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CN117843769A - Preparation and application of high-affinity anti-chicken infectious bursal disease virus scFv antibody - Google Patents

Preparation and application of high-affinity anti-chicken infectious bursal disease virus scFv antibody Download PDF

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CN117843769A
CN117843769A CN202310608000.9A CN202310608000A CN117843769A CN 117843769 A CN117843769 A CN 117843769A CN 202310608000 A CN202310608000 A CN 202310608000A CN 117843769 A CN117843769 A CN 117843769A
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scfv
infectious bursal
bursal disease
variable region
chain variable
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CN117843769B (en
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任桂萍
郭笑辰
孙文影
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Northeast Agricultural University
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Abstract

The invention discloses preparation and application of a high-affinity anti-chicken infectious bursal disease virus scFv antibody. The scFv antibody provided by the invention comprises a heavy chain variable region, a light chain variable region and a connecting region between the heavy chain variable region and the light chain variable region; the invention utilizes the bacterial display technology of antigen-antibody co-expression to screen and obtain 8 scFv antibodies, and has high affinity with IBDV VP2 protein and different IBDV virus strains, and can screen scFv antibodies with high affinity and neutralization activity by constructing a mutant library for preventing and treating IBDV; it can also be used for ELISA detection of IBDV pathogens.

Description

Preparation and application of high-affinity anti-chicken infectious bursal disease virus scFv antibody
Technical Field
The invention relates to preparation and application of a high-affinity anti-chicken infectious bursal disease virus scFv antibody, and belongs to the technical field of biology.
Background
Infectious bursal disease (Infectious Bursal Disease, IBD) is an acute, highly contagious disease caused by infectious bursal virus (Infectious Bursal Disease Virus, IBDV), which mainly infects chickens and young chickens of 3-12 weeks old, damaging the central immune organs of chickens, the bursa of Fabricius. Has the characteristics of high transmission speed, strong infectivity, high infection rate and high death rate. The disease is distributed worldwide at present, is one of the most important diseases in the poultry industry, and has huge economic loss caused by immune failure.
IBDV can rapidly multiply in lymphocytes, especially B lymphocytes, in chicken bursa, leading to immunosuppression, which can increase the susceptibility of the body to other pathogens and reduce the responsiveness to other vaccines. Antibody drugs are currently effective therapeutic drugs, and both the hyperimmune serum and the egg yolk antibody can have good effects in early stages of onset, but are limited due to poor controllability of industrial production, the existence of horizontally transmitted diseases, and the like. The invention utilizes the bacterial display technology of antigen-antibody co-expression to screen and obtain 8 scFv antibodies of anti-IBDV with higher affinity, which can screen scFv antibodies with high affinity and neutralization activity by constructing a mutant library for preventing and treating IBD; it can also be used in a double-antibody sandwich ELISA method for detecting the presence of pathogenic IBDV.
Disclosure of Invention
The invention aims to provide a high-affinity scFv antibody for resisting infectious bursal disease virus and application thereof. scFv antibodies consist of a heavy chain variable region, a light chain variable region and a connecting region between the heavy chain variable region and the light chain variable region, can be combined with IBDV VP2 protein with high affinity, and have important application in treating or preventing infectious bursal disease and ELISA kits for detecting infectious bursal disease virus.
Drawings
FIG. 1 is a SDS-PAGE map of scFv antibodies.
FIG. 2 shows the results of ELISA detection of the specificity and affinity of scFv antibodies for VP2 protein.
FIG. 3 shows the results of ELISA detection of scFv antibodies specific and affinity for different IBDV viruses.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
pET-27b (+) vector: purchased from Novagen company, cat.No.69337-3. Coli Rosetta: purchased from Novagen company, cat.No.71403-4. Chicken infectious bursal disease live vaccine (Gt strain): halon research vista, no. 080012122. Infectious bursal disease moderate virulence live vaccine (NF 8 strain): the Yangzhou Weike bioengineering Co., ltd, code 101042056. Chicken infectious bursal disease live vaccine (MB strain): ABIC, number 20621150B. Chicken infectious bursal disease live vaccine (B87 strain): the middle shore of the Hunan province is assigned the code 180022026 by the biological pharmaceutical industry Co., ltd.
Coating solution (ph 9.6): taking Na 2 CO 3 0.15g、NaHCO 3 0.293g, dissolved in water and fixed to a volume of 100mL with water.
