Preparation and application of high-affinity scFv antibody against infectious bursal virus of chicken

Abstract

The present invention discloses the preparation and application of high-affinity scFv antibodies against infectious bursal disease virus of chickens. The scFv antibodies provided by the present invention include a heavy chain variable region, a light chain variable region and a connecting region therebetween; the present invention screened and obtained 8 strains of scFv antibodies using bacterial display technology of antigen-antibody co-expression, and had high affinity with IBDV VP2 protein and different IBDV virus strains, and the scFv antibodies with high affinity and neutralizing activity can be screened out by constructing a mutant library for the prevention and treatment of IBD; the scFv antibodies can also be used for detecting IBDV pathogens by ELISA method.

Description

Technical Field

[0001] The invention relates to the preparation and application of high-affinity scFv antibodies against chicken infectious bursal virus, belonging to the field of biotechnology. Background Art

[0002] Infectious Bursal Disease (IBD) is an acute, highly contagious disease caused by the Infectious Bursal Disease Virus (IBDV). IBDV mainly attacks chicks and young chickens aged 3-12 weeks, damaging the bursa of Fabricius, the central immune organ of chickens. It has the characteristics of rapid transmission, strong infectivity, high infection rate and mortality rate. The disease is currently distributed worldwide and is one of the most important diseases in the poultry industry. The economic losses caused by immune failure are huge.

[0003] IBDV can rapidly multiply in lymphocytes, especially B lymphocytes, in the bursa of Fabricius in chicks, leading to immunosuppression, thereby increasing the body's susceptibility to other pathogens and reducing the responsiveness to other vaccines. Antibody drugs are currently effective therapeutic drugs. High-immune serum and egg yolk antibodies can have good effects in the early stages of the disease, but they are limited by poor controllability of industrial production and the existence of horizontally transmitted diseases. The present invention uses bacterial display technology of antigen-antibody co-expression to screen 8 strains of anti-IBDV scFv antibodies with high affinity. It can screen out scFv antibodies with high affinity and neutralizing activity by constructing a mutant library for the prevention and treatment of IBD; it can also be used for double antibody sandwich ELISA to detect the presence of pathogenic IBDV. Summary of the invention

[0004] The purpose of the present invention is to provide a high-affinity scFv antibody against chicken infectious bursal disease virus and its application. The scFv antibody is composed of a heavy chain variable region, a light chain variable region and a connecting region therebetween, which can bind to IBDV VP2 protein with high affinity and has important applications in the treatment or prevention of infectious bursal disease and in ELISA kits for detecting infectious bursal disease virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 The SDS-PAGE profile of the scFv antibody.

[0006] Figure 2 The results are shown in Figure 2, which are the results of ELISA testing the specificity and affinity of scFv antibodies for VP2 protein.

[0007] Figure 3The results are shown in Figure 2, which are the results of ELISA testing the specificity and affinity of scFv antibodies to different IBDV viruses. DETAILED DESCRIPTION

[0008] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0009] pET-27b(+) vector: purchased from Novagen, Cat.No.69337-3. Escherichia coli Rosetta: purchased from Novagen, Cat.No.71403-4. Live vaccine for infectious bursal disease of chicken (Gt strain): Harbin Veterinary Research and Development Co., Ltd., No.080012122. Live vaccine for infectious bursal disease of chicken with moderate virulence (NF8 strain): Yangzhou Weike Bioengineering Co., Ltd., No.101042056. Live vaccine for infectious bursal disease of chicken (MB strain): ABIC, No.20621150B. Live vaccine for infectious bursal disease of chicken (B87 strain): Hunan Zhongan Biopharmaceutical Co., Ltd., No.180022026.

[0010] Coating solution (pH 9.6): Take 0.15 g of Na2CO3 and 0.293 g of NaHCO3, dissolve them in water and make up to 100 mL with water.

[0011] PBS buffer: Take 8g NaCl, 0.2g KCl, 3.58g Na2HPO4.12H2O, and 0.24g KH2PO4 and dissolve them in 1L water.

[0012] Example 1. Obtaining scFv antibodies and their encoding genes

[0013] 1. Construction of antibody library

[0014] Take the spleen of chickens immunized with IBDV, extract RNA and reverse transcribe it into cDNA. According to the antibody sequence on GenBank, design the gene primers for cloning the variable region of the antibody, use cDNA as a template, and use the PCR method to clone the variable region gene of the antibody. Connect the VH and VL fragments into scFv genes and connect them to the bacterial display vector pBFD-Ab-VP2 to construct a bacterial surface display library for co-expression of anti-IBDV antigen and antibody. VH is about 380bp, VL is about 320bp, and VH-Tlinker-VL is about 740bp.

