变形链球菌pacA和gtfB-cat与ctxB嵌合表达质粒的构建及表达

Construction and Expression of pacA and gtfB-cat of Streptococcus Mutans Fused with ctxB in Prokaryotic Cells

目的:构建含变形链球菌表面蛋白A区编码基因(pacA)、葡糖基转移酶催化区编码基因(gtfB-cat)及霍乱毒素B亚单位编码基因(ctxB)的嵌合表达质粒并表达目的蛋白。方法:将目的基因pacA、gtfB-cat、ctxB克隆至原核克隆载体pET32-a(+)构建嵌合质粒pET-pacA-cat-ctxB,将其转入表达宿主菌E.coliBL21(DE3),并测量其表达情况。结果:构建的重组质粒经酶切、PCR鉴定及测序,证实目的基因均正确插入到载体pET-32a(+)中,插入的相位正确,未改变目的基因的阅读框架,经诱导后表达目的蛋白,分子质量大小与预期一致。结论:成功构建了嵌合质粒pET-pacA-cat-ctxB,且它们均在原核细胞内获得正确表达。

Objective： To construct the chimeric vector with the encoding gene of pacA of S.mutans, catalytic region in glucosyltransferase and cholera toxin B subnnit, and to testify its expression in Escherichia coll. Methods： The target genes pacA, gtfB-cat and etxB were cloned into procaryon cloning vectors pET32a （＋） and constructed chimeric plasmid pET-pa- cA-cat-ctxB, which was transferred to the expression host strain E.coliBL21（DE3）, and the expression was measured. Re- sults： The recombinant plasmid was inserted into the vector pET32a （＋） correctly through the tests of restriction enzyme diges- tion, PCR identification and sequencing respectively. The correct phase for the insertion was confirmed. The reading frame of target genes didn＇t change. The recombinant plasmid can express target protein in E.coli host strain BL21 （DE3） after being induced by IPTG, and the molecular weight of the protein was consistent with the prediction. Conclusion： The recombinant plasmid pET-pacA-cat-ctxB was successfully constructed and it can be expressed in prokaryotic cells.