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变形链球菌pacA和gtfB-cat与ctxB嵌合表达质粒的构建及表达

张剑1,赵秀1,刘建国2,杨德琴1,吴家媛1,顾瑜1   

  1. 1. 遵义医学院附属口腔医院
    2. 遵义医学院
  • 收稿日期:2011-07-29 修回日期:2012-01-16 出版日期:2012-06-15 发布日期:2012-06-15
  • 通讯作者: 张剑

Construction the vector of pacA and gtfB-cat of Streptococcus Mutans fused with ctxB，and expression in prokaryotic cells

  • Received:2011-07-29 Revised:2012-01-16 Published:2012-06-15 Online:2012-06-15

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摘要： 目的 拟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)，并做表达分析。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IPTG诱导后表达目的蛋白，分子量大小与预期一致。结论 成功构建了嵌合质粒pET-pacA-cat-ctxB, 且在原核细胞内正确表达。

关键词: 变形链球菌, 表面蛋白, 葡糖基转移酶, 霍乱毒素B亚单位, 原核表达

Abstract: To constructe 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 coli. Methods: Obtaining target genes pacA, gtfB-cat and ctxB with specific primers and templates of plasmids pcDNA3-pac, pET-gtfB and pBS-ctxB;Construction of intermediate vectors T-pacA, T-cat and T-ctxB through T-A cloning;T-ctxB and pET32-a(+) were digested with double restriction enzymes EcoRI and XhoI to construct the recombinant vector pET-ctxB, T-pacA and pET-ctxB were digested with KpnI and BamHI to construct the recombinant vector pET-pacA- ctxB. Finally, T-cat and pET-pacA-ctxB were digested with BamHI and EcoRI to construct the recombinant vector pET-pacA-cat-ctxB. Results: pacA, gtfB-cat and ctxB are all inserted into the vector pET32-a(+) correctly through the tests of restriction enzyme digestion, PCR and sequencing respectively. Their ORFs didn’t be changed. The recombinant plasmid can express target protein in E.coli host strain BL21 (DE3) after induced by IPTG, and the molecular weight of the protein were consistent with the prediction. Conclusion: The recombinant plasmid pET-pacA-cat-ctxB was successfully constructed and it can be expressed in prokaryotic cells.

Key words: Streptococcus mutans, surface protein, glucosyltransferase, cholera toxin B subunit, prokaryotic expression