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Infectious bursal disease virus (IBDV) polyprotein gene (VP2/VP4/VP3) eukaryon expressing plasmid and DNA vaccine

A polyprotein gene and DNA vaccine technology, applied in the direction of virus/phage, recombinant DNA technology, antiviral agents, etc., can solve the problems of difficult commercialization of genetically engineered vaccines, inability to solve antigenic variation, and high production costs, and achieve good results. The effect of immunogenicity, good immune efficacy, convenient storage and transportation

Inactive Publication Date: 2005-05-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the immunogenicity of genetically engineered vaccines depends on different expression systems, the ability to induce immune protection depends on the conformation of the epitope of the VP2 protein, the production cost is expensive, and the problem of antigenic variation cannot be solved, etc., genetically engineered vaccines are difficult to commercialize change

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  • Infectious bursal disease virus (IBDV) polyprotein gene (VP2/VP4/VP3) eukaryon expressing plasmid and DNA vaccine
  • Infectious bursal disease virus (IBDV) polyprotein gene (VP2/VP4/VP3) eukaryon expressing plasmid and DNA vaccine
  • Infectious bursal disease virus (IBDV) polyprotein gene (VP2/VP4/VP3) eukaryon expressing plasmid and DNA vaccine

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Effect test

Embodiment 1

[0026] This embodiment describes the method for obtaining the infectious bursal disease virus (IBDV) polyprotein (VP2 / VP4 / VP3) gene provided by the present invention, the cloning method is RT-PCR, comprising the following steps:

[0027] (1) Isolation and identification of infectious bursal disease virus (IBDV) ZJ2000 strain

[0028] Collect bursal disease material (code ZJ2000) from chickens with suspected IBD outbreaks in Hangzhou chicken farm in Zhejiang Province, grind it into a homogenate with sterilized normal saline at a ratio of 1:3, freeze and thaw repeatedly 3 times; 15000r / min, 40°C Centrifuge for 20min; take the supernatant and add chloroform (5%) for 30min; centrifuge at 15000r / min at 40°C for 20min; take the supernatant, add penicillin and streptomycin (1000IU / ml), overnight at 4°C, and store at -20°C spare.

[0029] The agar expansion test of the above-mentioned treatment and the SPF chicken (i.e. no specific pathogenic chicken) IBD standard positive serum show...

Embodiment 2

[0041] This embodiment describes the construction process and expression in mammalian cells of the eukaryotic expression plasmid (pCI-VP2 / VP4 / VP3) provided by the present invention, and includes the following steps:

[0042] (1) Construction process of eukaryotic expression plasmid (pCI-VP2 / VP4 / VP3)

[0043] The construction process of the eukaryotic expression plasmid pCI-VP2 / VP4 / VP3 is shown in Figure 2: the polyprotein gene fragment of 3.0kb was recovered by low-melting point gel, digested with EcoRI and KpnI; after extraction with phenol chloroform, dissolved in appropriate amount of TE in solution. The eukaryotic expression vector pCI was digested with EcoRI and KpnI, extracted with phenol and chloroform, and dissolved in an appropriate amount of TE solution. The target gene and the carrier were introduced into the ligation system at a molar ratio of 3:1, transformed into competent cells, cultured with shaking in LB medium for 1 h, and coated with LB plates containing am...

Embodiment 3

[0053] The present embodiment has described the infectious bursal disease virus (IBDV) polyprotein (VP2 / VP4 / VP3) gene DNA vaccine provided by the invention, and it comprises eukaryotic expression plasmid (pCI-VP2 / VP4 / VP3) and immunization Adjuvant, pCI-VP2 / VP4 / VP3: immune adjuvant = 1: 1-5 (mass ratio). The immune adjuvant can be immunostimulating complex (ISCOM), liposome, cytokine, Freund's incomplete adjuvant, propolis and the like. Its production methods include:

[0054] (1) Mass preparation of eukaryotic expression plasmids

[0055]Streak a single colony of pCI-VP2 / VP4 / VP3 containing ZJ2000 strain on an LB plate containing ampicillin and culture overnight; pick a single colony and shake it in 10ml of LB liquid medium containing ampicillin and culture overnight; press 1% Proportionally add 100ml of ampicillin-containing LB culture solution to expand the culture for 6h to an OD value of 0.7-0.8 at the place of λ=600nm; 800g centrifugation for 20min, collect the thalline,...

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Abstract

The present invention provides the recombinant vector pCI-VP2 / VP4 / VP3 of infectious bursal disease virus (IBDV) polyprotein, which is 10-40 times stronger than other conventional eukaryotic expression vectors, and the gene expression can be high. Enhance the effect of DNA vaccines. The present invention also provides a DNA vaccine of infectious bursal disease virus, comprising the above-mentioned eukaryotic expression plasmid and immune adjuvant, the mass ratio of which is 1:1-5, which is a safe, stable and effective infectious agent The bursal disease virus (IBDV) DNA vaccine has the advantages of simple preparation and convenient storage and transportation.

Description

technical field [0001] The invention relates to a eukaryotic expression plasmid and DNA vaccine of polyprotein VP2 / VP4 / VP3 gene. Background technique [0002] Infectious bursal disease (IBD) is an acute contagious disease caused by infectious bursal disease virus (IBDV). one. IBDV mainly infects the central immune organ of chicks—the bursa of Fabricius, resulting in immunosuppression of chicks. The importance of this disease lies not only in the direct economic losses caused by the outbreak of the disease, but also in the failure of immunization with other vaccines. At present, there are many studies on the main host protective antigen VP2 gene of IBDV, but the immune protection effect of VP2 protein on IBDV is not ideal. The traditional method of prevention and treatment of IBD is to inoculate attenuated vaccines and inactivated vaccines. However, attenuated vaccines tend to be more virulent and easily interfered by maternal antibodies. attack. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12A61K39/39A61P31/12C12N7/01C12N15/33C12N15/79
Inventor 于涟李建荣黄耀伟李龙
Owner ZHEJIANG UNIV
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