变形链球菌SBR基因表达载体的构建和表达

Construction of a vector expressing SBR gene

目的 :构建可用于大肠杆菌表达系统的含变形链球菌唾液结合区段 (SBR)基因的表达载体。方法 :采用定向克隆方法将变形链球菌唾液结合区段 (SBR)基因插入表达载体 pcMVT7,构建重组原核表达质粒pcMVT7 SBR ,转化大肠杆菌JM 10 9(DE3 ) ,IPTG诱导表达 ,亲和层析纯化目的蛋白。结果 :经酶切鉴定及DNA序列测定 ,重组质粒 pcMVT7 SBR序列及阅读框架正确 ,亲和层析纯化得到SBR融合蛋白。 结论 :SBR表达载体构建成功 ,为防龋疫苗的动物实验研究奠定基础。

Objective: To obtain a prokaryotic expression vector containing saliva binding region (SBR) gene of Streptococcus mutans. Methods: By directional cloning method, SBR gene fragment was cloned into the expression vector pcMVT7, the recombinant plasmid pcMVT7-SBR was transformed to E.coli JM109 (DE3). The gene expression was induced with IPTG. Restriction endonuclease and DNA sequencing techniques were used to identify the recombinant plasmid DNA, and finally target protein was purified by affinity chromatography. Results:The DNA sequence of SBR in the reconstructed vector pcMVT7-SBR was in corresponding with the initial design. The C-terminal 6×His tagged SBR fusion protein was expressed in JM109(DE3) and was purified by affinity chromatography. The expression rate of target protein was 29.73%. Conclusion:The recombinant expression plasmid pcMVT7-SBR was constructed.