Identification Of Advantage Antigen Domain Of DHV-â…  VP1and Development Of An Indirect LISA

Duck Viral Hepatitis(DVH) is an infectious disease caused by Duck Hepatitis Virus(DHV)manifesting a highly mortal and spreads rapidly disease. DHV was a member of picornavirus.Avian hepatitis virus genus. It prevailed in the world meaningwhile DHV-I is now worldwidedistribution. Neutralization test, ELISA and agar diffusion reaction were used world-widely todetect antibodies against DHV in serum for diagnosis of DVH.The antigencity and hydrophobicity of VP1gene was analysed by the software of DNA star,and the five parts of VP1gene with better antigen and hydrophilicity with the lengh of230~720bpunder the name of VP1-a~e/VP1were subcloned into pET-32a(+) prokaryotic expression vector.The positive clone of recombinant vector were identified and used to transform into theEscherichia coli Rosetta competent cell. The expression of the five parts of VP1proteins as well asthe purified part VP1-c were identified by SDS-PAGE. The result is the same as anticipatation. Theresult from the Western blot shows that the target part of protein was with great antigencity.The purified recombinant VP1-c protein was used as coating antigen to establish indirectELISA. A series of tests were carried out to determine the optimal coating situation; confiningsituation; dilution of serum and secondary antibody and reaction times. The standardization ofindirect ELISA is with the concentration of VP1-c2μg/mL;concentration of DHV is1:20; coatingsolution is0.05mol/L（pH=9.6）Na2CO3in37℃1h+4℃; blocking solution is5%milk in37℃90min;tested blood of DHV is37℃60min;HRP-labled antibody is1:15000in37℃60min; TMB solutionis37℃10min. The established ELISA’s cut-off was0.208; limit of detection was1:640.Intro-batch was0.38%～6.62%as well as the Inter-batch was3.68%～8.87%. Theestablished method could distinguish anti-serum against DHV from anti-serum of some commonpathogens of duck such as duck enteritis virus,gosling plague virus and Ana I aviadenovirus whichshows that the established ELISA was with great specificity.48serum samples were tested withthe established ELISA and the results were compared with the results of serum neutralization test.The total agreement between ELISA and neutralization test was86.9%. The results showed thatthe indirect ELISA established was an effective tool for diagnostic use of DHV infection of duck.