Prokaryotic Expression Of NS1 Protein Of Duck Tembusu Virus And The Establishment Of Indirect ELISA Method

Duck Tembusu virus disease(DTMUVD)is Duck Tembusu virus(DTMUV)infects ducks,causing decline in egg,retarded growth.In 2010,DTMUV was detected in Anhui,Zhejiang,Shandong,Jiangsu and Fujian.The virus has become a new pathogen that seriously affects the duck population in China.The NS1 protein is the non-structural protein of DTMUV,It is mainly related to viral RNA replication and pathogenicity.At the same time,NS1 protein has multiple epitopes that can induce the body to produce specific antibodies.In the present work,first,the specific primers were designed according to the full-length coding sequence of NS1 gene(GeneBank No.KM102539)of AH-F10 strain.After RT-PCR amplification,the amplimer was cloned into the vector pET-32 a to generate the recombinant plasmid pET32a-NS1.The vectors of pET32a-NS1 was transformed into E.coli BL21(DE3).One colony was selected and induced with IPTG.The SDS-PAGE analysis results indicate that the molecular weight of the recombined protein is in line with the expected size of 62 kDa,and mainly expressed in the sediment,after purified,renatured,concentrated.The results of SDS-PAGE and Western blotting showed that the recombined protein has high specificity reaction with DTMUV rabbit polyclonal antibody.An indirect ELISA was established using purified NS1 protein as coating antigen.Trying the different condition,the best condition found for indirect ELISA for rapid detection of antibodies against DTMUV(NS1-ELISA),follow as ELISA plates were coated with purified NS1 protein(40?g/ml)and incubated 37? for 60 min,the plates were sealed,and incubated for 90 min at 37?.Serum samples were diluted 1:400 in dilution buffer,and incubated for 30 min at 37?.Antibody was added to the wells at appropriate working concentration(1:2000),and incubated for 30 min at 37?,color was developed at room temperature at 20 min.The established NS1 protein antibody indirect ELISA method cannot detect Avian influenza,Newcastle disease,Duck Circovirus Disease,New duck parvovirus disease,Duck reovirus disease and Riemerella serovar Typhimurium positive serum,and was highly sensitive to 1:1600 dilution of Duck Tembusu virus positive serum.Intra-assay and inter-assay coefficient of variation is less than 5%.Compared with the IFA method,the NS1-ELISA method had a positive coincidence rate of 81%,a negative coincidence rate of 100%,and a total coincidence rate of 93.4%.Finally,a total of 147 duck serum samples derived from the different duck farms in Anhui province were tested using the NS1-I-ELISA assay,the results revealed the 91.16% samples is DTMUV positive.In summary,The indirect ELISA method based on NS1 proteins established in this study has specificity,sensitivity and reproducibility.The method provide a new serological rapid detection method for DTMUV epidemiological investigation.