弓形虫重组耻垢分枝杆菌M.S-P30-ROP2载体活疫苗构建及鉴定

目的构建和鉴定弓形虫1330-ROP2复合基因重组耻垢分枝杆菌M．s载体活疫苗。方法用亚克隆技术把P30-ROP2复合基因克隆入大肠杆菌／分枝杆菌穿梭质粒pSTM3，构建重组质粒pSTM3-P30-ROP2，电穿孔法导入耻垢分枝杆菌中，潮霉素筛选、PCR法鉴定阳性转化子，温度诱导表达鉴定。结果获得pSTM3-P30-ROP2重组穿梭表达载体，SDS—PAGE结果显示P30-ROP2复合基因表迭蛋白产物分子量约为66kDa。结论成功构建弓形虫复合抗原基因P30-ROP2重组耻垢分枝杆菌疫苗，为弓形虫病的防治提供了-种新方法。

Construction and identification of recombinant M.S-P30-ROP2 vaccine of Toxoplasma gondii

Objective To construct and identify living recombinant Mycobacterium smegmatis MC2155 vaccine containing P30 - ROP2 compound gene of Toxoplasma gondii. Methods P30- ROP2 compound gene was subcloned into the Escherichia coli/Mycobacterium shutde plasmid pSTM3 to attain a recombinant plasmid pSTM3- P30- ROP2, then this plasmid was transformed into MC^2 155 by electroporation. The transformant was cultured under hygromycin pressure, and positive clones were screened and identified by PCR from bacteria directly, then induced by temperature. The expressions of proteins were analyzed by SDS - PAGE. Resuits P30 - ROP2 compound gene was subcloned into pSTM3 correctly. Positive clones were obtained, the molecular weight of the expressed protein encoded by P30 - ROP2 was 66kDa on SDS - PAGE in accordance with the result of prestained protein marker. Conclusion The recombinant M. S- pSTM3 - P30- ROP2 is constructed successfully which provide a new way for prevention of Toxoplasma gondii infection.