Exploration Of Prokaryotic Expression Of MN Gene And Establishment Of M-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. It's 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 (orf6) and nucleocapsid protein gene (orf7) of PRRSV are subcloned into prokaryotic expression vector pBV220, the recombinant plasmid named pBVMN was constructed. By analyzing the characteristic of prokaryotic expression vector, the orf6 gene can be expressed correctly. The pBVMN was used to transform into E. coli DH5α.Induced by temperature (42℃), the orf6 gene was expressed successfully. The results of SDS-PAGE and West-blotting indicated that the membrane protein gene was expressed in a high level and the non-fusion protein, which was about 19kDa, had immunological reactive activity. At the same time, the nucleocapsid protein gene was not expressed because of the big distance from SD sequence. This study lay on foundation for the development of the diagnosis methods in serology for PRRSV.PRRSV the recombinant matrix membrane protein was expressed by E.coli and purified by simple method. Using purified matrix membrane protein, an indirect ELISA for detection of anti-PRRSV antibodies was developed and its optimal reaction conditions were determined: coating antigen for 37℃ 1 hour and 4℃ overnight at a concentration of 3.5μg/ml, serum sample(1:40) and HRP labeled SPA being incubated at 37'C 1 hour, the substrate for ELISA OPD reacted at common temperature for 5 minutes. The ELISA assay was confirmed to have a good reproducibility, specificity and sensitivity by reproducibility test, cross test and blocking test. And compared with the IDEXX test kit, the specificity and sensitivity of the ELISA is 96.3% and 93.5% respectively, coincidence is 89%,which showed no significant difference between the two assays. In addition, 168 serum samples collected from swine farms were detected by the assay, it was showed that the positive rate was 39.9% for antibody against PRRSV.