华支睾吸虫γ-谷氨酰转移酶7基因的原核表达及反应原性分析

Prokaryotic Expression Gamma-glutamyltransferase 7 Gene of Clonorchis sinensis and Analysis of Its Reactogenicity

γ-谷氨酰转移酶7(GGT7)是华支睾吸虫生长发育的各个时期都存在且高表达的蛋白,为探索其作为诊断抗原的潜能,依据GenBank中华支睾吸虫GGT7基因序列,设计了特异性引物,并构建了pET-32a-GGT7重组表达质粒转化至大肠埃希氏菌BL21(DE3)感受态细胞中,进行蛋白表达和反应原性分析。生物分子信息学分析显示,GGT7具有7个α-螺旋和6个β-折叠,B细胞抗原基因表位较多。扩增得到的华支睾吸虫GGT7基因大小为1773 bp,编码591个氨基酸,SDS-PAGE分析在65 ku分子处有明显的目的蛋白条带,Western blot检测表明该蛋白具有较好的反应原性。首次对华支睾吸虫GGT7基因进行了克隆表达,并证明目的蛋白具有良好的反应原性,为华支睾吸虫病诊断试剂的研发奠定了基础。

Gamma-glutamyltransferase 7(GGT7)is identified highly expressed in all stages of life cycle of C.sinensis.In order to analyze the potential of GGT7 gene as a diagnostic antigen for clonorchiasis,the specific primers were designed based on the sequence of GGT7 from GenBank dataset.The recombinant plasmid pET-32a-GGT7 was constructed and transformed into Escherichia coli BL21(DE3)competent cells to analyze the expression and reactogenicity of GGT7 protein.Bioinformatics analyses indicated that the number ofα-helices andβ-folds were seven and six,and many B cell antigen epitopes were found in GGT7 protein.The lenth of GGT7 nucleotide sequence amplified in this study was 1773 bp and encoded 591 amino acids.SDS-PAGE results showed that there was a single protein band,and the molecular weight was 65 ku.Western blot results showed that GGT7 protein had a good reactogenecity.GGT7 gene of C.sinensis was cloned and expressed for the first time in this study,and GGT7 protein had a good reactogenecity,which laid a foundation for the search of new diagnostic antigens for clonorchiasis.