH FR4 used in VH secondPCR), and Tm value, Under the reaction conditions shown in Tables 15 and 16, a VH region gene fragment to which a linker sequence was added was amplified.
(2) Second step
Using the overlap PCR first step product and VL second PCR product obtained in the first step of (1) above as templates, the primers described in Table 17 (PrimerF is a primer corresponding to VL FR1 used for VL secondPCR, PrimerR is VH second PCR The primer corresponding to VH FR4 used in the above) and the Tm value were used to link the VL region and VH region via a linker under the reaction conditions shown in Tables 18 and 19, thereby amplifying the entire scFv gene. The primer for the linker sequence contained sequences homologous to the 3 ′ end of the VL region and the 5 ′ end of the VH region amplified by second PCR.

<Phage display>
Phage display was performed by the following procedure using T7Select (registered trademark) 1-1 Cloning Kit (Novagen) and T7Select (registered trademark) Biopanning Kit (Novagen).

1. In vitro packaging
Pre- and 3rd scFv gene fragments treated with restriction enzymes EcoR I and HindIII are used as inserts, ligated with 1-1b vector, mixed with T7 Packaging Extracts and reacted at room temperature for 2 hours to introduce the target gene The vector was incorporated into the phage.

2. Plaque Assay
1) Reagent Each reagent was prepared so that it might become the following composition.
(1) TB (Per 500 ml)
450ml deionized water, 6g Bacto tryptone, 12g Yeast extract, 2ml glycerol, 50ml sterile K phosphate
(2) K phosphate (Per liter)
23.1g KH ₂ PO ₄ , 125.4g K ₂ HPO ₄
(3) M9TB liquid medium 5ml 20X M9 salts, 2ml 20% glucose, 0.1ml 1M MgSO ₄ , 100ml TB
(4) 20X M9 salts (Per liter)
20 g NH ₄ Cl, 60 g KH ₂ PO ₄ , 120 g Na ₂ HPO ₄・ 7H ₂ O
(5) Top agarose (Per 100 ml)
1g Bacto tryptone, 0.5g Yeast extract, 0.5g NaCl, 0.6g agarose, 100ml deionized water

2) Virus titer measurement (1) E. coli BLT5403 strain was inoculated into M9TB liquid medium and cultured with shaking at 140 rpm and 37 ° C. until the absorbance at a wavelength of 600 nm reached about 1.0.
(2) 10 μl each of pre and 3rd Packaging solutions were diluted 10 ² to 10 ⁶ with TB liquid medium, respectively, and 250 μl of the culture solution (1) was added to a 15 ml tube dispensed 100 μl each. Further, 3 ml Top agarose at about 45 to 50 ° C. was added and mixed, seeded uniformly on LB + Amp agar medium, and then statically cultured at 37 ° C. for 4 hours.
(3) The titer was calculated from the number of plaques produced (plaque number × dilution × 10 = pfu / ml * plaqueforming units).

3) Results pre: The titer calculated from 10 ² dilution (82 plaques) is 8.2 × 10 ⁴ (pfu / ml), 3rd: titer calculated from 10 ² dilution (55 plaques) Was 5.5 × 10 ⁴ (pfu / ml).
Since the packaging solution this time was 150 μl, the phage titer of the packaging solution was pre: 1.23 × 10 ⁴ (pfu) and 3rd: 8.25 × 10 ³ (pfu).
Since the phage titer of the Packaging solution was less than 5 × 10 ^6, it was decided to prepare a phage library by the Plate Lysate Amplification method.

3. Preparation of phage library The E. coli BLT5403 strain was inoculated into a 50 ml TB medium and cultured with shaking at 37 ° C. and 140 rpm until the absorbance at a wavelength of 600 nm reached 0.6 to 1.0. After adding the amount of culture solution calculated to 1 × 10 ⁶ phage per 10ml cells to the Packaging solution, Top agarose (45 to 45 times) of the mixture of the bacterial solution and Packaging solution within 20 minutes. And inoculated uniformly on LB + Amp agar medium, followed by stationary culture at 37 ° C. for 4 hours. After incubation, 10 ml Phage Extraction Buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO ₄ ) was added, and the mixture was allowed to stand overnight at 4 ° C. for reaction. Tilt the petri dish of LB + Amp agar medium and collect the phage library solution into one tube, add 500 μl chloroform and invert mix, collect the supernatant by centrifuging at 4 ° C, 3000 xg, 5 min, A phage library lysate was used. The phage library lysate was stored at 4 ° C.

