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Infectivity resistant bursal disease virus VP4 protein monoclonal antibody

A technology of monoclonal antibody and bursal disease, applied in the direction of antiviral immunoglobulin, can solve the problems of short course of disease, high incidence, failure of vaccine immunity, etc., and achieve simple preparation method, high purity and strong specificity Effect

Inactive Publication Date: 2008-02-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease has a high incidence and a short course. In addition to the disease itself can cause body damage, it can also lead to the failure of various vaccine immunity, which seriously endangers the healthy development of the poultry industry.

Method used

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  • Infectivity resistant bursal disease virus VP4 protein monoclonal antibody
  • Infectivity resistant bursal disease virus VP4 protein monoclonal antibody

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preparation example Construction

[0014] 1. Preparation of IBDV VP4 recombinant protein: design primers according to IBDV NB strain A segment nucleotide sequence, PCR amplify IBDV VP4 gene, clone it in prokaryotic expression vector pET-28a, transform Escherichia coli BL21 (DE3), through IPTG induced the expression of IBDV VP4 protein; the expressed bacteria were collected, frozen and thawed three times, and the inclusion bodies were separated by centrifugation. The expressed protein was dissolved with 8M urea solution, and purified by nickel chelate affinity chromatography to obtain IBDV VP4 recombinant protein.

[0015] 2. Preparation of anti-IBDV VP4 protein monoclonal antibody: Emulsify and purify VP4 recombinant protein with an equal amount of Freund's adjuvant, immunize 6-8 week-old BALB / c mice, and take the splenocytes and myeloma cells SP2 / 0 in the Cell fusion was carried out under the action of polyethylene glycol, and HAT medium was used for selective culture; the purified VP4 recombinant protein and I...

Embodiment 1IB

[0017] Construction of embodiment 1 IBDV VP4 recombinant expression plasmid

[0018] Design and synthesize specific primers according to the nucleotide sequence of segment A of IBDV NB strain (EF517528.1), upstream primer 5'-CGTCGT CCATGG CCGACAAGGGGTACGAGGTAGTC-3' (NcoI site is underlined), downstream primer 5'CGTCGT CTCGAG CATGGCAAGGTGGTACTGGCGTCC-3'((the underline is the XhoI site). The IBDV VP4 gene was amplified from the IBDVA segmental plasmid by PCR, and the NcoI / XhoI restriction site was introduced. After the PCR product was digested with NcoI / XhoI double enzymes, ligated Between the NcoI / XhoI sites of the prokaryotic expression vector pET-28a, transform Escherichia coli Top10, extract the plasmid of transformed bacteria, identify the recombinant expression plasmid by PCR (Figure 1), and obtain the IBDV VP4 recombinant expression plasmid pET28a- VP4.

Embodiment 2I

[0019] Expression and purification of embodiment 2 IBDVVP4 recombinant protein

[0020] The recombinant expression plasmid pET28a-VP4 was transformed into Escherichia coli BL21(DE3) by the calcium chloride method, and the positive clones were picked and inoculated in LB liquid medium (containing 50 μg / mL Kan). Inoculate in a 100mL Erlenmeyer flask containing 10mL of fresh LB medium at a ratio of :100, culture at 37°C with shaking at 300r / min, when 0D 600 When = 0.6, add a final concentration of 1mM IPTG to induce expression for 2h, centrifuge at 12000r / min at 4°C for 30sec, collect the cells, freeze-thaw three times, centrifuge at 12000r / min at 4°C for 10min, analyze the supernatant and precipitate of the lysed cells by SDS-PAGE, The expression of IBDV VP4 protein was analyzed ( FIG. 2 ). Add 5 times the volume of 8M urea to dissolve the precipitate, oscillate until the solution is clear, centrifuge at 12000r / min at 4°C for 10min, discard the precipitate, transfer the superna...

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Abstract

The anti-infectious bursal disease virus (IBDV) VP4 protein monoclonal antibody of the invention belongs to the field of biotechnology and relates to genetic engineering. The IBDV VP4 gene was cloned into the prokaryotic expression vector pET-28a, induced and expressed in Escherichia coli BL21(DE3); the inclusion body was isolated, dissolved with 8M urea, and purified by nickel chelate affinity chromatography to obtain the VP4 recombinant protein. BALB / c mice were immunized with VP4 recombinant protein, and the splenocytes were taken for cell fusion with myeloma cell SP2 / 0. After selective culture in HAT medium, cells infected with VP4 recombinant protein and IBDV were screened by double ELISA and limited dilution The hybridoma cell lines secreting anti-IBDV VP4 protein monoclonal antibody were obtained by cloning by the method. The ELISA titers of 4C3, 6A4 and 6H8 ascites were 6.4×105, 2.6×106 and 5.2×106, respectively, all of which could specifically recognize IBDV The viral protein that infects cells has an Ig subtype of IgG1 for the heavy chain and κ for the light chain.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to antibody engineering technology, in particular to anti-infectious bursal disease virus VP4 protein monoclonal antibody. Background technique [0002] Infectious bursal disease virus (IBDV) is the pathogen of infectious bursal disease (IBD), mainly invading the central immune organs of the bursa of 3-6 week-old chicks, destroying the sIgM on the surface B lymphocytes, and cause apoptosis of spleen, thymus and peripheral blood lymphocytes, leading to immunodeficiency and immunosuppressive diseases mainly characterized by lymphocyte exhaustion and necrosis. The disease has a high incidence rate and a short course. In addition to the damage to the body caused by the disease itself, it can also lead to the failure of various vaccines and seriously endanger the healthy development of the poultry industry. IBDV belongs to the genus Avibirnavirus of the Birnaviridae family (Birnaviridae). It h...

Claims

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Application Information

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IPC IPC(8): C07K16/08
Inventor 周继勇王永志郭军庆
Owner ZHEJIANG UNIV
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