CN117051002B - Nanobodies against SpyCatcher003 protein and their applications - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to an anti-SpyCatcher 003 protein nanobody and application thereof, wherein the nanobody comprises at least one of SC-N2, SC-N3 or SC-N4, the nucleotide sequence of the SC-N2 is shown as SEQ ID NO.1, the nucleotide sequence of the SC-N3 is shown as SEQ ID NO.2, and the nucleotide sequence of the SC-N4 is shown as SEQ ID NO. 3. The application of the nano antibody in preparation and detection of SpyCatcher003 protein products. The invention discloses a high-affinity nano antibody aiming at SpyCatcher003 protein, which has the advantages of small molecular weight, high stability, low production cost and the like, is used for detecting the existence and the location of the SpyCatcher with high sensitivity and specificity, and can be applied to the fields of laboratory research, biosensor development, clinical diagnosis and the like.

Description

Nanobodies against SpyCatcher003 protein and their applications

Technical Field

The present invention relates to the field of genetic engineering, and in particular to a nano antibody against SpyCatcher003 protein and an application thereof.

Background technique

SpyCatcher is an important tool for protein engineering and labeling. It is derived from the second immunoglobulin-like collagen adhesin domain (CnaB2) of fibronectin (FbaB) of Streptococcus pyogenes. It can specifically recognize the homologous SpyTag polypeptide and bind to form a covalent link. The SpyTag/SpyCatcher system has many advantages: 1) Rapid binding: SpyTag and SpyCatcher can be quickly bound within a few minutes at room temperature without the need for other auxiliary molecules; 2) Strong binding: The covalent binding of SpyTag and SpyCatcher forms a very strong isopeptide bond that is not easy to dissociate; 3) Reversibility: Despite the strong binding force, the binding of SpyTag and SpyCatcher can be broken and re-bound under appropriate conditions. Due to the many advantages of the SpyTag/SpyCatcher system, such as simplicity, flexibility and controllability, the system is currently widely used in biomedical research and biotechnology.

However, there is a lack of antibodies against SpyCatcher on the market, and only Bio-Rad (USA) provides a human-rabbit chimeric IgG subtype antibody. Usually, the acquisition of such traditional antibodies requires a high time and cost, and the large molecular weight and complex structure of traditional antibodies also limit their use in certain applications. At present, the detection of SpyCatcher and its fusion protein relies more on other indirect means, which also introduces uncertainty to downstream experiments and limits the scope of application and convenience of the system.

In summary, although SpyCatcher has wide applications in protein engineering, nanomaterial assembly and drug delivery, antibodies against SpyCatcher are currently scarce and very necessary.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide nanobodies against SpyCatcher003 protein and applications thereof.

The object of the present invention is achieved through the following technical scheme: a nanobody against SpyCatcher003 protein, wherein the nanobody includes at least one of SC-N2, SC-N3 or SC-N4, the nucleotide sequence of SC-N2 is shown in SEQ ID NO.1, the nucleotide sequence of SC-N3 is shown in SEQ ID NO.2, and the nucleotide sequence of SC-N4 is shown in SEQ ID NO.3.

Furthermore, the amino acid sequence of SC-N2 is shown in SEQ ID NO.4, the amino acid sequence of SC-N3 is shown in SEQ ID NO.5, and the amino acid sequence of SC-N4 is shown in SEQ ID NO.6.

Furthermore, the nanobody comprises a complementarity determining region, which consists of CDR1, CDR2 and CDR3.

Furthermore, the amino acids of CDR1, CDR2 and CDR3 of the nanobody SC-N2 are SEQ ID NO.4 from the 26th to 33rd, 51st to 58th and 97th to 108th positions from the N-terminus, respectively; the amino acids of CDR1, CDR2 and CDR3 of the nanobody SC-N3 are SEQ ID NO.5 from the 26th to 33rd, 51st to 57th and 96th to 107th positions from the N-terminus, respectively; the amino acids of CDR1, CDR2 and CDR3 of the nanobody SC-N4 are SEQ ID NO.6 from the 26th to 33rd, 51st to 58th and 97th to 108th positions from the N-terminus, respectively.

