Expression And Application Of Truncated Vp3Gene Of Duck Hepatitis Virus Type â… 

Duck viral hepatitis (DHV) is an acute infection of ducklings, which shows rapidspread, high mortality and typical hemorrhages in the liver. The disease causes an infectionin ducklings following four weeks of age and especially following three weeks of age. It isone of the most serious infectious disease in duck.VP3is the one of the major structural proteins of DHV, which located on surface ofthe virus capsid. The VP3contains the main antigenic determinant, which can induce theimmune response. The expression of truncated VP3protein can lay the foundation for thestudy of its immune function and for the research of gene engineering vaccine of DHVtype Ⅰ.In this study a fragment of1-468bits of vp3gene of DHV-Ⅰwas randomly selected,truncated and expressed, then the fusion protein was purified and used as antigen toestablish indirect ELISA method, which was applied in some duck farms of Anhuiprovince. Briefly as follows:First, a pair of special primers according to the complete genome of DHV-Ⅰ fromGeneBank (DQ886445) was designed to amplify truncated vp3gene (1-468). Truncatedvp3gene fragment was recombined to vector pET-32a(+) to construct recombinant plasmidpET-VP468. The universal primer T7promoter primer (#69348-3) and T7terminatorprimer (#69337-3) of pET system were used to identify the recombinant plasmids toextract appraisal right recombinant plasmid. Then the pET-VP468and pET-32a(+) weretransformed into E. Coli Rosetta-gami (DE3) pLysS, and were induced by IPTG toobtained fusion protein Trx-VP468. Ni2+-NTA affinity chromatography column was usedto purify Trx-VP468and BCA Protein Quantitation Kit of different company was used fordetermination of concentration of fusion protein. Trx-VP468was examined by SDS-PAGEand Western-blot. The results showed that the truncated recombinant protein Trx-VP3(1-156) was about36kDa and the fusion protein with an insoluble because of forminginclusion body. Western-blot test showed that Trx-VP468got the correct expression.Secondly, SPF duck was immunized with the purified DHV-ⅠA66strain for thepreparation of hyperimmune serum as positive serum. First immunization was carried outwith complete freund’s adjuvant and virus equal mixture. The second and thirdimmunization were done with l incomplete freund’s adjuvant and virus equal mixingemulsification. The immunized antigen dose was about300μg per individual. SPF duck was immunized three times and every time interval of ten days. After1weeks of secondand third immunization, the seren were collected and detected for the antibody titre. Somepositive seren were prepared as positive samples. With the fixed virus-serum dilutionmethod and the known toxic virus value detected by the positive sample the serumantibody titre ultimately measured was1:90.51.Finally, Trx-VP468was used as coating antigen in the plate to establish a indirectELISA method for the detection of DHV-Ⅰantibodies. With the criss-cross serial-dilutionanalysis the optimum concentration of reagents in the ELISA was determined, and alsoother optimum conditions such as incubation time of first antibody, reaction time of secondantibody, chromogenic time of substrate were optimized. After detecting156healthy duckserum with the optimal conditions the standard of critical value of positive and negativeserum and the result judgement were calculated. The criss-cross serial-dilution todetermine the optimal coating antigen amount was10μg/ml, serum dilution for1:100,rabbit anti duck antibody dilution of1:1000. Incubation time of first antibody, reactiontime of second antibody, chromogenic time of substrate were1h,0.5h, and15min; thecritical value was0.372. Establishment of indirect ELISA method was applicated to detectantibody of clinical duck serum in some areas of Anhui province.331of duck serum fromparts of Anhui for DHV-Ⅰwere detected, the result of the positive rate was73.1%(242/331).The results showed that the truncated vp3gene was correctly expressed in E. coli, therecombinant protein had a high purity after preliminary purification, which could be usedthe indirect ELISA method. The duck serum samples from parts of Anhui were collectedfor testing DHV-Ⅰantibody. The results showed that DHV-Ⅰstructural protein could beused in immunological function research and genetic engineering vaccine research anddevelopment foundation.