A族链球菌类胶原样蛋白Scl1的原核表达及纯化

Prokaryotic expression and purification of Scl1 protein

目的构建A族链球菌Scl1的重组体并进行原核表达及表达蛋白的纯化,为进一步研究该蛋白的空间结构及其与A族链球菌所引起的多种疾病之间的关系奠定基础。方法根据A族链球菌M1型国际标准菌株SF370基因组设计Scl1基因扩增引物,通过聚合酶链反应(PCR)获得Scl1目的基因,与原核表达载体pet28a分别通过EcoRⅠ和HindⅢ双酶切后进行连接,构建重组质粒Scl1-pet28a,并进行序列测定,与原序列比对确定序列的一致性。将Scl1-pet28a转入E.coli BL21中,用IPTG诱导后经MALDI-TOF-MSMS质谱鉴定Scl1的表达,用AKTA全自动蛋白纯化系统对表达蛋白进行纯化。结果获得序列正确的Scl1基因及原核重组表达质粒Scl1-pet28a,MALDI-TOF-MSMS证实重组质粒能够在E.coli BL21中表达,纯化的表达产物为分子质量单位约50ku的单一Scl1蛋白。结论成功构建了Scl1原核表达载体,在E.coli中表达出目的蛋白并获得纯化蛋白Scl1。

Objective To construct a recombinant plasmid expressing Scl1 protein and to purify the Scl1 protein.Methods The complete gene sequence coding for Scl1 was obtained by PCR from M1 type strain SF370,and the gene was cloned into the expression vector pet28a.Recombinant expression plasmid was transformed into BL21 and induced by IPTG to express recombinant Scl1 protein.The expressed protein was identified using MALDI-TOF-MSMS and purified by AKTA automated protein purification system.Results pet28a-Scl1,a recombinant Escherichia coli-expressing plasmid,was constructed.MALDI-TOF-MSMS analysis confirmed that E.coli BL21 transformed with recombinant Scl1 plasmid expressed recombinant Scl1 protein and the protein was successfully purified.Conclusion A prokaryotic expression vector of Scl1 was successfully constructed.The recombinant protein was expressed in E.coli BL21 and purified to obtain specific target protein.