Furthermore, the amino acid sequence of the SpyCatcher003 protein is shown in SEQ ID NO.7.

Furthermore, the nucleotide sequence of the upstream primer for amplifying the nanobody is shown in SEQ ID NO.8, and the downstream primer is shown in SEQ ID NO.9.

The present invention also provides an expression vector, wherein the vector contains the above-mentioned nanobody.

In one embodiment, the initial vector of the expression vector is pFUSE-hIgG1-Fc2.

The present invention also provides a kit for detecting SpyCatcher003 protein, comprising the above-mentioned nano antibody.

The present invention also provides the use of nano antibodies in preparing a product for detecting SpyCatcher003 protein.

The present invention has the following advantages: The present invention discloses a high-affinity nanobody for SpyCatcher003 protein, which has many advantages such as small molecular weight, high stability, and low production cost. It is the first nanobody for the protein, filling the gap in the market and providing more and better options for downstream applications. It has many application potentials and advantages. It is used to detect the presence and location of SpyCatcher with high sensitivity and specificity, and can be applied to laboratory research, biosensor development or clinical diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the BLI detection curve of SpyCatcher003 positive clone VHH-hFc.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and embodiments, and the protection scope of the present invention is not limited to the following:

Phage display nanobody library:

Nanobody VHH is a heavy chain antibody variable region derived from camelids that naturally lacks light chains, with a molecular weight of about 15kDa, which is 1/10 of the molecular weight of conventional antibodies. Nanobodies are the smallest naturally occurring fragments that can bind to antigens, and they have many advantages, such as high specificity, small size, and easy labeling and engineering, which make them have broad application prospects in diagnosis and treatment.

Phage display antibody library technology is a method of displaying proteins on the surface of phage. The phage display nano antibody library used in the present invention is a fusion expression of the alpaca-derived nano antibody VHH gene with the P3 protein on the surface of the phage and displayed on the surface of the progeny phage. The library was purchased from AlpVHHS (Cat. No.: AlpSDAb-P).

SpyCatcher003 Protein:

The SpyTag003/SpyCatcher003 mutant is the fastest covalently bound SpyTag/SpyCatcher system. The SpyCacher003 protein (with a His tag at the N-terminus) was purchased from Bio-Rad (Cat. No. TZC025).

The sequence of the SpyCacher003 protein is shown in SEQ ID NO.7, specifically:

VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT

Example 1: Screening of anti-SpyCatcher003 protein nanobodies

The solid phase screening method is used to screen specific antibodies. The specific steps are as follows:

1) Antigen plating: SpyCatcher003 protein (5 μg/ml, diluted in PBS) of appropriate concentration was coated on 4 wells of Maxsorp 96-well plates at 4°C overnight.

2) Host bacteria culture: Take the host bacteria TG1 and inoculate it into a suitable volume of fresh 2YT medium, shake it at 37°C, 250 rpm for 3-4 hours until OD600 = 0.5, and place it at room temperature for use.

3) Remove the liquid from the wells and wash three times with PBS.

4) Use MPBS (final concentration 5% skim milk, diluted in PBS) to block the plate wells and phage display nanoantibody library AlpVHHS (Input), and incubate at room temperature for 1 to 2 hours.

5) Remove the blocking solution and wash the wells three times with PBS.

6) Add the antibody library and incubate for binding at room temperature with shaking (low speed) for 2 hours.

7) Wash the wells several times with PBST and PBS respectively.

8) Elution: Add freshly prepared 100 mM TEA and incubate at room temperature with shaking (low speed) for 20 min.

9) Neutralization: Pipette vigorously 10 times, add the eluate into a shaking tube containing 1/2 volume of 1M Tris-HCl (pH 7.5), and incubate at room temperature for 5 minutes to neutralize TEA.

