日本血吸虫含EF-手型结构域钙结合蛋白的克隆表达与免疫诊断分析

Cloning,Expression and Immunodiagnostic Analysis of Schistosoma japonicum Calcium-binding EF-hand Domain Containing Protein

Objective To clone and expression Schistosoma japonicum calcium-binding EF-hand domain containing protein(SjEFCAB),purify the expressed protein,and evaluate its antigenicity and diagnostic value.Methods The positive clone screened from egg cDNA library was used as template to amplify the SjEFCAB gene by PCR.The target fragment was cloned into prokaryotic expression vector pGEX-4T-1.The positive recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for expression of the protein.The recombinant protein was purified with GST-tag affinity chromatography.Western blotting was used to analyze the antigenicity.The purified protein was used as coating antigen for indirect ELISA to evaluate its diagnostic effect.Serum samples from patients with schistosomiasis japonica(78 cases),clonorchiasis sinensis(5 cases),cysticercosis(10 cases),paragonimiasis westermani(6 cases),trichinosis(9 cases) and healthy persons(50 cases) were examined.Results The recombinant plasmid pGEX-4T-1-SjEFCAB was constructed and the SjEFCAB recombinant protein(Mr 8 200) was expressed in E.coli.The soluble fusion protein was purified with affinity chromatography.Western blotting analysis showed that the recombinant protein was recognized by sera of infected rabbits and pooled sera of schistosomiasis japonica patients.The sensitivity and specificity of ELISA for diagnosis of schistosomiasis japonica were 82.1%(64/78) and 95.0%(76/80),respectively.The cross reaction with sera of clonorchiasis sinensis,cysticercosis,and trichinosis patients were 1/5,1/10,and 1/9,respectively.There was no cross reaction with sera of paragonimiasis westermani patients.Conclusion The recombinant SjEFCAB antigen has potential diagnostic value for schistosomiasis japonica.