Establishment Of Indirect ELISA Diagnose Method For Duck Plague Virus Based On Capsid Protein PUL17

Duck plague is an acute infectious disease with high morbidity and mortality caused by duck plague virus(DPV),which can cause serious harm to the duck industry in China.Due to the lack of specific drugs to treat duck plague,effective immunization procedures,accurate diagnosis and monitoring techniques are of great significance for the prevention and control of duck plague.As a member of the herpesvirus family,the capsid and tegument of duck plague virus are the major structural components.In this thesis,pUL17,a subunit of capsid associated-tegument complex,was selected as the antigen of indirect ELISA method to diagnose duck plague virus and this method was applied to monitor the antibody levels in ducks that have been immunized with the duck plague attenuated vaccine or challenged by the wild-type strain.It provided an effective technical method for monitoring and controlling duck plague.The research results are as follows:1.Acquisition of UL17 recombinant proteinIn this study,the antigenic epitope of DPV pUL17 was predicted,and found it had a great potential to induce immune responses.According to the sequence of DPV UL17 gene sequence,we constructed a prokaryotic expression plasmid,pET32a(+)-UL17.After inducing expression,it was analyzed and determined that UL17 recombinant protein was largely expressed in inclusion body.In optimizing the induction conditions,it was found maximum expression amount of UL17 recombinant protein could be obtained when the concentration of IPTG reached 0.6 mmol/L at 37 ℃ for 4 h.The high-purity UL17 recombinant protein obtained after gel cutting purification could produce a strong immune reaction with duck plague virus positive serum,this result showed pUL17 could be used as the antigen to diagnose duck plague virus.2.Establishment of an indirect ELISA method based on duck plague virus capsid protein pUL17In this study,we initially established an indirect ELISA method for diagnosing duck plague virus which was based on DPV capsid protein pUL17,and determined optimal conditions for this method after a series of optimization tests: the coating concentration of antigen was 50 μg/m L and coated at 4 ℃ for 12 h,the optimum blocking condition was using 5% skimmed milk powder to block for 1 h at 37 ℃,serum was diluted at 1:80 to react for 1 h at 37 ℃,enzyme-labeled secondary antibody was diluted at 1:1000 to react for 1 h at 37 ℃.The determination cut-off value of pUL17 indirect ELISA method was 0.354,specificity test results showed that this method could specifically detect duck plague virus antibody,and had no cross-reactivity with duck tembusu virus(DTMUV),Escherichia coli(E.coli),Riemerella anatipestifer(RA)and Salmonella enteritidis(SE),which proved this method had good specificity.The sensitivity test results showed that pUL17 indirect ELISA method could detect 3200 times diluted duck plague positive serum,which indicated this method had good sensitivity.The repeatability test results showed that the coefficient of variation of intra-plate test was between 2.62% and 6.81%,and the coefficient of variation of inter-plate test was between 1.96% and 6.30%,which indicated the repeatability of this method was satisfactory.3.Application of pUL17 indirect ELISA methodThe pUL17 indirect ELISA was applied to detect serum antibody level of two groups of ducks vaccinated with DPV attenuated vaccine or infected with DPV CHv on 0 d,3 d,7 d,14 d,21 d,28 d,35 d,and 42 d.The results showed found that DP antibody could be effectively detected on 14 d,and this antibody level continued to rise until 42 d.pUL17 indirect ELISA method and neutralization test were used to detect 50 duck serum samples,and the results showed pUL17 indirect ELISA had 94% overall coincidence rate with virus neutralization test,so the two methods had a good consistency.In summary,the indirect ELISA diagnosis method,based on duck plague virus capsid protein pUL17,was established in this study.This method had good specificity,high sensitivity,and satisfactory repeatability,and could be applied to clinical detection of duck plague virus and dynamic monitoring of duck plague antibody levels,which provided an effective method for the diagnosis and control of duck plague.