10) The mixture from the previous step was added to 10 times the volume of the host bacteria and cultured at 37°C with shaking at 250 rpm for 1 h.

11) Phage titration: Take a small amount of the culture from the previous step and make a 10-fold gradient dilution with 2YT. Take samples of appropriate dilutions for titration on 2YT plates and incubate at 37°C overnight.

12) Transfer the remaining culture to a 50 ml centrifuge tube, centrifuge at 2500 g for 10 min, discard the supernatant, add the original volume of 2YTA resuspended bacterial solution, and continue culturing at 37°C, 250 rpm for 2 h.

13) Add M13KO7 helper phage to a final concentration of 10 ¹⁰ phage/ml and infect at 37°C, 250 rpm for 30 min.

14) Transfer the culture to a 50 ml centrifuge tube, centrifuge at 2500 g for 10 min, discard the supernatant, add the original volume of 2YTAK/IPTG (IPTG final concentration 0.2 mM) medium, and incubate at 30°C, 280 rpm overnight.

15) The next day, count the number of infectious monoclonal bacteria titrated in 11) and calculate the number of phages bound in this round (Output)

16) At the same time, collect Phage from the overnight culture supernatant as the second round of input phage for the next round of screening.

17) Calculate the enrichment constant R = Output/Input for each round.

When the R value increases significantly, it can be determined that the screening is enriched. This screening showed obvious enrichment in the third round. The experimental results are shown in Table 1.

Table 1 Enrichment constants of SpyCatcher003 protein in three rounds of screening

Enrichment constant First round R1 Second round R2 Round 3 R3 Output/Input 1.64×10 ^-7 1.25× ^10-7 5.89×10 ^-3

Example 2: Identification of positive monoclonal anti-SpyCatcher003 protein nanoantibody--Phage ELISA

1) The Output phage from the last round in which significant enrichment occurred was selected to infect TG1 bacteria. As shown in Example 1, significant enrichment occurred in the third round, so the Output phage from the second round was used.

2) Use an appropriate amount of Outputphage to infect the TG1 host bacteria, spread on LB/Amp100 plates, and culture at 37°C overnight.

3) The next day, several (eg, 192) single clones were picked from the LB/Amp100 plate and added to a 96-well deep-well plate containing 200 μL/well 2YT/Amp100/2% Glucose medium, and cultured overnight at 250 rpm and 37°C.

4) Inoculate the overnight culture from the previous day into a 96-well deep-well plate containing 200 L/well 2YT/Amp100/2% Glucose medium at a ratio of 1:50, at 250 rpm, 37°C, until the bacterial solution OD600 ≈ 0.5 (the bacteria are in the logarithmic growth phase).

5) Add M13K07 helper phage at a final concentration of about 10 ¹⁰ phage/ml and infect at 37°C, 250 rpm for 30 min.

6) Centrifuge at 3000g, RT for 10 min, invert and discard the supernatant, add 400 L/well 2YT/Amp100/Kan50, and resuspend the cells.

7) Incubate at 37°C, 250 rpm overnight. Coat the 96-well plate used for ELISA tomorrow with 2 μg/ml SpyCatcher003 protein, 100 L/well; coat at 4°C overnight.

8) Take out the 96-well ELISA plate coated yesterday, remove the antigen, and wash 3 times with PBS, 200L/well.

9) Add MPBS to block the ELISA plate, 200 L/well, at room temperature for 2 h.

10) Take out the 96-well deep-well plate in 3) and centrifuge at 3000 g for 10 min.

11) Pipette 60 L/well supernatant into a clean 96-well plate, add an equal volume of 5% skim milk-PBS solution and pre-blocked phage-containing culture supernatant, and incubate at RT for 1-2 h.

12) Discard the blocking solution in the ELISA plate and wash 4 times with PBST, 200 L/well.

13) Add the culture supernatant pre-blocked in step 14, 100 L/well, and incubate at room temperature with gentle shaking for 1 hour.

14) Wash 8 times with PBST, 200 L/well.

