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Cloning and Expression of GP5 Gene JL/07/SW Strain of Porcine Reproductive and Respiratory Syndrome Virus and Establishment of Indirect ELISA Diagnosis Method

Author: BaiJing
Tutor: ZuoGenQiang；DingZhuang
School: Shihezi University
Course: Preventive Veterinary Medicine
Keywords: Porcine reproductive and respiratory syndrome virus GP5 gene Clone Expression Indirect ELISA
CLC: S858.28
Type: Master's thesis
Year: 2010
Downloads: 15
Quote: 0
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Abstract


According to the gene sequences of porcine reproductive and respiratory syndrome virus in the Americas strain VR -2332 strains designed for the GP5 gene-specific primer P1/P2 as a template to PRRSVJL/07/SW strain cell cultures by RT- PCR method was successfully amplified the GP5 gene GP5 gene cloning , then pMD18-T as the carrier . The sequencing results showed that the the GP5 gene is 603bp, encoding 201 amino acids ; Sequence analysis showed that GP5 gene with the VR-2332 strains and LV strain nucleotide homology of 91.6% and 48.7% , respectively , encoding amino acid homology 87.6% and 60.0% . The GP5 subcloned into pCI-neo expression vector to construct the expression plasmid pCI - GP5 Marc-145 cells transfected with the recombinant plasmid , indirect immunofluorescence assay for identification of the expression of the target protein , SDS-PAGE . The results express the fusion protein of approximately 25 KDa , Western-blot Western blot analysis , recombinant protein with positive serum anti-PRRSV -specific reaction , indicating that its antigen activity . The recombinant expression of fusion GP5 protein as coating antigen , Western-blot analysis showed that the recombinant protein with PRRSV -positive serum reaction and have a good reaction , this purified recombinant protein indirect ELISA method for diagnosis of PRRS . Experimentally determined the optimum antigen concentration of 2.30μg/mL PRRSV positive serum dilution of 1:100 . PRRS and swine by indirect ELISA method other common diseases have no cross-reactivity , and specificity . This thesis PRRSV GP5 gene cloning and analysis , application of molecular biology techniques connected the PRRSV GP5 genes and the eukaryotic expression vector pCI-neo and the establishment of an indirect ELISA method , characteristics of of PRRSV JL/07/SW strains pathogenic molecular biology research , and the establishment of a preliminary diagnosis . The research is of great significance for PRRS study of etiology , epidemiology investigation and diagnosis prevention . Laid the foundation for further study of the structural gene function and its immunogenicity .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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