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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 pvMSP1-19 gene of Plasmodium vivax in vitro and construct Escherichia coli shuttle plasmid PMV261 / pvMSP1-19 and electroporation Mycobacterium smegmatis. Methods Polymerase chain reaction (PCR) was used to amplify pvMSP1-19 gene of Plasmodium vivax in vitro and cloned into PMD19-T vector to construct subclone shuttle plasmid of PMV / pvMSP1-19 Escherichia coli - Mycobacterium. Transformation Methods PMV / pvMSP1-19 shuttle plasmid was transformed into M. smegmatis. The recombinant Mycobacterium tuberculosis was induced by heat, the expression of pvMSP1-19 protein was observed by SDS-PAGE electrophoresis and the immunological activity was identified by Western blot. Results The P. vivax pvMSP1-19 gene was successfully amplified and the PMV / pvMSP1-19 shuttle plasmid was correctly constructed and M. smegmatis was transformed. Westernblot was used to confirm that the recombinant pMMSP1-19 protein expressed by M. smegmatis was recognized by the sera of patients with Plasmodium vivax. Conclusion The recombinant Mycobacterium smegmatis can express pvMSP1-19 protein, which is immunoreactive and provides the necessary basis for the development of recombinant BCG vaccine of pvMSP1-19 gene.