15) Add HRP-anti-M13 antibody, 100 μL/well, and incubate with gentle shaking at room temperature for 30 min.

16) Wash 6 times with PBST and then wash 2 times with PBS, 200 μL/well.

17) Add TMB substrate, 50 L/well, incubate at room temperature (25°C) or 37°C in the dark for 5-10 min, and observe the color development (blue).

18) Add an equal volume of stop solution (1 M sulfuric acid solution), 100 L/well, to terminate the reaction. The reaction solution in the well changes from blue to yellow.

19) Within 30 minutes after terminating the reaction, measure the absorbance at 450 nm using an enzyme reader.

20) Select positive clones (OD450 value ≥ 0.5) for sequencing.

21) The monoclonal sequences were compared to obtain positive monoclonal clones (Unique) with different CDR3 sequences. A total of three specific monoclonal clones (named SC-N2 to SC-N4) were obtained in this screening.

The nucleotide sequence of SC-N2 is shown in SEQ ID NO.1, specifically:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCATTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGGATCTATGCCGTGAATTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTAGCACGTATTACCTGGACTACTGGTAGTGTATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAGGACAACTCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAATATGAGGACACGGCCGTTTATTACTGCGCATTCCGCTCCGTGGGCGACTCCAAATATTCCGGCTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA-3’

The nucleotide sequence of SC-N3 is shown in SEQ ID NO.2, specifically:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCATTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGGATCTATGCCGTGAATTGGTCCCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCCCGTATTAGTCGTAGTGGCAGCACATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAAAGACAATACCGAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCCCGGGGTGCTGGTTCCCCCCATTATGACTATTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA-3’

The nucleotide sequence of SC-N4 is shown in SEQ ID NO.3, specifically:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCATGGGGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGAAGCATCAGCAGTATCTATGCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGGAGCTACTAGCTGGAGTGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTTCAGCTAGACCGGTCTCGACCCCAAGGTGGGACTACTGGGGCCAGGGGACCCAGGTCA CTGTCTCCTCA-3’

The amino acid sequence of SC-N2 is shown in SEQ ID NO.4, specifically:

The amino acid sequence of SC-N3 is shown in SEQ ID NO.5, specifically:

The amino acid sequence of SC-N4 is shown in SEQ ID NO.6, specifically:

In the above amino acid sequence, the bold part is CDR1, the single underlined bold part is CDR2, and the double underlined bold part is CDR3.

Example 3: Verification of the binding activity of anti-SpyCatcher003 protein nanoantibodies

To further confirm the binding activity of the positive single clones, these three positive clones were expressed in eukaryotes and their binding to the SpyCatcher003 protein was further verified.

1. Eukaryotic Expression

The eukaryotic expression is in the form of nanobody (VHH) fused with the Hinge-Fc (hFc) fragment of human IgG1 subtype antibody, and the expression product is VHH-hFc dimer. The specific expression steps are as follows:

1) Construction of recombinant vector:

The eukaryotic expression vector pFUSE-hIgG1-Fc2 ((purchased from InvivoGen, catalog number: pfuse-hg1fc2)) was double-digested with EcoRV and NcoI;

The positive VHH clone fragments were amplified by PCR, and the specific reaction conditions are shown in Table 2:

Table 2 PCR reaction system and reaction conditions

*Primer sequences are as follows:

VHH-Fc F:

5'-GCACTTGTCACGAATTCGATACTCAGGTGCAGCTCGTGGAG-3'SEQ ID NO.8

VHH-Fc R:

5'-TGAGTTTTGTCAGATCTAACTGAGGAGACRGTGACCTGG-3'SEQ ID NO.9

The positive VHH fragment was purified by a PCR product recovery kit and then connected to the restriction linearized vector by homologous recombination. The ligation product was transformed into DH5α competent cells, and 500 μL SOC was added to resuspend the cells. After activation at 37°C, 220 rpm for 30 minutes, 100 μL of bacterial solution was applied to LB agarose plates containing a final concentration of 50 μg/mL zeocin and incubated at 37°C overnight. The next day, 4 clones were randomly selected and sent to Qingke Company for sequencing.

