Analysis Heredity And Variation Of Structural Proteinin Genes Of Two PRRSV Strains And Expression Of The M Gene

Porcine reproductive and respiratory syndrome is a infectious disease of swine characterised by reproductive disorders in sows and by respiratory problems in piglets. It was caused by porcine reproductive and respiratory syndrome virus(PRRSV). Since it was reported, PRRS has been causing tremendous economic losses for the swine industry throughout the world. It is rellay urgent to control this epidemic in production, and the key is to produce effective vaccines. However, because of great variation of PRRSV, the development of effective vaccine is very difficult.This topic seeks to isolate strains from local pig farms in Hebei Province, comparing with the structural genes of strains which has been published in GenBank, and then to analyze the genetic variation from molecular level, determine the sub-type position of PRRSV strains within Hebei Province, thus provide theoretical basis for diagnosis. Moreover, M protein is the most conservative structural protein among strains in North American and Europen. It is the main target of humoral immunity in infected pigs, and it has a strong immunogenicity. This paper selects HB-3(cz) strain to express the M gene, which would be providing basis for the further development of new vaccines and diagnostic reagents of PRRSV.In this study, two PRRSV were isolated from different parts of Hebei Province, and the structural protein genes amplified by RT-PCR was cloned into pMD-T vector respectively. The recombinant plasmids were sequenced and compared with other strains. Homology and phylogenetic analysis revealed: The two strains belong to the American genotype; the prevalence PRRSV strains of Hebei which isolated in recent years including TS and HB-3(cz) are in the same subtype, and they are closed with HB-1(sh) strain. In addition, PRRSV epidemic in the country did not show obviously regional.In the experiment, the M gene of HB-3 (cz) strains was cloned into the prokaryotic expression vector pET-32a(+), through optimizing the expression terms, thus successfully induced the protein. SDS-PAGE demonstrated the molecular weight of recombinant protein about 36.9kDa. Western-blotting showed that the recombinant protein could react with the porcine polyclonal antibody against PRRSV, and indicated that the protein could be used as antigen in diagnostic for the detection of antibodies against PRRSV. It would be lay theoretical foundation for further study of strain-specific vaccine and diagnostic kit against Hebei Province PRRSV.