[Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences].

作者：Qian Liu, Xue-Nian Xu, Yan Zhou, Na Cheng, Yuting Dong, Huajun Zheng, Yongqiang Zhu, Yong-Qiang Zhu · 发表于：PubMed · 年份：2013 · 被引用次数：1 · 研究领域：Biology、Molecular biology、Virology、Genetics、Immunology

OBJECTIVE: To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins. METHODS: The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences. RESULTS...