间日疟原虫pvMSP_(1-19)基因重组耻垢分枝杆菌疫苗的构建与鉴定  

Construction and identification of a recombinant Mycobacterium smegmatis vaccine expressing the Plasmodium vivax pvMSP_(1-19) gene

作　　者：陈勇[1] 夏惠[2] 陶志勇[2] 刘丹[2] 高颖[2] 方强[2] 李光友[2] 

机构地区：[1]蚌埠医学院科研中心,安徽蚌埠233030 [2]蚌埠医学院病原生物学教研室,安徽蚌埠233030

出　　处：《中国病原生物学杂志》2011年第7期507-510,共4页Journal of Pathogen Biology

基　　金：卫生部寄生虫病预防与控制技术重点实验室开放课题(No.WK008-04)

摘　　要：目的体外扩增间日疟原虫pvMSP1-19基因,构建大肠埃希菌-穿梭质粒PMV261/pvMSP1-19和电转化耻垢分枝杆菌。方法采用聚合酶链反应(PCR),体外扩增间日疟原虫的pvMSP1-19基因,克隆入PMD19-T载体,构建亚克隆PMV/pvMSP1-19大肠埃希菌-分枝杆菌穿梭质粒;电转化方法将PMV/pvMSP1-19穿梭质粒转化到耻垢分枝杆菌中。并热诱导此重组分枝杆菌,SDS-PAGE电泳观察pvMSP1-19蛋白的表达,Western blot鉴定其免疫学活性。结果成功扩增了间日疟原虫pvMSP1-19基因,并且正确构建PMV/pvMSP1-19穿梭质粒和转化耻垢分枝杆菌。采用Westernblot证实该重组耻垢分枝杆菌表达的pvMSP1-19蛋白能被间日疟患者的血清识别。结论构建的重组耻垢分枝杆菌能表达pvMSP1-19蛋白,该蛋白具有免疫反应性,为pvMSP1-19基因重组BCG疫苗的研制提供了必要的基础。Objective To amplify the gene coding for the C-terminal region of merozoite surface protein 1-19（pvMSP1-19） from Plasmodium vivax;construct PMV261/pvMSP1-19,an E.coli-Mycobacterium smegmatis shuttle plasmid;and then introduce the plasmid into M.smegmatis by electroporation.Methods The pvMSP1-19 gene was amplified by PCR and cloned into a PMD19-T vector plasmid,and then the E.coli-Mycobacterium shuttle plasmid PMV261/pvMSP1-19 was constructed and introduced into M.smegmatis by electroporation.The recombinant M.smegmatis was induced by heating,and then the expression and immunologic activity of the target protein were analyzed with SDS-PAGE and Western blot.Results The pvMSP1-19 gene was amplified successfully,and the PMV/ pvMSP1-19 plasmid was constructed and correctly introduced into M.smegmatis.The recombinant pvMSP1-19 protein antigen was successfully recognized by specific serum antibodies from patients infected with Plasmodium vivax according to Western blot.Conclusion The recombinant pvMSP1-19 protein from M.smegmatis containing the PMV261/ pvMSP1-19 plasmid was immunoreactive,providing the required basis for preparation of a recombinant pvMSP1-19-BCG vaccine.

关 键 词：疟原虫 间日 pvMSP1-19 耻垢分枝杆菌 表达 免疫反应性 

分 类 号：R382.31[医药卫生—医学寄生虫学]