[0015] 2. Screening of antibody library

[0016] All clones after transformation were collected, induced with IPTG (0.25 mmol / L) for 4 h, treated with EDTA-MgCl2, incubated with rabbit anti-VP2 protein polyclonal antibody (i.e., the polyclonal antibody solution prepared in Example 4, with a working concentration of 1:5,000 dilution) and FITC-labeled goat anti-rabbit secondary antibody (purchased from Sigma, with a working concentration of 1:500 dilution) for 1 h each, washed with PBS, and screened using a flow cytometer.

[0017] After three rounds of screening, eight scFv monoclonal antibodies with high binding ability to VP2 protein were obtained, and they were named scFv-11, scFv-12, scFv-13, scFv-14, scFv-15, scFv-16, scFv-17 and scFv-18 antibodies.

[0018] The amino acid sequences of the eight scFv antibodies are shown in Sequences 1 to 8 in the sequence listing, and the nucleotide sequences thereof are shown in Sequences 9 to 16 in the sequence listing.

[0019] Example 2. Preparation of 8 scFv antibodies

[0020] 1. Using the plasmid screened and obtained in Example 1 and capable of expressing eight monoclonal antibodies with high binding ability as a template, eight scFv antibody genes were amplified by PCR using primers consisting of F1 and R1 to obtain PCR amplification products.

[0021] F1: 5'–CGC CATATG GCCGTGACGTTGGACGAG-3′;

[0022] R1: 5'–CCC AAGCTT TTAACCTAGGACGGTCAGGG-3'.

[0023] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases Nde I and Hind III, and recover the digestion product.

[0024] 3. Double-digest the pET-27b(+) vector with restriction endonucleases Nde I and Hind III to recover a vector backbone of approximately 5367 bp.

[0025] 4. Connect the digestion product of step 2 and the vector backbone of step 3 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recombinant plasmid is described as follows: the double-stranded DNA molecules shown in sequences 9 to 16 of the sequence list are inserted between the Nde I and Hind III restriction sites of the pET-27b(+) vector.

[0026] 5. Introduce the recombinant plasmid obtained in step 4 into Escherichia coli Rosetta to obtain recombinant bacteria.

[0027] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / ml kanamycin, and cultured at 37°C and 100 rpm until OD 600nm =0.3; add IPTG to a concentration of 0.25mmol / L, and culture at 37°C and 100r / min for 4h.

[0028] 7. Take 15 L of the culture system completed in step 6, centrifuge at 4°C and 4000 r / min for 30 min and collect the bacterial precipitate.

[0029] 8. Take the bacterial precipitate obtained in step 7, resuspend it with PBS buffer, add lysozyme solution (purchased from Amresco) and make the lysozyme concentration be 1 mg / ml, let it stand at 4°C for 1 h, then perform ultrasonic disruption (25 watts power, 3 min), centrifuge at 4°C, 10000g for 30 min, and collect the precipitate.

[0030] 9. Purify the target protein using AKTA Purifier 100 protein chromatography system (purchased from GE)

[0031] Take the precipitate obtained in step 8, dissolve it with 100 ml of dissolution buffer (8 mol / L urea aqueous solution, pH 8.0), and then load it on a HiLoad 16 / 60 Superdex75 pg column (purchased from GE), then elute it with 500 ml of refolding buffer (2 mol / L urea aqueous solution, pH 8.0) and collect the eluate after passing through the column, and then dialyze it in PBS buffer overnight to obtain a solution of 8 scFv antibody solutions. All steps were performed at 4 ° C. The SDS-PAGE patterns of the 8 scFv antibody solutions are shown in Figure 1 , showing a band of approximately 28 kD, consistent with expectations.

[0032] Example 3: ELISA to detect affinity and specificity of scFv antibodies

[0033] 1. ELISA to detect the specificity and affinity of several scFv antibodies to VP2 protein

[0034] 1. Coat the ELISA plate with a scFv antibody solution having a protein concentration of 10 μg / ml (i.e., the scFv antibody solution prepared in Example 2, with the protein concentration adjusted with the coating solution) at 4° C. overnight, and then wash 3 times with PBST buffer, each time for 2 min.

[0035] 2. Add 100 μl of VP2 protein solution with a protein concentration of 40 μg / ml to each well, incubate at 37°C for 1 hour, and then wash three times with PBST buffer, each time for 2 minutes.

[0036] 3. Add rabbit anti-VP2 polyclonal antibody, incubate at 37°C for 1 h, and then wash 3 times with PBST buffer, 2 min each time.

[0037] 4. Add HRP-labeled goat anti-rabbit antibody (purchased from R&D system, working concentration is 1:8000 dilution), incubate at 37°C for 1 hour, and then wash 3 times with PBST buffer, 2 minutes each time.

