Gene Analysis Of E And NS1of DTMUV And Preliminary Application Based On The Recombinant Proteins

Duck Tembusu virus (DTMUV) is a recently emerged pathogen associated with severe egg drop syndrome and high morbidity and mortality. It has rapidly spread around major duck-producing regions in China. The fection has caused a serious economic loss to the poultry industry in China.To provide a basis for the follow-up vaccine effect evaluation and epidemiological survey of DTMUV, our study successfully expressed envelope protein E and non-structural protein NS1. Based on the expressed proteins, indirect ELISA methods were established to detect DTMUV antibody in duck serum. Compared with the serum neutralization test, indirect ELISA saving time and effort is suitable for the detection of large numbers of samples. To provide a basis for disease prevention and control technology research, the study also carried out sequence analysis of E gene and NS1gene.1. The sequence analysis of E gene and NS1gene of DTMUVUsing program MegAlign of DNAstar software, sequence alignment analysis of E gene and NS1gene of DTMUV was carried out. We found that the E gene and NS1gene of DTMUV highly correlated with Tembusu virus isolated form mosquito in Malaysia and Sitiawan virus isolated form commodity chicken in Malaysia, with amino acididentities more than93%. Two phylogenetic trees were created from the alignments of E gene and NSlgene nucleotide sequences. Phylogenetic trees also indicated that DTMUV was closely related to Tembusu viru(JX477685) and Sitiawan viru(JX477686),but DTMUV was far with Dengue virus and Yellow fever virus.2. Cloning and Expression of E Gene of DTMUV and Preliminary ApplicationThe whole cDNA of E gene was amplified by PCR from DTMUV (strain LC), and cloned into the pMD18-T vector. The fragment was identified by restriction enzymes digestion, and then was cloned into the pET-28a (+) vector. The recombinant expression plasmid PET28a-E was obtained and transformed into BL21(DE3). After the recombinant bacteria were induced by optimal concentration of IPTG, the E fusion protein was proved toget exact expression and have good immunogenicity by SDS-PAGE and Western blotting.Based on the expressed protein an indirect ELISA was established to detect DTMUVantibody in duck serum. The indirect ELISA shared95.0%coincidence rate with theneutralization test, which showed that this method had a good prospect on the detection ofDTMUV antibody.3. Cloning and Expression of NS1Gene of DTMUV and Preliminary ApplicationWhen Flavivirus infects cells, nonstructural protein NS1is produced by Flavivirus. Theantibodies of ducks which infected with virus or inactivated virus can be distinguished bynonstructural protein NS1. The whole cDNA of NS1gene was amplified by PCR fromDTMUV (strain LC) and the recombinant plasmid PET28a-NS1was successfully constructed.NS1protein of DTMUV was expressed by Escherichia coli expression system. After therecombinant bacteria were induced by IPTG, the NS1protein was proved to get exactexpression and have good immunogenicity by SDS-PAGE and Western blotting. Base on theexpressed protein an indirect ELISA was established to detect DTMUV antibody in duckserum. Judging whether DTMUV antibody is produced by naturally infection or immuneresponse after vaccination can be achieved in this way.