PBS buffer: 8g NaCl, 0.2g KCl and Na are taken 2 HPO 4 .12H 2 O 3.58g、KH 2 PO 4 0.24g, dissolved in 1L water.
Example 1 acquisition of scFv antibodies and genes encoding same
1. Construction of antibody libraries
The spleen of the chicken immunized with IBDV is taken, RNA is extracted and then is reversely transcribed into cDNA. According to the antibody sequence on GenBank, a gene primer for cloning the variable region of the antibody is designed, cDNA is used as a template, and a PCR method is used for cloning the variable region gene of the antibody. The VH and VL fragments were linked to scFv genes and to the bacterial display vector pBFD-Ab-VP2 to construct a bacterial surface display library co-expressed with anti-IBDV antigen antibodies. About 380bp for VH, about 320bp for VL and about 740bp for VH-Tlinker-VL.
2. Screening of antibody libraries
Collecting all clones after transformation, inducing for 4h by IPTG (0.25 mmol/L), and passing through EDTA-MgCl 2 Treatment, incubation with polyclonal antibodies against rabbit anti-VP 2 protein (i.e., polyclonal antibody solution prepared in example 4, working concentration 1:5 times diluted) for 1h each with FITC-labeled goat anti-rabbit secondary antibody (purchased from sigma company, working concentration 1:500 times diluted), washing with PBS, and screening with flow cytometry.
After three rounds of screening, 8 scFv monoclonal antibodies with high binding capacity to VP2 protein were obtained, which were designated scFv-11, scFv-12, scFv-13, scFv-14, scFv-15, scFv-16, scFv-17 and scFv-18, respectively.
The amino acid sequences of the 8 scFv antibodies are shown as sequences 1-8 in the sequence table, and the nucleotide sequences are shown as sequences 9-16 in the sequence table.
Example 2 preparation of 8 scFv antibodies
1. The 8 scFv antibody genes were PCR amplified with primers consisting of F1 and R1 using the plasmid capable of expressing 8 monoclonal antibodies with high binding ability obtained by the screening in example 1 as a template, respectively, to obtain PCR amplified products.
F1：5'–CGCCATATGGCCGTGACGTTGGACGAG-3'；
R1：5'–CCCAAGCTTTTAACCTAGGACGGTCAGGG-3'。
2. The PCR amplification product of step 1 was digested with restriction enzymes NdeI and HindIII, and the digested product was recovered.
3. The pET-27b (+) vector was digested with restriction enzymes NdeI and HindIII, and the vector backbone of about 5367bp was recovered.
4. And (3) connecting the enzyme digestion product of the step (2) with the vector skeleton of the step (3) to obtain the recombinant plasmid. Based on the sequencing results, the recombinant plasmid was structurally described as follows: double-stranded DNA molecules shown in sequences 9 to 16 of the sequence table are inserted between Nde I and Hind III cleavage sites of the pET-27b (+) vector.
5. And (3) introducing the recombinant plasmid obtained in the step (4) into escherichia coli Rosetta to obtain recombinant bacteria.
6. Inoculating the recombinant strain obtained in step 5 into LB liquid medium containing 50 μg/ml kanamycin, shake culturing at 37deg.C and 100r/min to OD 600nm =0.3; IPTG was added thereto and the mixture was cultured at 37℃and 100r/min with shaking at a concentration of 0.25mmol/L for 4 hours.
7. Taking 15L of the culture system which completes the step 6, centrifuging at 4 ℃ and 4000r/min for 30min, and collecting bacterial precipitate.
8. Taking the thallus sediment obtained in the step 7, re-suspending the thallus sediment by using PBS buffer solution, adding lysozyme solution (purchased from Amresco) and enabling the concentration of the lysozyme to be 1mg/ml, standing for 1h at 4 ℃, then performing ultrasonic crushing (power of 25W, 3 min), centrifuging for 30min at 4 ℃ and 10000g, and collecting the sediment.
9. Purification of proteins of interest using the AKTA Purifier 100 protein chromatography System (available from GE Co.)
Taking the precipitate obtained in the step 8, fully dissolving the precipitate by using 100mL of dissolving buffer solution (8 mol/L urea aqueous solution, pH 8.0), then loading the solution on a HiLoad 16/60Superdex75 pg column (purchased from GE company), eluting by using 500mL of renaturation buffer solution (2 mol/L urea aqueous solution, pH 8.0), collecting the eluate after column passing, and dialyzing in PBS buffer solution overnight to obtain the solution, namely 8 scFv antibody solutions. All steps were carried out at 4 ℃. SDS-PAGE patterns of the 8 scFv antibody solutions are shown in FIG. 1, showing bands of approximately 28kD, as expected.
Example 3 ELISA detection of affinity and specificity of scFv antibodies
1. ELISA (enzyme Linked immunosorbent assay) for detecting specificity and affinity of several scFv antibodies to VP2 protein
1. The ELISA plate was coated with scFv antibody solution having a protein concentration of 10. Mu.g/ml (i.e., scFv antibody solution prepared in example 2, adjusted in protein concentration with coating solution), overnight at 4℃and then washed 3 times with PBST buffer for 2min each.
