| Objective:Since April in 2010,the outbreak of new infectious disease in the main producing areas of duck was found in China.The main symptoms were fever,loss of appetite,pulling the yellow-green loose stools,egg drop or even stop,neurological and movement disorders.Duck Tembusu virus(DTMUV)was identified as a pathogen of the disease and has caused huge economic losses for duck farming.It is necessary to establish a sensitive,rapid and specific diagnosis method to prevent the disease.Besides,E protein is a structural protein of DTMUV,which has strong antigenicity.It was important for diagnosis,vaccine development to prevent the disease.Methods:Experiment one:According to the conserved sequence of E gene in GenBank,one pair of specific primers were designed and the amplification fragment size was to 1020 bp.The annealing temperature for RT-PCR reaction was selected.Six different kinds of virus were tested to verify the specificity of the detection method.Meanwhile,DTMUV positive allantoic fluid was made at intervals of 10-fold dilution and detected by RT-PCR method to determine the sensitivity.The stability and reproducibility of the method were tested with the detection of viral positive samples between groups and within groups.Finally,the feces,cloacal swabs and blood of clinically suspected cases were detected to determine whether the diagnostic method was suitable for the early detection of clinical living body.Experiment two:According to the gene sequence of the E protein major antigenic domains,the specific primers were designed for E gene amplification.The E gene specific amplification products and the carrier were digested by BamH I and Hind III enzyme,and them were connected by T4 ligase,and the recombinant plasmid was transformed into BL21(DE3).The recombinant plasmid was idntificated by PCR,restriction enzyme digestion and sequencing.The IPTG induction concentration and time were studied.Protein was analyzed by SDS-PAGE.The immunogenicity of recombinant proteins was detected by Western blot.Results:Experiment one:The results showed that the E gene can be successfully amplified.The optimization annealing temperature was 55?.Specific test results showed that the DTMUV only was anplified with 1020 bp target band,while the amplification results of other five kinds of virus were negative.The sensitivity test results showed that the minimum detectable amount of nucleic reached to 967.5 × 10-7 ng/?L.87 suspected clinical samples were carried out to detect,and in which there were 40 positive,The consistent with high detection rate with latex agglutination assay rate was 72.49%.Clinically suspected cases of feces,cloacal swabs and blood were each 30 parts by RT-PCR,the positive rates were to 33.33%,36.67%,26.67%.Experiment two:The expression vector pET32a(+)was digested by BamH I and Hind?.E gene and pET32a(+)were connected by T4 DNA ligase,then were transformed into competent cells.Three recombinant plasmids were identified by PCR,results showed that the specifically 1020bp fragment was amplified.Restriction enzyme digestion and sequencing results showed that the prokaryotic expression vector pET32a(+)-E was successfully constructed.Induction of recombinant protein expression conditions results showed that the optimum concentration of IPTG was 1.3mmol/mL,the optimal induction time is 8h.SDS-PAGE analysis showed that the recombinant protein was mainly present in the form of inclusion bodies.Purified recombinant protein test results showed that the optimum concentration of imidazole was 80mM.Western blot analysis showed that the recombinant E protein was correctly expressed,and it had good immunogenicity.Conclusion:1.In this test we successfully established the diagnosis technology of DTMUV by RT-PCR.The method can be used to diagnose DTMUV and its molecular epidemiology.2.E protein of DTMUV is correctly expressed in vitro,which would help to rearch the control method of DTMUV infection. |
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