Cloning, Sequencing And Expression Of ORF5 Gene Of Porcine Reproductive And Respiratory Syndrome Virus Of PM And ZMD Strains

Two pairs of oligonucleotic primers were designed according to the published sequences of PRRSV strain BJ-4,i.e. A/B. Two fragments of 696bp and 534bp were amplified by RT-PCR from RNA which extracted from pathologic organs from Hanan, The 696bp fragment was cloned into PMD18-T vector and sequenced. It was found that the two ORF5 fragments have 100% homology , and have 99.0% and 97.5% homologies with VR2332 ( A North American isolate) in nucleotide and amine acid level,respectively, whereas it has only 59.3% and 53.2% homologies with those of Europen isolates,and only 90.7% and 90.0% homologies with the referenced strain BJ-4.It is concluded that strains of Henan is genetically closer to strains isolated from North American than those from Europen, strains of Henan and strains isolated from North American might share a commen ancestor.,and there might be some variance between strain from Henan and BJ-4.The sequences have been published in Genbank with numbers of AY613348 and AY613349.The ORF5 Gene was digested with EcoRâ… and Hindâ…¢,and cloned into pET-32a digested with EcoRâ… and Hindâ…¢ .The right recombinant plasmid was screened out and identified by enzyme digestion electrophoresis.The special protein was induced by IPTG and expressed ,which is about 40kDa.The intention we had this study is to inspect the PRRS in Henan by using RT-PCR , to supervise the relation of PRRSV in Henan with those in the world and the epidemic in Henan,and to provide bases for the prevention and cure of PRRS.