Research On Indirect ELISA For Detection Of Antibody Against Subgroup K Avian Leukosis Virus

Avian leukosis(AL)is a general designation of avian benign or malignant neoplastic disease caused by avian leukosis virus.Since the report,AL has caused greater economic losses to poultry industry around the world.So far,there is no effective drug and available vaccine against ALV.The most reliable method to reduce and purify exogenous ALV is eliminating positive flocks.ALV is the reverse transcriptase virus and easy to mutate,its gp85 gene determines subgroup specificity.The ALV was divided into 10 subgroups,ALV-A to ALV-J,according to envelope protein characteristics.In recent years,a new subgroup has been reported in China and named ALV-K.However,there are few studies on ALV-K and there is no commercial kit for detection of ALV-K antibody.In this study,the c DNA of an ALV-K isolate strain(GDFX0603)was used as a template to amplify its gp85 gene by specific primer PCR.The sequencing results showed that the target gene was the same as the gp85 sequence of GDFX0603 strain published by Gene Bank.Then the gp85 gene and the p ET32 a vector was ligated together using T4 ligase.The recombinant plasmid p ET-GDFX0603-gp85 was constructed and identified.Then the recombinant protein expression conditions were screened and optimized to obtain a large number of recombinant proteins.SDS-PAGE analysis showed that the optimal induction time was 6h,37°C was the optimal induction temperature,the best concentration of IPTG was 0.7 m M.And the expressed products were in the presence of inclusion bodies and its size was about 53 ku.To get high purity protein,the expressed protein was purified by Ni column.Western-Blot analysis showed that recombinant protein had good immunogenicity.The indirect ELISA method was established for detecting ALV-K-gp85 antibody.The ALV-K gp85 protein was used as coating antigen and the optimal reaction conditions were screened by indirect ELISA method.The results showed that the coating solution selected PBS buffer(p H=7.6),the concentration of antigen was 5?g,the optimal serum and secondary antibody dilution was 1: 200 and 1: 3000,the best serum reaction time was 60 min,and the secondary antibody reaction time was 45 min,the last reaction time for substrate was 10 min.The cutoff values of the negative and the positive sera was 0.325.The intra-and inter-assay demonstrated that the coefficient of maximum variation was 10.41% and 12.57% respectively.The specific tests showed that there were no cross reaction to the anti-sera against ALV-B,ALV-J,AIV,NDV,IBDV,MDV,REV,Escherichia coli and Salmonella,while it had weak cross reaction with the serum of ALV-A.32 serum samples from ALV-K artificial infection test were tested and the seropositivity rate of ALV-K was 56.25%.Therefore the indirect ELISA method of this study was beneficial to the detection for antibody against ALV-K in serology and the development of ALV-K antibody detection kit.