Recombinant yeast engineering strain coexpressed by proteins of chicken anaemia viruses VP1 and VP2, construction method thereof and application thereof

A technology for recombinant yeast and chicken anemia, which is applied in the field of biomedicine, can solve the problems of only partial secretion and no immune response induced by the expressed protein, and achieve the goal of reducing the cost of breeding, high activity of the expressed product, and great economic and social benefits Effect

Active Publication Date: 2012-01-11
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
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Problems solved by technology

Lin Huan, a member of our laboratory, expressed CAV VP1 and VP2 genes in Pichia pastoris, but the expressed proteins did not induce a good immune response, because VP1 and VP2 can only be formed through the interaction during expression Neutralizing epitopes that induce the production of neutralizing antibodies; based on the above basis, Chen Shaoping, a member of our laboratory, co-transformed the pPIC9k-VP1 and pPIC9k-VP2 vectors constructed by Lin Huan, and obtained the co-expression of CAV VP1 and VP2. Recombinant yeast strain, the expressed protein has good immunogenicity and can induce the production of CAV-specific antibodies, but the VP1 protein can only be partially secreted during the expression process

Method used

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  • Recombinant yeast engineering strain coexpressed by proteins of chicken anaemia viruses VP1 and VP2, construction method thereof and application thereof
  • Recombinant yeast engineering strain coexpressed by proteins of chicken anaemia viruses VP1 and VP2, construction method thereof and application thereof
  • Recombinant yeast engineering strain coexpressed by proteins of chicken anaemia viruses VP1 and VP2, construction method thereof and application thereof

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Embodiment 1

[0043] The construction of embodiment 1 recombinant yeast engineering strain

[0044] 1 Materials and methods

[0045] 1.1 Plasmids, strains, cells: pBluemCAV recombinant plasmids, anti-CAV VP1 monoclonal antibody (MAb2F3), anti-CAV VP2 monoclonal antibody (MAbE11C9) are preserved by our laboratory. Escherichia coli competent cells DH10F' were produced by our laboratory. Pichia pastoris strain GS 115 and vector pAO815 were purchased from Invitrogen. The CAV M9905 virus strain was isolated from the tissues of 13-day-old broiler chickens with anemia in our laboratory. The strain (M9905) has been preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms, and its strain preservation number is: CGMCC 5016 ; pPICZαB and pPICZαC vectors were purchased from Invitrogen Company; HRP-labeled rabbit anti-mouse IgG antibody and FITC-labeled rabbit anti-mouse secondary antibody were purchased from Sigma Company. MDCC-MSB1 cells were preserved by...

Embodiment 2

[0097] The detection of embodiment 2 recombinant protein immunogenicity

[0098] 1 Expression of recombinant protein

[0099] Get the broken culture, first measure the protein concentration with a UV spectrophotometer, the concentration is 48g / L, then carry out SDS-PAGE, and use thin-layer gel scanning to determine that the percentage of the target protein accounting for the total protein is 25%. The expression level of the target protein was 12g / L.

[0100] 1.1 Immunization of animals

[0101] Preparation of chicken infectious anemia recombinant antigen vaccine: Dilute fermented bacterial protein to 200μg / 0.6ml, 400μg / 0.6ml, 2000μg / 0.6ml with PBS (PH7.4) according to the content of VP 1, and add an equal volume of French adjuvant ((ISA50V2) purchased from SEPPIC company) emulsification.

[0102] Take 50 1-week-old SPF chickens and divide them into 5 groups, 10 in each group. Groups 1, 2, and 3 are expression protein immunization groups: the immunization dose of group 1 is...

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Abstract

The invention discloses a recombinant yeast engineering strain coexpressed by proteins of chicken anaemia viruses VP1 and VP2, a construction method thereof and an application thereof. The preservation number of the recombinant yeast engineering strain is CGMCC 5045. A large scale induction is carried out on the engineering strain, and the expression level of the VP1 reaches 12g/L. The detection of SDS-PAGE and Western-blot proves that the recombinant coexpressing proteins have the biological activity and the antigenicity of the natural proteins of the chicken anaemia viruses. Results of experiments that a recombinant antigen vaccine which is prepared from the recombinant proteins is use to immunize SPF chickens show that: the chicken infective anaemia recombinant antigen vaccine can effectively induce a body to generate a specific humoral immune response and the immune chicken to obtain the protect of anti-CAV attack, and can effectively prevent the propagation of the viruses in bodies.

Description

technical field [0001] The present invention relates to a recombinant yeast genetically engineered strain constructed by genetic engineering technology, in particular to a recombinant yeast genetically engineered strain co-expressed with chicken anemia virus VP1 and VP2 proteins, also relates to its construction method, and further relates to the expressed recombinant protein and its application in the development of a novel recombinant antigen vaccine against chicken anemia virus. The invention belongs to the field of biomedicine. Background technique [0002] Chicken infectious anemia is caused by chicken anemia virus (Chicken Anemia Virus, CAV), an important viral disease that can lead to immunosuppression in chickens, characterized by aplastic anemia in chicks, generalized lymphoid tissue atrophy, subcutaneous and muscle hemorrhage and accompanying immunity. Characterized by inhibition, chicken infectious anemia seriously endangers the poultry industry in the world. Wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C07K14/01A61K39/12A61P31/20C12R1/84
Inventor 王笑梅高宏雷祁小乐高玉龙秦立廷王永强
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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