Development And Application Of The Nested PCR Assay For Duck Tembusu Virus And Recombinant Protein Indirect ELISA Assay

From April through November2010, duck infectious disease characterized by a suddenonset caused by a newly emerged flavivirus is widely spreading in southeast China. It isnecessary to establish a rapid and specific diagnosis method. The development of molecularbiological technique provided new strategies for the design of flavivirus rapid diagnosis.Flavivirus universal primer pairs and nested PCR were developed as a efficient methodto detect flavivirus, especially new type flavivirus. Four primers Flav P1-Flav P4located inconserved region of the NS5gene were designed after alignment of the flavivirus genome andthen nested PCR was developed assay for DTV. Nested PCR was1000times more sensitivethan the conventional PCR. The clinical samples detection showed that the universal primersand nested PCR were highly specificity and sensitivity. Phylogenetic analysis showed thatnew type flavivirus belongs to mosquito-born flavivirus, NTAV group, similar with Tembusuand BYD isolated. The results showed that the nested PCR was applicable for rapid laboratorydiagnosis and epidemiologic surveillance, in addition, the station of evolution of DTV wasclarified.The envelope glycoprotein (E protein) of flavivirus, is associated with viral attachmentand fusion with host cells, determines the virus,s hemagglutination ability, cellular tropismand viral virulence, and induces protective immune response. In order to analysis thebiological characteristics of the E protein, envelop proteins genes of six DTV isolated frombroilers were cloned by polymerase chain reaction (PCR). The constructed plasmids weredigested with EcoRⅠand XhoⅠ, the obtained fragments were ligated directly to theexpression vector pET-21a(+) which was digested with EcoRⅠand XhoⅠ. The recombinantvector was transformed into the expression competent cells Escherichia coli BL21. By PCRidentification, recombinant vector digeston and sequence analysis, it showed that therecombinant vector was successfully constructed. Then the target protein expression wasinduced by the recombinant vector at different temperature, different time points, and different concentrations of IPTG. E gene was comprised of1503nucleotides, encoding501aminoacid. By SDS-PAGE, the molecular weight of E protein was54.3kDa expressed inpET21a/BL21(DE3) system. Western-blot analysis showed that the purified fusion proteinscould both react with duck serum containing antibody against DTV. The fusion protein waspurified by metal-ligand affinity chromatography, which should be used as diagnostic reagentof DTV.Currently，the neutralizing test (NT) is the most specific procedure for diagnosis offlavivirus inefetion. However，NT takes several days and requires a containment laboratoryfor virus manipulation. An indirect ELISA was established for rapid detection of antibodiesagainst DTV using the purified recombinant proteins as coating antigen. The reactionconditions were optimized, including7μg/mL coating antigen of the purified recombinantproteins,1∶320dilution of testing serum and1∶1000dilution of HRP conjugated anti-duckIgG with cut off-value of0.3244(OD450nm). The results revealed the indirect ELISA could beused for laboratory diagnosis and serosurvey for DTV infection.