Eukaryotic Expression And Indirect ELISA Development Of MDRV σC Gene

PURPOSE: Muscovy duck reovirus disease (MDRVD) is a high morbidity and mortality rate of infectious disease caused by Muscovy duck reovirus, which mainly occurs between 10 to 30 days old Muscovy duckings. The morbidity and mortality is between 30% to 90%, and between 60% to 80% respectively. The disease is characterized clinically by weak foot, diarrhea, stunt in growth, and pathologically by hepatic and splenic necrosis and cellulose pericarditis. There is still no Rapid Diagnostic Kit that can be used to diagnose MDRV disease. It is necessary to establish a practical and rapid diagnostic method of MDRVD.MEHTOUD: Two sets of oligonucletide primers were designed according to the sequences of MW9710 strain of MDRV. The viral was propagated in special antibody negative (SAN) Muscovy duck embryos, and its RNAs were extracted and used in reverse transcription-polymerase chain reaction (RT-PCR) for amplifying the S4 cDNA of virus gene. The cDNA was then cloned into pMD18-T vector, and positive plasmids were sequenced. And than, theσC gene was subcloned into pPIC9K, the expression vector of Pichia Pastoris and transformed into E coli DH5a.The recombinant expression plasmid pPIC9K -σC was isolated from positive plasmids. The pPIC9K-σC was then linearized by SacI and transformed into Pichia Pastoris GS115 strain by electroporation. Five pPIC9K-σC positive recombinants were isolated by PCR detection and G418 resistance selection. One of them was further selected for expression by methanol induction. SDS - PAGE analysis showed that the target protein was expressed in the supernatant of the pPIC9K-σC transformant after inducing, but no in non-recombinant pPIC9K vetor. The target protein molecular weight was consistent with the estimation.RESULTS: (1) The eukaryotic yeast expression system of MDRVσC gene of MW9710 strain was established. (2) The targetσC protein can be expressed and secreted into the medium. (3) The immunocompetence ofσC protein was detected by Western-Blotting assay using the first polyclone-antibody of positive MDRV serum and the second polyclone-antibody of the anti-muscovy duck poly-antibody of goat marked with AP. (4) A high specific indirect ELISA assay was developed by usingσC protein as the coating antigen.CONCLUSION: The results of this study is essential for developing a Rapid Diagnostic Kit for MDRV disease.