Establishment and Application of an Indirect ELISA with Recombinant M Protein of Porcine Reproductive and Respiratory Syndrome Virus

To set up a sensitive,specific,rapid and high throughput serological antibody detection method for porcine reproductive and respiratory syndrome virus(PRRSV),M protein were expressed using prokaryotic expression technology,an indirect ELISA antibody detection method for PRRSV were set up using the purified recombinant M protein as coating antigen.Based on published PRRSV M genome sequence,one pairs of specific primers were designed,amplified 435 bp M gene fragment by RT-PCR,the fragment was cloned into pET32a(+) expression vector,and induced by IPTG,the recombinant M protein was expressed in inclusion body forms,after recombinant protein purification,Western blotting showed the protein had good antigenicity and specificity.After optimization of reaction conditions,using the purified recombinant M protein as antigen established an indirect ELISA method for PRRSV,the established M-ELISA detected six kinds of swine disease(CSFV,JEV,TEDV,PRV,PPV,PCV2) positive sera were negative,the coefficient variation of the method intra-assay and inter-assay repetitive tests were less than 5% and 10%,compared with IDEXX ELISA kit,the coincidence rate of the method was 95.3%.The established M-ELISA detection method provided a simple,rapid,high throughput serological antibody detection method for the PRRS vaccinated pigs antibody level monitoring,and the wild PRRSV infection diagnosis and epidemiological investigation.