2) Transfection: The recombinant expression vector was transfected into 293F cells using PEI.

3) Culture: The cells were cultured and expressed for 5 days.

4) Antibody separation: The culture supernatant was collected and VHH-hFc was separated and purified using Protein A.

5) Use ultrafiltration tubes to replace and concentrate the antibody solution.

6) Determine the concentration, package and store in an ultra-low temperature refrigerator.

Example 5: Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of nanobodies

The affinity of VHH-hFc to SpyCatcher003 protein was determined by ELISA. The specific method is as follows:

1) Coating: Dilute SpyCatcher003 protein to 2 μg/mL using PBS, 100 μL/well, and allow to bind overnight at 4°C.

2) Washing: Wash 3 times with PBS, 200 μL/well.

3) Blocking: 5% MPBS, 200 μL/well, incubate at room temperature for 2 h.

4) Primary antibody incubation: VHH-hFc antibody with serial dilutions, 100 μL/well, binding at room temperature for 1 hour.

5) Washing: Wash 6 times with PBST, 200 μL/well.

6) Secondary antibody incubation: Use horseradish peroxidase (HRP)-labeled anti-human IgG-Fc antibody, 1:5000 dilution, 100 μL/well, and bind at room temperature for 30 minutes.

7) Washing: Wash with PBST for 6 times and PBS for 3 times, 200 μL/well.

8) Color development: Add TMB substrate, 50 μL/well, and react at room temperature for about 5 minutes.

9) Stop: Add 1 M dilute sulfuric acid, 50 μL/well.

10) Read OD value: Use a microplate reader to measure the OD450 signal value.

11) Data analysis is shown in Table 3.

Table 3 Affinity of SpyCatcher003 positive clones (ELISA)

serial number SC-N2 SC-N3 SC-N4 EC50(μg/mL) 0.012 0.253 0.450

The ELISA results showed that all three monoclones had very high affinity.

Example 6: Determination of Nanobody Affinity by Bio-Layer Interferometry (BIL)

Using Sartorius BLI Systems used SpyCatcher003 protein (with 6×his tag, 5μg/mL) as ligand and 2-fold gradient diluted VHH-hFc as analyte (10μg/mL, 5μg/mL, 2.5μg/mL, 1.25μg/mL, 0.625μg/mL, 0.3125μg/mL, 0.15625μg/mL, 7 concentrations in total) to measure the affinity of VHH-hFc. The experimental results are shown in Figure 1 and Table 4.

Table 4 VHH-hFc affinity (BLI) of SpyCatcher003 positive clones

serial number SC-N2 SC-N3 SC-N4 KD(nM) 0.45 1.37 1.41

It can be seen from Table 4 and Figure 1 that all three clones have very high affinities (nM level), among which SC-N2 has the highest affinity.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims (6)

1. A nanobody against SpyCatcher003 protein, characterized in that the nanobody is at least one of SC-N2, SC-N3 or SC-N4, the amino acid sequence of SC-N2 is shown in SEQ ID NO.4, the amino acid sequence of SC-N3 is shown in SEQ ID NO.5, the amino acid sequence of SC-N4 is shown in SEQ ID NO.6, and the amino acid sequence of the SpyCatcher003 protein is shown in SEQ ID NO.7. 2. A nucleic acid molecule, characterized in that the nucleotide sequence is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and its expression product corresponds to SC-N2, SC-N3 or SC-N4 described in claim 1, respectively. 3. An expression vector, characterized in that the vector contains any nucleic acid molecule described in claim 2. The expression vector according to claim 3 , characterized in that the initial vector of the expression vector is pFUSE-hIgG1-Fc2. 5. A kit for detecting SpyCatcher003 protein, characterized in that it contains the nanobody according to claim 1. 6. Use of the nanobody according to claim 1 in the preparation of a product for detecting SpyCatcher003 protein.