弓形虫SAG2基因体外扩增、克隆及在耻垢分支杆菌中的表达

目的体外扩增弓形虫RH株表面抗原2(SAG2)基因,并构建大肠杆菌-分支杆菌穿梭质粒ps3000-SAG2,构建重组耻垢分支杆菌,免疫接种小鼠,通过检测免疫小鼠血清中的弓形虫抗体,证明SAG2重组蛋白在耻垢分支杆菌中有表达。方法采用聚合酶链式反应(PCR)方法,体外扩增弓形虫RH株的SAG2基因,克隆入pGEMT载体,然后构建亚克隆ps3000-SAG2大肠杆菌-分支杆菌穿梭质粒,经电转化方法,将SAG2基因的穿梭质粒转化到耻垢分支杆菌(Mycobacterium smegmatismc2155)中,用重组耻垢分支杆菌免疫小鼠,Western-blotting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SAG2基因;正确构建穿梭质粒ps3000-SAG2,免疫印迹分析,免疫小鼠血清能所识别SAG2重组蛋白。结论耻垢分支杆菌在小鼠体内表达出了重组蛋白SAG2,具有抗原性,刺激小鼠机体产生出了抗弓形虫的抗体,为下一步弓形虫重组BCG(Bacilli Calmette-Guérin)疫苗的研究奠定了基础。

In vitro amplification and cloning of Toxoplasma gondii SAG2 gene and its expression in Mycobacterium smegmatis

In the present study,the gene encoding the surface antigen-2 of Toxoplasma gondii RH strain(SAG2) was amplified in vitro by PCR,cloned to vector pGEMT and then the E.coli-Mycobacterium shuttle plasmid ps3000-SAG2 was constructed.This shuttle plasmid was then transformed to M.smegmatis mc~(2)155 through electroporation,and the recombinant SAG2-M.smegmatis mc~(2)155 protein was inoculated to BALB/c mice,whose serum was analyzed by Western blotting to detect the anti-toxoplasma antibodies.It was found that,the recombinant protein pET-SAG2 was recognized by mouse immune serum.The recombinant protein with antigenicity could be expressed on M.smegmatis in mice and could induce the producton of mouse anti-toxoplasma antibodies.These results may provide basis for the further studies of the recombinant BCG vaccine.