Abstract:M gene of RV LEP-Flury strain was amplified by reverse transcriptase—polymerase chain reaction(RT-PCR).The product was purified and cloned into the prokaryotic expression vector pET-32a(+) to obtain the cloning expressed plasmid pET-M,double digestion and sequence analysis were carried afterwards.The recombinant plasmid was identified by PCR, double enzyme digestion and sequence analysis. They were transformed into Rosetta competent cells for expression. SDS-PAGE was performed to analyze the M protein expression production.Results showed that M gene was highly expressed in E.coli,the molecular weight of the protein was 42ku.Western-Blotting analysis showed that the recombinant protein was recognized specifically by positive serum of RV. Based on the purified RV M protein, an indirect ELISA method for the detection of the RV antibodies was established.The optimal concentration of RV M coating the ELISA plate was 6μg/mL.The optimal concentration of serum samples and SPA-HRP was 1:200 and 1:2000 respectively.Compared with commercially available ELISA kit,the coincidence rate of indirect ELISA was 89.2% .Our results show that the developed indirect ELISA based on the RV M was useful for the detection of the RV antibody in clinical samples.