4). Plaque Assay
1) Titer measurement of phage library lysate M9TB liquid medium was inoculated with E. coli BLT5403 strain, and cultured with shaking at 140 rpm and 37 ° C. until the absorbance at a wavelength of 600 nm reached about 1.0. 10 μl of Pre and 3rd phage library lysates were diluted 10 ⁵ to 10 ⁸ with TB liquid medium, respectively, and 250 μl of the above culture solution was added to a 15 ml tube dispensed by 100 μl. Further, 3 ml Topagarose at about 45 to 50 ° C. was added and mixed, seeded uniformly on LB + Amp agar medium, and then statically cultured at 37 ° C. for 4 hours. The titer was calculated from the number of plaques produced (number of plaques × dilution × 10 = pfu / ml * plaque forming units).

2) Results pre: The titer calculated from 10 ⁸ dilution (174 plaques) is 1.74 x 10 ¹¹ (pfu / ml), 3rd: titer calculated from 10 ⁸ dilution (263 plaques) Was 2.63 × 10 ¹¹ (pfu / ml).
In addition, since the MOI (infection efficiency) of T7 phage is 10 ^-3 to 10 ^-2 , the infection efficiency is pre: 1.74 × 10 ⁸ to 1.74 × 10 ⁹ (pfu), 3rd: 2.63 × 10 ⁸ to 2.63 × Calculated as 10 ⁹ (pfu).

5). Biopanning 1) Preparation of EGFP coating plate F96 MAXISORP NUNC-IMMUNO PLATE (Thermo) is a target protein diluted with deionized water after washing 2-3 times with deionized water and dehydrated to 10 μg / ml EGFP was dispensed at 100 μl / well and allowed to stand at 4 ° C. overnight to immobilize. After washing 3 times with 300 μl / well deionized water, 5% Blocking Reagent solution was added to 200 μl / well and blocking was performed at room temperature for 1 hour. After washing 5 times with 300 μl / well of deionized water, the deionized water was dispensed to 200 μl / well and stored at 4 ° C. until use.
Next, the amount of phage lysate to be reacted was determined. When all amino acid numbers (n) displayed by the target protein are determined at random, the total sequence pattern is n ²¹ . The amount of phage serving as an index is calculated by n ²¹ × titer × MOI. Thus, when the number of amino acids in the hypervariable region of the goldfish antibody variable region was estimated, n = about 70. However, since the instructions attached to the kit stated that a phage amount of 1.0 × 10 ⁸ or more in one biopanning was not suitable, this time, 1.0 × 10 ⁸ lysate was prepared and this was the first panning. Used for.

2) Biopanning (first time)
200 μl of phage lysate was added to the EGFP coating plate and allowed to stand at 4 ° C. for 16 hours for reaction. After washing the ELISA plate 5 times with TBST 200 μl / well, 200 μl / well of T7 Elution Buffer was added and the phage lysate was eluted by reacting at room temperature for 10-20 minutes. E. coli BLT5403 strain is inoculated into a 50 ml LB + Amp liquid medium, and the recovered eluate is added to the culture solution shake-cultured at 37 ° C. and 140 rpm until the absorbance at 600 nm reaches 0.5 to 0.6, and then at 37 ° C. and 140 rpm. Shake culture for 1-3 hours until cell lysis was observed. The supernatant was collected by centrifugation at 4 ° C., 8000 × g for 10 minutes, and stored at 4 ° C. until the next biopanning.

3) Biopanning (2nd to 4th)
100 μl of phage lysate that had been subjected to biopanning (first time) was added to the EGFP coating plate and allowed to react at room temperature for 30 minutes. After washing the ELISA plate 5 times with 200 μl TBST, T7 Elution Buffer was added to 200 μl / well, and the mixture was reacted at room temperature for 10 to 20 minutes for elution. E. coli BLT5403 strain is inoculated into a 50 ml LB + Amp liquid medium, and the total amount of the eluate is added to the culture medium shaken at 37 ° C. and 140 rpm until the absorbance at 600 nm reaches 0.5 to 0.6, and 1 to 37 ° C. at 140 rpm. Shake culture for 3 hours until cell lysis was observed. The supernatant was collected by centrifugation at 4 ° C., 8000 × g for 10 minutes, and stored at 4 ° C.

