Prokaryotic Expression And Application Of Duck Tembusu Virus NS1Protein

Duck Tembusu virus(DTMUV) is arecently emerged pathogen associated withsevere egg drop syndrome and high morbidity and mortality.It has rapidly spread aroundmajor duck-producing regions in China.The infection has caused a serious economic lossto the poultry industry in China. It is very important establishing an effective andconvenient method to diagnose DTMUV.Molecular cloning of the complete coding region of the NS1gene, andbioinformatics analysis, prokaryotic expression of NS1were performed to establish ofthe NS1Ab-capture ELISA detection method and applie to clinical samples.1. Cloning and Bioinformatics Analysis of DTMUV NS1geneNS1gene was cloned into pMD18-T vector, restriction enzyme digestion and DNAsequencing, gene fragment1050bp, including the complete coding region of the NS1gene. Analysis found that the experimental strains of non-structural protein NS1gene hasthe highest homology as99.9%with DTMUV ZJ-GH2strain, and DTMUV ZC-1nucleotide homology with the lowest high as97.1%, and Sitiwan virus (JX477686)nucleotide homology as high as86.5%.2. Expression and purification of DTMUV NS1proteinThe nonstructural protein gene NS1of DTMUV is subcloned into prokaryoticexpreesion vector pET-32a(+),the recombinant plasmid named pET-32a-NS1wasconstructed. By analyzing the characteristic of prokaryotic expression vector,the NS1gene can be expressed correctly. The pET-32a-NS1was used to transform into E.coliBL21(DE3).Induced by temperature (37℃) for4h, the NS1gene was expressedsuccessfully. The results of SDS-PAGE and West-blotting indicated that the NS1proteinwas expressed in a high level non-fusion and fusion protein, which was about62kD, hadimmunological reactive activity. This study lay on foundation for the development ofdiagnosis methods in serology for DTMUV.3. Establishment and application of DTMUV NS1Indirect Ab-capture ELISA methodThe recombinant NS1protein was expressed by E.coli and purified by HisPur Ni-NTA Purification Kit.Using the purified NS1protein, an indirect ELISA for detectionof anti-DTMUV antibodies was developed and its optimal reaction conditions weredetermined;coating antigen for4℃overnight at a concentration of2.5μg/mL, serumsample(1:100) and HRP labeled SPA being incubated at37℃for90min, the substrate forELISA TMB reacted at37℃for5mins. The ELISA assay was confirmed to have a goodreproducibility and specificity by reproducibility test and cross test.