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Recombinant yeast strain for expressing chicken IBDV (infectious bursal disease virus) VLPs (virus-like particles), protein expressed by recombinant yeast strain and application

A chicken infectious and bursal disease technology, applied in the direction of viral peptides, antiviral agents, viral antigen components, etc., can solve the problems of inability to clear viruses, inability to eliminate immunity, inability to provide histopathological protection, etc.

Active Publication Date: 2016-07-13
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above vaccines can only provide clinical protection, but cannot provide good histopathological protection, nor can they remove the virus in the body, and cannot achieve the purpose of eliminating immunity

Method used

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  • Recombinant yeast strain for expressing chicken IBDV (infectious bursal disease virus) VLPs (virus-like particles), protein expressed by recombinant yeast strain and application
  • Recombinant yeast strain for expressing chicken IBDV (infectious bursal disease virus) VLPs (virus-like particles), protein expressed by recombinant yeast strain and application
  • Recombinant yeast strain for expressing chicken IBDV (infectious bursal disease virus) VLPs (virus-like particles), protein expressed by recombinant yeast strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Synthesis and Cloning of Chicken Infectious Bursal Disease Virus optiVP2

[0038] 1. Extraction of viral RNA

[0039] The SPF chicken bursa infected with chicken infectious bursal disease virus supervirulent Gx (vvIBDV-Gx) strain was ground with a grinder. Take 200 μl of disease material and add TE (PH8.0) to 500 μl, add 5 μl of proteinase K and 50 μl of 10% SDS, and bathe in water at 56°C for 3 hours. Add an equal volume of phenol / chloroform for extraction three times, and an equal volume of chloroform for extraction once. Remove the supernatant to another 1.5ml centrifuge tube, add 1 / 10 volume of NaAc (3M, pH 5.2), and equal volume of isopropanol, and precipitate at -20°C for 2 hours. Centrifuge at 12000 rpm for 15 minutes at 4°C and wash once with 75% ethanol. Dry in vacuo and redissolve RNA in RNase-free deionized water.

[0040] 2. Obtaining the RF243 gene of the genome A segment

[0041] Carry out RT-PCR on the extracted viral RNA, apply random prim...

Embodiment 2

[0051] Construction and Identification of Embodiment 2 Recombinant Yeast

[0052] 1. Construction of recombinant yeast vector

[0053] The optiVP2 gene (shown in SEQ ID NO: 1) and the yeast vector pAO815 (Invitrogen, CA, USA) were respectively digested with EcoRI and recovered with an agarose gel recovery kit. The vector is dephosphorylated with CIAP. T for foreign fragments and vectors 4 DNA ligase was used for ligation reaction, and the ligated product was transformed into Escherichia coli competent cells to construct a recombinant plasmid pAO815optiVP2*1 containing one copy of optiVP2 gene. The recombinant plasmid was extracted by alkaline lysis, and the complete expression cassette was excised with BglⅡ and BamHI; the recombinant plasmid pAO815optiVP2*1 was digested with BamHI, and dephosphorylated with CIAP. by T 4 DNA ligase performs the ligation reaction. The ligated product was transformed into Escherichia coli competent cells, and a recombinant plasmid pAO815opti...

Embodiment 3

[0065] Embodiment 3 Determination of immunogenicity of recombinant protein of the present invention

[0066] The virus-like particles (IBD-sVP) prepared in Example 2 were divided into two groups: Group 1 was diluted to 20 μg / ml with sterilized water, Group 2 was emulsified with oil adjuvant 1:1, and the protein concentration after emulsification was 20 μg / ml .

[0067] Take 4-week-old SPF chickens and divide them into groups with 10 chickens in each group. Groups A, B, C, and D are virus-like particle groups of the present invention: group A is immunized with IBD-sVP 20 μg / 1ml / body, without adjuvant, and immunized twice; group B is immunized with IBD-sVP 20 μg / 1ml / body, with adjuvant, Immunized twice; group C was immunized once with IBD-sVP20μg / 1ml without adjuvant; group D was immunized once with IBD-sVP20μg / 1ml with adjuvant. Group E is the routine inactivated vaccine (inactivated chicken infectious bursal disease vaccine, Veken Biotechnology Development Company, Harbin Ve...

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Abstract

The invention discloses a recombinant yeast strain for expressing chicken IBDV (infectious bursal disease virus) VLPs (virus-like particles), a protein expressed by the recombinant yeast strain and an application. The recombinant yeast strain contains an optimized chicken IBDV VP2 gene sequence and can efficiently express a VP2 protein, and the expressed recombinant VP2 protein can be self-assembled into the VLPs. A vaccine is prepared from the chicken IBDV VLP protein expressed by the recombinant yeast strain to immunize chickens, and an experiment result indicates that the subunit peptide vaccine can effectively induce organisms to produce specific humoral immune response, so that the immunized chickens acquire 100% resistance to highly pathogenic vv IBDV fatal attack, residual viruses in the organisms can be cleared, and the purpose of sterilizing immunity is achieved. Therefore, one novel technical means is provided for prevention and control of chicken infectious bursal disease.

Description

technical field [0001] The present invention relates to a recombinant yeast strain and its expressed protein and application, in particular to a recombinant yeast strain expressing chicken infectious bursal virus virus-like particles, and also to chicken infectious bursal virus expressed by the yeast strain. Bursal virus virus-like particles and their use in the prevention of chicken infectious bursal disease. The invention belongs to the technical field of medicine or veterinary medicine. Background technique [0002] Chicken infectious bursal disease (Infectious bursal disease, IBD) is caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV). Traditional vaccines have played a very important role in the history of the prevention and treatment of the disease. With the new changes of the disease in recent years-the emergence of serotype variants and super-virulent strains, the prevention and treatment of the disease has become more and more difficul...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/81C12N1/19C07K14/08C12P21/02A61K39/12A61P31/14
CPCA61K39/00C07K14/08
Inventor 王笑梅高宏雷祁小乐高玉龙王永强
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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