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Cloning and Prokaryotic Expression of GP5 Gene of of Porcine Reproductive and Respiratory Syndrome Virus

Author: NieWei
Tutor: LiGuoPing；ZhouLunJiang
School: Fujian Agriculture and Forestry University
Course: Clinical Veterinary Medicine
Keywords: Porcine reproductive and respiratory syndrome virus ORF5 Western Blotting
CLC: S852.659.6
Type: Master's thesis
Year: 2009
Downloads: 79
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Abstract


Applications Marc-145 cells to amplify porcine reproductive and respiratory syndrome Fuzhou strains of virus ( PRRSV FZ ) was designed based on the nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSV) has published a pair of specific primers ( P1 / P2), full-length PRRSV FZ strain was amplified by RT-PCR method glycosylated envelope protein gene ( ORF5 ) the PCR products were ligated into pMD 18 simple T Vector and transformed into E. coli DH5α , screened positive The recombinant plasmid sequencing and analysis . The results showed that the nucleotide homology the FZ of strains of PRRSV AY885248.1 strain ORF5 gene is 100%, and 99.5% nucleotide homology with the BJ-4 strain , with the American strain VR-2332 nucleotide the homology was 99.3 %, while the homology of the strains with the European type (LV) compared to 27.1%, and Therefore , this virus strain belongs to the type strain of the Americas . Positive recombinant plasmid was cloned into the prokaryotic expression vector pGEX-6p-1 () on the positive clones were screened by restriction enzyme and sequence identification , the results showed that the recombinant expression vector was constructed successfully . Identified as positive recombinant plasmid was transformed into expression in bacteria E.coli BL21 identified as positive by IPTG induced expression of prokaryotic expression products by SDS-PAGE electrophoresis and Western Blotting analysis of expression characteristics of the products , the results showed that the expression of the fusion the GP5 protein molecular weight of 48.5 kDa, the expression products exist in the form of inclusion bodies , the recombinant protein with PRRSV monoclonal antibody immune reactogenicity .

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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 > Underlying the single - stranded RNA viruses
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