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目的体外扩增弓形虫RH株致密颗粒蛋白4(GRA4)基因,构建大肠埃希菌-分枝杆菌穿梭质粒ps3000-GRA4,重组耻垢分枝杆菌,鉴定重组蛋白的抗原性。方法用聚合酶链式反应(PCR)方法从弓形虫RH株基因组DNA中扩增GRA4基因,克隆入pGEM-T载体,然后亚克隆法构建ps3000-GRA4大肠埃希菌-分枝杆菌穿梭质粒,经电转化方法,将GRA4基因片断的穿梭质粒转化到耻垢分枝杆菌(Mycobacterium smegmatismc2155)中,用重组耻垢分枝杆菌免疫小鼠,Western blot方法检测免疫小鼠血清中的弓形虫抗体。结果成功扩增了弓形虫的GRA4基因并构建克隆质粒pGEMT-GRA4、穿梭质粒ps3000-GRA4,Western blot分析显示,GRA4重组蛋白能被重组质粒免疫小鼠血清识别,分子质量单位约为40.0 ku。结论重组耻垢分枝杆菌在小鼠体内表达出了重组蛋白GRA4,具有抗原性,刺激小鼠机体产生了抗弓形虫的抗体,为弓形虫重组BCG(Bacillus Calmette-Guerin)疫苗的研究奠定了基础。
Objective To amplify the GRA4 gene of RH strain Toxoplasma gondii in vitro and construct Escherichia coli - mycobacterium shuttle plasmid ps3000-GRA4 and recombinant Mycobacterium smegmatis to identify the antigenicity of the recombinant protein. Methods The GRA4 gene was amplified from genomic DNA of RH strain Toxoplasma gondii by polymerase chain reaction (PCR) and cloned into pGEM-T vector. The shuttle plasmid pSEM-T of ps3000-GRA4 was constructed by subcloning, The electroporation method was used to transform the shuttle plasmid of GRA4 gene into Mycobacterium smegmatismc2155. The recombinant plasmid was immunized with recombinant M. smegmatis and the Toxoplasma gondii antibody was detected by Western blot. Results The GRA4 gene of Toxoplasma gondii was successfully amplified and the cloned plasmid pGEMT-GRA4 was constructed. The shuttle plasmid ps3000-GRA4 was successfully amplified. Western blot analysis showed that the GRA4 recombinant protein was recognized by the serum of the recombinant plasmid-immunized mice with molecular mass of about 40.0 kU. Conclusion Recombinant Mycobacterium smegmatis expressed recombinant protein GRA4 in mice, which has antigenicity and stimulated the body of mice to produce antibodies against Toxoplasma gondii. It laid the foundation for the research of the recombinant BCG vaccine against Bacillus Calmette-Guerin basis.