Cloning Expression,Histological Localization And Immunological Characteristic Study Of Cysteine Protease From Clonorchis Sinensis

Object Through the study of the function and characteristics of Clonorchis sinensis cysteine protease(CsCysP38.3),it is analyzed that CsCysP38.3 plays an important role in the relationship between parasit and host,and to evaluate its potential as candidate diagnostic antigen and candidate vaccine molecule.Methods Two pairs of specific primers were designed and synthesized according to the sequence of the cysteine protease genes of Clonorchis sinensis in GenBank(DQ902586.1),the total RNAs of adult worms of Clonorchis sinensis were extracted,the cDNA was synthesized by reverse transcription,and the PCR was amplified simultaneously by using cDNA as a template so as to gain the target gene.The target gene was ligated with prokaryotic expression plasmid pET-28 a,and the recombinant plasmid pET-28a-CsCysP38.3 was constructed.The recombinant plasmid was transformed into E.coli BL21 for expression,then the recombinant protein CsCysP38.3(rCs CysP38.3)was purified and renatured.The localization of CsCysP38.3 in adult,larva and metacercaria was observed by fluorescence immunohistochemical method.rCsCysP38.3 as antigen to detect specific IgG subclasses and IgE in sera of patients with Clonorchis sinensis by ELISA method,at the same time,detection of IgG1 and Ig G4 were used to evaluate the detection rate and cross reaction of clonorchiasis.Sprague-Dawley rats were immunized with rCsCysP38.3 and infected with metacercaria when the titer of anti-rCsCys P38.3 IgG was the highest.The immune protective effect was evaluated by counting the number of eggs in feces and the number of adult worms.Results The recombinant plasmid of pET-28a-CsCysP38.3 was successfully constructed,and rCsCysP38.3 was purified and renatured to obtain the soluble protein with concentration of 750 ? g/mL.The results of fluorescence immunohistochemistry showed that CsCys P38.3 was located in the intestinal branches in adult worms,in intestinal,cortical as well as cortical cells in larva,and in cortical cells in metacercaria.r CsCysP38.3 mainly detected IgG1 and IgG4 in sera of patients with Clonorchis sinensis.The detection rate of specific IgG1 was 33.3%(8/24 cases),and that of IgG4 was 58.3%(14/24 cases),which mainly had cross reactions with schistosomiasis and paragonimiasis.The level of specific IgG increased significantly after immunization with rCsCysP38.3 in rats,and reached its peak at 6 weeks after primary immunization,the titer is 1: 1638400.There was no significant difference in EPG(egg count per gram fecal worm)and the number of adults between the immunized group and the control group.The results showed that there was no obvious immune protective effect after immunization with rCsCysP38.3.Conclusion 1.the localization of CsCysP38.3 in adult and larva indicated that the cysteine protease may be involved in nutritional metabolism,and the location of CsCysP38.3 in metacercaria may be related to encystation and excystation.2.detection of specific IgG4 in sera of patients with Clonorchis sinensis by rCs CysP38.3 was effective and rCsCysP38.3 could be used as a candidate antigen for diagnosis.3.the immunoprotective effect of rCsCys P38.3 on metacercaria infection was not obvious.4.rCsCysP38.3 has preferable antigenicity and immunogenicity.