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Construction of Attenuated Salmonella Typhimurium Strain Expressing Cap Protein of PCV2 and Animals Immunization

Author: ZhaoHongNi
Tutor: TongDeWen；XuXinGang
School: Northwest University of Science and Technology
Course: Basic Veterinary Science
Keywords: PCV2 Cap protein Attenuated Salmonella typhimurium Live vector vaccine
CLC: S855
Type: Master's thesis
Year: 2011
Downloads: 62
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Abstract


The purpose of this study is to build an expression of PCV2 Cap protein attenuated Salmonella typhimurium live vector vaccine, to build vaccine evaluation by animal immune test. Cap protein PCV2 ORF2 gene encoding the major structural protein and immune protective proteins, this study chose truncated nuclear localization of the N-terminal 41 amino acids of the District the ORF2 encoding gene as a target gene, the expression of balanced lethal system using attenuated Salmonella typhimurium ORF2 gene optimized live vector vaccine construct expressing Cap protein, and the biological characteristics of the recombinant vaccine and immunological characteristics evaluated. Study achieved the following results: 1 express the porcine circovirus Cap protein Salmonella typhimurium live vector vaccine construct synthetic specific primers P1, P2, PCR amplification of ORF2 gene was inserted into the BamH I and Pst I digested Asd the constitutive expression vector pYA3341 to construct the recombinant expression vector pYA3341-ORF2; recombinant vector was transformed into the missing aspartate-β-semialdehyde dehydrogenase (Δasd) adenylate cyclase (Δcya), and the ring Purinergic receptors protein gene (Δcrp), attenuated Salmonella typhimurium the intermediate host bacteria X3730 and final host bacteria X4550, the X4550 (pYA3341-ORF2) recombinant live vector vaccine strain. Biological characteristics of recombinant live vector vaccine strain detection of recombinant live vector the Phytophthora vaccine X4550 (pYA3341-ORF2) in vitro stability characteristics, growth characteristics, oral security and mice given value distribution. The results show that the CAP protein can have high levels of expression in X4550, and stability in vitro genetic recombinant bacteria grew well oral safe non-toxic and having a good distribution characteristics of a fixed value in the organism. The 3 recombinant live vector vaccine strain Immunogenicity of Recombinant live vector vaccine strain X4550 (pYA3341-ORF2) the orally immunized Balb / C mice as immunohistochemistry to oral recombinant strain X4550 (pYA3341) mice as a negative control group, The mice orally PBS, as the blank control group. ELISA method to detect the vaccine immune mice serum antibody titer reached 1:1920; indirect immunofluorescence assay vaccine immunized mice serum antibody titer and can be reached 1:19.2; MTT assay mice immunized lymphocytes mitotic index can reach 4.26; the flow cytometry vaccine immunized mice CD4, CD8 T lymphocytes were approximately the number 29.9,7.52, CD4 / CD8 T cell ratio of about 3.99. Recombinant live vector vaccine strain X4550 (pYA3341-ORF2) the immunized pigs ELISA assay vaccination pig serum antibody titers 1:57.6; vaccines immunohistochemistry pig serum neutralizing antibody titer can be reached 1:11.2; vaccine group lymphatic The mitotic index reached 3.82. Recombinant vaccine X4550 (pYA3341-ORF2) capable of stably expressing Cap protein, has good immunogenicity and provide reference for further vaccine research and clinical applications.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Infectious Diseases
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