6). Isolation of phage The same method as the plaque assay was performed. In order to isolate the phages, the dilution was adjusted and the cells were cultured so that there were not more than 100 plaques per plate. E. coli BLT5403 strain was inoculated into M9TB liquid medium, and cultured with shaking at 37 ° C. at 140 rpm until the absorbance at 600 nm was about 1.0. 10 μl of 3rd phage lysate was diluted 10 ^{8 with} TB liquid medium, and 250 μl of the above culture solution was added to a 15 ml tube into which 100 μl was dispensed. Further, 3 ml Top agarose at about 45 to 50 ° C. was added and mixed, seeded uniformly on LB + Amp agar medium, and then statically cultured at 37 ° C. for 4 hours. After culturing, plaques were isolated, PCR (KOD) was performed using the T7 UP primer and T7 DOWN primer included in the kit, gel extraction was performed after agarose gel electrophoresis, and the PCR product was purified.

7). Introduction into pET22b (+) vector The 3rd scFv gene fragment (scFv 9, 10, 12, 13) obtained by the phage display method was subjected to restriction enzyme treatment with EcoRI (TaKaRa) and HindIII (TaKaRa), and pET22b (+ ) Ligation was performed on the EcoRI-HindIII site of the vector (Novagen) using a DNALigation Kit (TaKaRa). Escherichia coli DH5α strain was transformed to obtain a plasmid in which the gene sequence of the VL region and VH region was linked by a linker sequence, and the scFv gene with the His tag sequence added to the C-terminal side was subcloned.

FIG. 2 shows a schematic diagram of the scFv recombinant amino acid sequence and the recombinant protein.
A of FIG. 2 shows the deduced amino acid sequence when the scFv fragment cloned into the multicloning site EcoRI-HindIII of the pET22b (+) vector (Novagen) is incorporated. By using the restriction enzyme NotI and SalI sites, it was possible to incorporate the VL region alone or the VH region alone, and to change the combination of the VL region and the VH region.
FIG. 2B shows the presumed structure of the recombinant protein when the plasmid vector A is transformed into E. coli for protein expression. It was estimated that 11 amino acids derived from the vector were added to the scFv structure, 7 amino acids on the N-terminal side, and 7 His-tags were added to the C-terminal side.

8). scFv gene sequence The base sequence of the scFv gene fragment introduced into the pET22b (+) vector was confirmed by DNA sequencing reaction and GENETYX (software development) by CEQ2000 DNA Analysis System (Beckman). Primers were T7 UP primer and T7 DOWN primer.

9. scFv protein expression 1) Production and purification of scFv protein The scFv protein was produced using the prepared pET22b (+)-scFv plasmid. First, Escherichia coli BL21 strain was transformed, inoculated into LB + Amp liquid medium, and cultured with shaking at 37 ° C. and 140 rpm. When the absorbance at a wavelength of 600 nm reached 0.5, it was rapidly cooled to 15 ° C., IPTG was added to a final concentration of 1.0 mM, and the expression of scFv protein was induced by shaking culture at 15 ° C., 140 rpm for 24 hours. After induction, the bacterial cells were collected by centrifugation at 4 ° C., 5000 × g for 10 minutes, and the liquid medium components remaining in PBS were removed. Subsequently, Ni binding buffer (20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 5 mM Imidazole, pH 7.4) was added, and the cells were disrupted on ice by a micro ultrasonic cell disrupter (MICROSON XL 2000, MISONIX). This cell disruption solution was centrifuged at 15000 × g for 30 minutes at 4 ° C., and the supernatant was collected. Contaminants were removed from the collected supernatant with a 0.45 μm filter (ADVANTEC) to obtain an Escherichia coli-expressed scFv extract. The resulting Escherichia coli-expressed scFv extract was applied to a His TrapHP column (GE Healthcare), and His tag was added by Ni binding buffer and Ni elution buffer (20 mM Na ₂ HPO ₄ , 0.5M NaCl, 500 mM MImidazole, pH 7.4). The scFv protein was purified. As a confirmation of expression and purification, the results of SDS-PAGE, Western blotting with CBB staining and anti-His antibody (GE Healthcare) are shown in FIG.

2) Results As shown in FIG. 3, a band estimated to be scFv concentrated by Ni purification was confirmed at the target size of about 31 kDa. This Ni-purified fraction was used as an scFv protein extraction fraction for subsequent ELISA detection.
In FIG. 3, “before” indicates the fraction before purification of the Ni column, and “after” indicates the fraction after purification of the Ni column, and the red arrow indicates the size of about 31 kDa estimated to be scFv. is there.

