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The Eukaryotic Expression of Foot-and-mouth Disease Virus Structural Protein VP4 Gene

Author: WangRuo
Tutor: WangJiaZuo
School: Agricultural University of Hebei
Course: Preventive Veterinary Medicine
Keywords: Foot-and-mouth disease virus VP4 gene Eukaryotic expression Cells transfected HeLa cells
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 63
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Abstract


Foot-and-mouth disease (foot-and-mouth disease, FMD) is cloven-hoofed animals caused by an acute infection by foot-and-mouth disease virus (FMD virus, FMDV), hot and extremely rapid spread of contagious diseases. Serious harm to the quality of the productivity of livestock and livestock products not only caused great economic losses to livestock production, import and export trade, and a direct threat to the human food security, international trade, and the image of the country, and thus by the World Animal Health Organization (OIE) classified as Class A infectious animal diseases, and are subject to the general concern of the people around the world. , For the prevention of foot-and-mouth disease outbreak and spread of the disease, most developing countries, the main method of injection of inactivated foot-and-mouth disease virus vaccine. Inactivated vaccine is not completely inactivated and the short period of protection. Therefore, the new foot-and-mouth disease vaccines and immunization strategy research has become the focus of problem to be solved. Foot-and-mouth disease virus capsid structural proteins VP1, VP2, VP3, VP4 each 60 copies composed of a peptide vaccine against foot-and-mouth disease virus Asia I, characterized by Asia Ⅰ FMDV VP1 and VP4 B cell epitopes The DNA sequence of the T cell epitopes of amino acid series individually encoded epitope protein or the contiguous encoding epitope fusion protein was obtained with a carrier macromolecule, wherein the B cell epitopes with the Asia Ⅰ type foot-and-mouth disease virus structural protein of VP1 80 ~~ 101,133 ~~ 160 and amino acids 200 to 213 of the T cell epitopes including Asia Ⅰ type VP4 20 ~ 34 amino acid or of VP1 20 ~ 34 and 35 ~ 48 amino acids. In this experiment, the plasmid pUC57-VP4 as a template FMDV VP4 gene after EcoRI and BamHI double digestion and gene sequencing of target genes and the eukaryotic expression vector pcDNA3.1 () were the same restriction enzymes EcoRI and The BamHI digested by T4 ligase to construct the recombinant plasmid transformed into E. coli E. coli DH5α get recombinant plasmid pcDNA3.1-VP4, after restriction endonuclease correctly. sequencing, the recombinant plasmid pcDNA3.1-VP4. The results showed that the VP4 gene was cloned into the expression vector pcDNA3.1. VP4 gene published in GenBank sequence homology comparisons prove the Institute of strains with the Foot-and-mouth disease virus O isolate O / NYOO gene coding sequence (NCBI accession number were AY333431.1) homology highest up to 100%. The results prove the successful construction of recombinant plasmid pcDNA3.1-VP4. Whether for the identification of the pcDNA3.1-VP4 gene expression in eukaryotic cells, the present study with the recombinant plasmid pcDNA3.1-VP4 transfected HeLa cells, the HeLa cells transfected with empty vector was used as a negative control. SDS-PAGE analysis results show that, in the molecular weight of about 8 kD at a distinct protein bands, while the empty vector in the corresponding position no protein bands. The results prove that the plasmids were successfully constructed and expressed VP4 protein in vitro, which laid a solid foundation for further VP4 as the study of structural protein vaccine antigens, as well as exploring a possible vaccine antigen modification and adjuvant screening.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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