2. 100 μl of VP2 protein solution with a protein concentration of 40 μg/ml was added to each well, incubated for 1h at 37deg.C, and then washed 3 times with PBST buffer for 2min each.
3. Rabbit anti-VP 2 polyclonal antibody was added, incubated for 1h at 37℃and then washed 3 times with PBST buffer for 2min each.
4. HRP-labeled goat anti-rabbit antibody (available from R & D system company at a working concentration of 1:8 fold dilution) was added, incubated for 1h at 37 ℃ and then washed 3 times with PBST buffer for 2min each.
5. TMB substrate color development liquid is added, and color development is carried out at 37 ℃ in a dark place for 5min.
6. 50 μl of 2mol/L H was added to each well 2 SO 4 The OD of the aqueous solution was measured with an enzyme-labeled instrument at a wavelength of 450 nm.
An equal volume of PBS buffer was set to replace the PBS set of VP2 protein solution in step 2 and the rabbit anti-VP 2 polyclonal antibody in step 3. When the ELISA plate is coated with scFv antibody solution with the protein concentration of 10 mug/ml in the step 1: setting a control group 1 without adding VP2 protein solution in the step 2, a control group 2 without adding rabbit anti-VP 2 polyclonal antibody in the step 3, a control group 3 without adding VP2 protein solution in the step 2 and without adding rabbit anti-VP 2 polyclonal antibody in the step 3, and replacing a control group 4 of VP2 protein solution with an equal volume and equal protein concentration of BSA solution.
3 duplicate wells were set per treatment.
The results are shown in FIG. 2.ELISA results show that all 8 scFv antibodies can be specifically combined with VP2 protein, and have high affinity.
2.ELISA detection of scFv antibody specificity and affinity for different IBDV viruses
1. The ELISA plate was coated with scFv antibody solution having a protein concentration of 10. Mu.g/ml (i.e., scFv antibody solution prepared in example 2, adjusted in protein concentration with coating solution), overnight at 4℃and then washed 3 times with PBST buffer for 2min each.
2. Mu.l IBDV virus solution (virus dose 10) was added to each well 6.2 TCID 50 ) Incubation was carried out for 1h at 37℃and then washed 3 times with PBST buffer for 2min each.
3. Rabbit anti-VP 2 polyclonal antibody was added, incubated for 1h at 37℃and then washed 3 times with PBST buffer for 2min each.
4. HRP-labeled goat anti-rabbit antibody (available from R & D system company at a working concentration of 1:8 fold dilution) was added, incubated for 1h at 37 ℃ and then washed 3 times with PBST buffer for 2min each.
5. TMB substrate color development liquid is added, and color development is carried out at 37 ℃ in a dark place for 5min.
6. 50 μl of 2mol/L H was added to each well 2 SO 4 The OD of the aqueous solution was measured with an enzyme-labeled instrument at a wavelength of 450 nm.
The experiments described above were performed with the following IBDV strains, respectively: gt strain, NF8 strain, MB strain, and B87 strain.
An equal volume of PBS buffer was set to replace the PBS set of IBDV virus solution in step 2 and rabbit anti-VP 2 polyclonal antibody in step 3. A control group 1 without adding IBDV virus liquid in the step 2, a control group 2 without adding rabbit anti-VP 2 polyclonal antibody in the step 3, a control group 3 without adding IBDV virus liquid and without adding rabbit anti-VP 2 polyclonal antibody in the step 3 are set, and an equal volume of newcastle disease liquid with equal titer is used for replacing a control group 4 of IBDV virus liquid.
3 duplicate wells were set per treatment.
The results are shown in FIG. 3.ELISA results show that 8 scFv antibodies can be specifically combined with different IBDV strains and have different affinities for the different IBDV strains.

Claims (5)

1. A single chain antibody scFv-13 of chicken infectious bursal disease virus, which consists of a heavy chain variable region, a light chain variable region and a connecting region between the heavy chain variable region and the light chain variable region;
the scFv-13 is as follows (a): (a) Protein consisting of amino acid residues 1-248 from the N terminal of a sequence 3 in a sequence table.
2. A gene encoding the single chain antibody of claim 1.
3. The gene of claim 2, wherein:
in the gene, the DNA molecule encoding the scFv-13 is as follows (1): (1) DNA molecules shown in 1 st-747 th nucleotide from the 5' end of the sequence 11 in the sequence table.
4. An expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising a gene according to any one of claims 2 to 3.
5. Use of the single chain antibody of claim 1, the gene of claim 2 or 3, or the expression cassette, recombinant vector, transgenic cell line or recombinant bacterium of claim 4 in the preparation of a product; the function of the product is as follows (I) and/or (II) and/or (III) and/or (IV): detecting infectious bursal disease virus; (II) assisted identification of infectious bursal disease virus; (iii) preventing and/or treating infectious bursal disease in chickens; (IV) preventing and/or treating diseases induced by infectious bursal disease virus in chickens.
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