10. ELISA detection 1) ELISA detection In order to examine the affinity for the target protein EGFP in the prepared scFv, detection was carried out by ELISA method in which EGFP was directly immobilized. First, 100 μl of EGFP solution diluted to 50 μg / well with carbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) was poured into MAXISORP NUNC-IMMUNOPLATE and left at 4 ° C. overnight. And solidified. After washing twice with 200 μl / well of PBST, 1% BSA / PBS was dispensed at 200 μl / well and blocked at 37 ° C. for 1 hour. After washing with PBST and diluting 10-fold, 100-fold and 1000-fold with Can Get Signal Solution 1 (TOYOBO), the Ni-purified scFv protein extract fraction was dispensed at 100 μl / well and reacted at 37 ° C. for 1 hour. After washing with PBST, anti-His tag antibody diluted 5000 times with Can Get Signal Solution 1 (TOYOBO) was dispensed at 100 μl / well and reacted at 25 ° C. for 2 hours. After washing with PBST, anti-mouseIgG HRP-labeled antibody diluted 20000 times with Can Get Signal Solution 2 (TOYOBO) was dispensed at 100 μl / well and reacted at 37 ° C. for 1 hour. After washing 4 times with PBST, TMB substrate (SurModics) returned to room temperature was dispensed at 100 μl / well, allowed to stand at room temperature for 30 minutes for reaction, and then the reaction stop solution (0.1 N HCl, 0.6 N H ₂ SO ₄ ) Was added at 100 μl / well to stop the reaction, and the absorbance at a wavelength of 450 nm was measured with a plate reader.

2) Results FIG. 4 shows the detection results of anti-EGFP activity of scFv-His by ELISA. When the 10-fold diluted scFv protein extract was reacted, specific binding to EGFP could be confirmed in the scFv10, scFv12 and scFv13 samples. Of the four types of scFv, scFv13 had the highest affinity.

3) scFv sequence results The scFv 9 sequence results are shown in Fig. 5, SEQ ID NO: 31 (base sequence) and SEQ ID NO: 35 (amino acid sequence). The scFv 10 sequence results are shown in FIG. 6, SEQ ID NO: 32 (base sequence) and SEQ ID NO: 36 (amino acid sequence). The scFv 12 sequence results are shown in FIG. 7, SEQ ID NO: 33 (base sequence) and SEQ ID NO: 37 (amino acid sequence). The scFv 13 sequence results are shown in FIG. 8, SEQ ID NO: 34 (base sequence) and SEQ ID NO: 38 (amino acid sequence). In FIGS. 5 to 8, region names (VL-FR, VH-CDR, etc.) are shown at the start points of the regions.

According to the present invention, it is possible to obtain base sequence information and / or amino acid sequence information of the VH region and / or VL region as information on the antibody specific to the target protein antigen. It is also possible to create an antibody based on the acquired information and obtain a test / diagnostic drug or antibody drug.

Claims (10)

A method for obtaining information on an antigen-specific antibody comprising the following steps (A) to (D):
(A) A step of administering an antigen to a fish having blisters and producing an antibody (B) A step of collecting a blister fluid containing an antibody from the fish of (A) (C) A blister fluid sampled in (B) above Step of constructing a more antigen-specific polyclonal antibody gene library (D) Step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in (C) above The method according to claim 1, wherein the antigen-specific antibody information is antigen-specific VH region and / or VL region base sequence information and / or amino acid sequence information. 3. In construction of an antigen-specific polyclonal antibody gene library, a primer containing the base sequence set forth in SEQ ID NO: 1 and / or a primer containing the base sequence set forth in SEQ ID NO: 2 is used. The method described. The method according to any one of claims 1 to 3, wherein the fish having a water bubble is a water bubble eye or ranchu. Information on the base sequence of an antigen-specific antibody obtained by the method according to any one of claims 1 to 4. Amino acid sequence information of an antigen-specific antibody obtained by the method according to any one of claims 1 to 4. A phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody obtained through the following steps (A) to (E).
(A) A step of administering an antigen to a fish having blisters and producing an antibody (B) A step of collecting a blister fluid containing an antibody from the fish of (A) (C) A blister fluid sampled in (B) above Step of constructing a more antigen-specific polyclonal antibody gene library (D) Step of phage-displaying the antigen-specific polyclonal antibody gene library constructed in (C) (E) Step of (D) Screening a phage-displayed recombinant protein showing all or part of an antigen-specific polyclonal antibody that binds to an antigen from a phage library constructed by The phage-displayed recombinant protein according to claim 7, which is encoded by the base sequence set forth in any one of SEQ ID NOs: 31 to 34 or represented by the amino acid sequence set forth in any of SEQ ID NOs: 35 to 38 in the sequence list . A primer comprising the base sequence set forth in SEQ ID NO: 1 and / or a primer comprising the base sequence set forth in SEQ ID NO: 2 The primer according to claim 9 for the construction of an antigen-specific polyclonal antibody gene library.

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