[0038] 5. Add TMB substrate colorimetric solution and develop the color at 37°C in the dark for 5 minutes.

[0039] 6. Add 50 μl of 2 mol / L H2SO4 aqueous solution to each well and detect the OD value at a wavelength of 450 nm using an enzyme reader.

[0040] Set up a PBS group in which an equal volume of PBS buffer is used to replace the VP2 protein solution in step 2 and the rabbit anti-VP2 polyclonal antibody in step 3. When the scFv antibody solution with a protein concentration of 10 μg / ml is used to coat the ELISA plate in step 1: set up a control group 1 in which the VP2 protein solution is not added in step 2, a control group 2 in which the rabbit anti-VP2 polyclonal antibody is not added in step 3, a control group 3 in which the VP2 protein solution is not added in step 2 and the rabbit anti-VP2 polyclonal antibody is not added in step 3, and a control group 4 in which the VP2 protein solution is replaced by an equal volume of BSA solution with an equal protein concentration.

[0041] Three replicate wells were set up for each treatment.

[0042] Results Figure 2 ELISA results showed that all eight scFv antibodies could bind specifically to VP2 protein with high affinity.

[0043] 2. ELISA to detect the specificity and affinity of scFv antibodies to different IBDV viruses

[0044] 1. Coat the ELISA plate with a scFv antibody solution having a protein concentration of 10 μg / ml (i.e., the scFv antibody solution prepared in Example 2, with the protein concentration adjusted with the coating solution) at 4° C. overnight, and then wash 3 times with PBST buffer, each time for 2 min.

[0045] 2. Add 100 μl IBDV virus solution (virus dose is 10 6.2 TCID 50 ), incubated at 37°C for 1 h, and then washed three times with PBST buffer, each time for 2 min.

[0046] 3. Add rabbit anti-VP2 polyclonal antibody, incubate at 37°C for 1 h, and then wash 3 times with PBST buffer, 2 min each time.

[0047] 4. Add HRP-labeled goat anti-rabbit antibody (purchased from R&D system, working concentration is 1:8000 dilution), incubate at 37°C for 1 hour, and then wash 3 times with PBST buffer, 2 minutes each time.

[0048] 5. Add TMB substrate colorimetric solution and develop the color at 37°C in the dark for 5 minutes.

[0049] 6. Add 50 μl of 2 mol / L H2SO4 aqueous solution to each well and detect the OD value at a wavelength of 450 nm using an enzyme reader.

[0050] The above experiments were carried out using the following IBDV strains: Gt strain, NF8 strain, MB strain, and B87 strain.

[0051] A PBS group was set up in which an equal volume of PBS buffer was used to replace the IBDV virus solution in step 2 and the rabbit anti-VP2 polyclonal antibody in step 3. A control group 1 was set up in which no IBDV virus solution was added in step 2, a control group 2 was set up in which no rabbit anti-VP2 polyclonal antibody was added in step 3, a control group 3 was set up in which no IBDV virus solution was added in step 2 and no rabbit anti-VP2 polyclonal antibody was added in step 3, and a control group 4 was set up in which an equal volume of Newcastle disease virus solution of equal titer was used to replace the IBDV virus solution.

[0052] Three replicate wells were set up for each treatment.

[0053] Results Figure 3 ELISA results showed that all eight scFv antibodies could specifically bind to different IBDV strains and had different affinities for different IBDV strains.

Claims

1. A single-chain antibody scFv-16 against infectious bursal virus of chickens, the single-chain antibody scFv-16 consisting of a heavy chain variable region, a light chain variable region and a connecting region therebetween; The single-chain antibody scFv-16 is as follows: (a) a protein consisting of amino acid residues 1 to 246 from the N-terminus of sequence 6 in the sequence list.

2. A gene encoding the single-chain antibody scFv-16 according to claim 1.

3. The gene according to claim 2, characterized in that: In the gene, the DNA molecule encoding the single-chain antibody scFv-16 is as follows (1): (1) the DNA molecule represented by nucleotides 1 to 741 from the 5' end of sequence 14 in the sequence listing.

4. An expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing the gene according to any one of claims 2 to 3.

5. Use of the single-chain antibody scFv-16 according to claim 1, the gene according to claim 2 or 3, or the expression cassette, recombinant vector, transgenic cell line or recombinant bacteria according to claim 4 in the preparation of a product; the function of the product is as follows (I) or (II) or (III): (I) detection of infectious bursal disease virus; (II) assisting in the identification of infectious bursal disease virus; (III) prevention and / or treatment of infectious bursal disease.

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