Cloning and expression of the Clonorchis sinensisspecific glycine rich antigen 2a genes and its immunodiagnosis value
1. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention,
WHO collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China；
2. Guangxi Center for Disease Control and Prevention, Nanning 530028, China
Abstract：To research and identify new specific antigen gene of Clonorchis sinensis, a λ ZAP cDNA library of adult Clonorchis sinensis was immunoscreened with pooled sera of clonorchiasis cases or sera of mice immunized with excretorysecretory antigen of C. sinensis. The positive clones obtained were sequenced and analyzed. The coding sequence of the target gene was subcloned into prokaryotic plasmid pET28a(+) with a nonfusion expression strategy. The recombinant protein was purified by Hisbind affinity column (NiNTA) and its immunodiagnosis value was evaluated by indirect ELISA to detect the level of specific antibodies in the sera, including 35 samples of human clonorchiasis sera whose egg were positive by stool examination, 36 healthy control sera samples, 15 sera samples from patients infected with Schistosoma japonicum, 15 sera samples from Paragonimus westermani and 13 sera samples from Taenia solium patients. The sensitivity, specificity and total coincidence rate of ELISA were 80.0%, 97.2% and 88.7%, respectively. And false positive rates for sera samples from S. japonicum, P. westermani and T. solium patients were 6.7%, 6.7% and 7.7%, respectively. Sequence analysis revealed that these
GRA2a antigens were C. Sinensisspecific and their functions have not been identified in literatures. In this study, the GRA2a gene family of C. sinensis was identified. The expression plasmid pET28aCs4DSPM was constructed and soluble recombinant protein of pET28aCs4DSPM was purified successfully and it indicated high diagnostic value for clinical detection.
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