Characterisation Of Antigen Genes Of Chonorchis Sinensis

Clonorchiasis caused by Clonorchis sinensis is an important fish-borne zoonoticparasitic disease and predominantly leads to hepatobiliary diseases. Chronic infectioncan produce severe complications, including gallstone, cholecystitis, cholangitis andhepatomegaly. The most severe complication is associated with cholangiocarcinoma(CCA). C. sinensis was reclassified as group Ⅰb io-carcinogen by the InternationalAgency of Cancer Research in2009. Most of the recombinant proteins were foundsensitive and specific for serodiagnosis of clonorchiasis but not specific enough toreplace the crude extract that we use for now. This is a hot topic of the further studies.cDNA expression library of Clonorchis sinensis adult had been successfullyestablished and three genes GsG1,CsG2and CsG6were screened by immunologicalmethod in our lab. Three genes were cloned and expressed in prokaryotic expressionsystem. Three kinds of recombinant protein was used for the preparation of polyclonalantibody and monoclonal antibodies. Then immunolocalization in Clonorchis sinensisof polyclonal antibody and monoclonal antibodies were carried out. Finally we usedrecombinant protein as diagnosis antigen to grope for the best antigen concentrationand the best dilution multiple of serum, and then ELISA immunological diagnosticmethods was established.At last the sensitivity and specificity was identificated toevaluate immune value of the recombinant protein.Results showed that three kinds of antigen gene recombinant expression plasmidwere successfully constructed and got the efficient expression.The protein molecularweight is about37kDa,34kDa and30kDa; The recombinant protein can be specificidentification by positive serum of people infected C.sinensis by western blot.Polyclonal antibody and monoclonal antibody was successfully achieved.ThePolyclonal antibody mainly distributes in oral sucker, ventral sucker and monoclonalantibody mainly distributes in the oral sucker, ventral sucker and eggs in uterus.Results showed CsG1(P/N=7.91), CsG2(P/N=4.15) and CsG6(P/N=5.29) antigenhas diagnostic value(P/N≥2is meaning). The optimal density of CsG1, CsG2andCsG6antigen is6.4μg/mL,6.0μg/mL and3.2μg/mL and the optimal serum dilution is1:100,1:80and1:80by chessboard titration.64negative samples and210positivesamples was detected by established ELISA method. Sensitivity of CsG1,CsG2and CsG6was100%(210/210),95.7%(201/210) and100%(210/210) respectively;specificity of CsG1, CsG2and CsG6is92.2%(59/64),81.25%（52/64）and85.94%（55/64） respectively.Characterisation of antigen genes of Chonorchis sinensis and three kinds ofimportant clonorchiosis diagnosis candidate protein molecules were screened and theELISA immunological diagnostic methods was successfully established. Thereforethe screened recombinant proteins is of great importance to diagnosis and preventionof clonorchiosis and has great application value.