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

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

Inactive Publication Date: 2004-01-14
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 e...

Method used

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

Examples

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Embodiment 1

[0028] 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, which includes the following steps: (1) Infectious Isolation and Identification of Bursal Disease Virus (IBDV) ZJ2000 Strain

[0029] 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.

[0030] The agar expansion test of the above-mentioned treatment and the SPF chicken (i.e. no specific pathogenic chicken) IBD standard positive serum shows: be...

Embodiment 2

[0037] 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: (1) eukaryotic expression plasmid (pCI-VP2 / VP4 / VP3) build process

[0038] 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 ampicillin. A singl...

Embodiment 3

[0044] 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 method includes: (1) large-scale preparation of eukaryotic expression plasmid

[0045]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, add...

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Abstract

A polymer protein (VP2/VP4/VP3) gene of infectious Fabricius bursa virus (IBDV) for intensifying the immunogenicity of vaccine and a recombinant carrier using pCI as eukaryon expression vector and containing said coding sequence are disclosed. The expression ability of said recombinant carrier increase by 10-40 times than normal eukaryon carrier. A DNA vaccine for infectious Fabricius bursa virus is also disclosed, which contains said eukaryon expression plasmid and immunological adjuvant and features high safety, stability and effect.

Description

technical field [0001] The invention relates to a polyprotein (VP2 / VP4 / VP3) gene, a eukaryotic expression plasmid and a DNA vaccine. 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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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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