Exploration Of Prokaryotic Expression Of N Gene And Establishment Of N-ELISA Diagnosis Method For Reproductive And Respiratory Syndrome Virus

PRRS is one of the most important diseases in the pig industry.It has caused great damages since its being recognized. Its characteristic is rapid transmission and great damage. It is very important to establish an effective and convenient method to diagnose PRRSV.The Membrane protein gene (ORF7, nucleocapsid protein gene) of PRRSV is subcloned into prokaryotic expreesion vector pET-32a, the recombinant plasmid named pET-32a-N was constructed.By analyzing the characteristic of prokaryotic expression vector,the ORF7 gene can be expressed correctly. The pET-32a-N was used to transform into E.coli DH5α.Induced by temperature (28℃), the ORF7 gene was expressed successfully. The results of SDS-PAGE and West-blotting indicated that the nucleocapsid protein was expressed in a high level non-fusion and fusion protein, which was about 34kDa, had immunological reactive activity. This study lay on foundation for the development of diagnosis methods in serology for PRRSV.PRRSV the recombinant nucleocapsid protein was expressed by E.coli and purified by kit。Using purified nucleocapsid protein, an indirect ELISA for detection of anti-PRRSV antibodies was developed and its optimal reaction conditions were determined;coating antigen for 4℃overnight at a concentration of 51.7μg/ml, serum PRRSV; N Gene; Prokaryotic Expression...