Establishment And Application Of Indirect ELISA Based On VP0 Protein For Detecting Type 1 And 3 DHAV Antibodies

Duck Viral Hepatitis(DVH)is an acute and fatal disease of ducklings caused by Duck Hepatitis Virus(DHV).Because of its rapid morbidity and high mortality,it has caused huge economic losses to duck industry.Duck viral hepatitis in China is mainly caused by DHAV-1 and DHAV-3,and mixed infection of two types is more popular.Therefore,a simple,rapid,low-cost and comprehensive detection method of antibodies is urgently needed.In this study,the recombinant plasmids p MD18T-DHAV-1/2 and p MD18T-DHAV3-1/2 containing VP0/VP3/VP1 genes of type 1 and type 3 were used as templates.DHAV-1/3-VP0,DHAV-1/3-VP3 and DHAV-1/3-VP1 genes were amplified by PCR and cloned into p ET-30a(+)and p ET-32a(+)prokaryotic expression vectors,and the recombinant prokaryotic proteins were produced.Then the recombinant proteins were purified.Western Blot was used to analyze the cross immunoreactivity of the purified recombinant proteins VP0/VP1/VP3 with duck serum antibodies of DHAV(type 1 and 3)respectively.The results showed that the recombinant proteins VP0/VP1 could react with duck serum antibodies of the same t ype and different type,while the recombinant protein VP3 could only react with duck serum antibodies of the same type.The cross immunoreactivity of the recombinant proteins VP0/VP1/VP3 were detected by ELISA using purified recombinant protein as coated antigen respectively.The results showed that the recombinant proteins VP0/VP3/VP1 could react with the same and different serum antibodies simultaneously.The reaction intensity of recombinant VP0 and VP3 proteins with the same antibody was stronger than that with the different type antibody.The intensity of the recombinant VP1 protein with the same and different type reaction were similar.Western Blot and ELISA analysis showed that recombinant proteins VP0 and VP1 were more suitable to detect group specific antigens.Based on the above results,an indirect ELISA method was established to detect serum antibodies of DHAV(type 1 and 3)simultaneously with purified recombinant protein DHAV-1 VP0 as antigen.The optimum reaction conditions were as follows: 0.2 5 ?g/m L DHAV-1 VP0 recombinant protein was incubated at 37? for 2 hours and then coated overnight at 4?;5% skimmed milk was blocking at 37? for 90 minutes;serum was diluted at 1:100 times and incubated at 37? for 1 h;Goat anti-duck Ig G labeled with HRP was diluted at 1:400 times and incubated at 37? for 1 h;TMB color reacted to avoid light for 15 minutes.By detecting 118 serum samples of negative ducks,the cutoff value of negative and positive was 0.307.The specificity assay showed that the ELISA reacted with DHAV-1 and DHAV-3 positive sera only,and had no cross-reaction with duck-derived gosling plague antibody,duck enteritis antibody,avian influenza H9N2 subtype antibody,duck tambusu antibody;The method the lowest detection rate was 1:320,and the coefficient variability of intra-plate and inter-plate was less than 10%.A batch of clinical serum samples were simultaneously detected by VP0-ELISA and DHAV-Ab qualitative kit.The coincidence rate of the two methods was as high as 95.7%.This method c an be applied to the detection of duck hepatitis A virus antibody,and provide technical support for the detection of DHAV infection and the evaluation of the immune effect of bivalent vaccine in the future,so as to realize the rapid serological diagnosis of duck viral hepatitis and promote the healthy development of duck industry.