Prokaryotic Expression Of Sigma C Protein Of Novel Duck Reovirus And The Establishment Of Indirect ELISA Method

New Duck Reovirus(NDRV),is a newly emerging pathogenic virus which is characterized by irregular necrosis and hemorrhage of liver and spleen and caused huge economic losses to the duck industry.in this study an indirect ELISA method by using purified Sigma C protein of duck reovirus expressed in prokaryotic cells as coating antigen was developed for the rapid detection of NDRV antibodies.(1)Prokaryotic expression and purification of sigma C protein of duck reovirus.Using a novel duck reovirus(NDRV?SY strain)genome as template,the sigma C gene was amplified by RT-PCR.The amplified product was cloned into pET-32a(+)vector and the recombinant plasmid veetor pET?3 2a?sigma C was obtained.The reeombinant plasmid vector pET-32a-sigma C was transformed into BL21(DE3)and expressed by IPTG.The recombinant protein with molecular weight of 55KDa was obtained.After denaturation,the purity of sigma C protein was 85%.The antigenicity of sigma C protein was preliminarily analyzed by Western blotting.The results showed that the new duck reovirus Sigma C protein had good reactivity.(2)Establishment of a new indirect ELISA method for detection of reovirus.The purified Sigma C recombinant protein of duck reovirus was used as coating antigen.The reaction conditions were optimized and an indirect ELISA method for NDRV antibody detection was established.The optimun conditions were as follows:the coating concentration of sigma C protein was 4.5ug/ml;the serum dilution was 1:12800;the dilution of enzyme-labeled antibody was 1:500;and the substrate was colored at 37 C for 10 minutes.The method has good specificity and no cross-reactions to the positive sera of Riemerella duck plague(RA),H9 subtype,duck viral hepatitis-1(DHV-1),duck tembusu virus(DTMUV)and duck plague virus(DVE);it can detect 1:12800 diluted NDRV positive serum with good sensitivity;the variation coefficients of intra-batch and inter-batch repeat tests are less than 10%.It has good repeatability.169 serum samples from immunized duck flocks in Jiangsu and other places were detected.The positive rate of the samples was 44.97%.In summary,the sigma C-ELISA developed in this study offers a specific,sensitive and rapid.The method provide a new serological rapid detection method for NDRV epidemiological investigation.