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Infectivity resistant bursal disease virus VP4 protein monoclonal antibody

A monoclonal antibody, bursal disease technology, applied in antiviral immunoglobulin and other directions, can solve the problems of vaccine immunization failure, high morbidity, short course of disease, etc., and achieve high specificity, high purity and simple preparation method. Effect

Inactive Publication Date: 2010-07-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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
  • 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 IBDV A segment plasmid by PCR, and the NcoI / XhoI restriction site was introduced. After the PCR product was digested with NcoI / XhoI double enzymes, Connected to the NcoI / XhoI site of the prokaryotic expression vector pET-28a, transformed Escherichia coli Top10, extracted the plasmid of transformed bacteria, and identified the recombinant expression plasmid by PCR ( figure 1 ), through sequence determination verification, the recombinant expression plasmid pET28a-VP4 of IBDV VP4 was obtained.

Embodiment 2I

[0019] Expression and purification of embodiment 2 IBDV VP4 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 OD600=0.6, add a final concentration of 1mM IPTG to induce expression for 2h, centrifuge at 12000r / min at 4°C for 30sec, The bacteria were collected, frozen and thawed three times, centrifuged at 12000r / min at 4°C for 10 minutes, the supernatant and precipitate of the lysed bacteria were analyzed by SDS-PAGE, and the expression of IBDV VP4 protein was analyzed ( figure 2 ). Add 5 times the volume of 8M urea to dissolve the precipitate, shake until the solution is clear, centrifuge at 12000r / min at 4°C for 10min, discard the precip...

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Abstract

The invention relates to an anti-infectious bursal disease virus (IBDV) VP4 protein monoclonal antibody, belonging to the area of biotechnology; the invention relates to the genetic engineering. The IBDV VP4 gene is cloned on the prokaryotic expression carrier pET-28a and the expression is induced in the coli BL21 (DE3); the inclusive body is separated to be dissolved with the 8 M urea; the VP4 recombinant protein can be made after the affinity of the nickel chelation and the purification. The VP4 recombinant protein can be used to cultivate the immune BALB / c mice; the spleen cell and the myeloma cell SP2 / 0 are taken to make with the cell fusion to be selectively cultivated with the HAT culture medium; the double ELISA screening and cloning of the limited dilution is done on the VP4 recombinant protein and the IBDV; the hybridoma cell line of the secreted antibody IBDV VP4 protein monoclonal antibody can be got; therein, the effective value of the ELISA in the C3, 6A4 and 6 H8 ascites are respectively 6.4 multiplied by 105, 2.6 multiplied by 106 and 5.2 multiplied by 106, all of which can identify the virus protein of the IBDV infected cells; the isoform heavy chain of Ig is the IgG1; the light chain is the kappa.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08
Inventor 周继勇王永志郭军庆
Owner ZHEJIANG UNIV
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