小鼠组织因子原核质粒的构建和重组蛋白的表达

目的 体外扩增小鼠组织因子基因cDNA序列,构建小鼠组织因子重组原核表达质粒,在大肠杆菌中诱导表达.方法 根据基因库提供的基因序列设计克隆扩增小鼠组织因子cDNA基因序列的引物,利用原位反转录聚合酶链式反应(RT-PCR)技术扩增获得小鼠组织因子cDNA基因,通过内切酶酶切和T7酶连接,将小鼠组织因子cDNA序列插入原核表达质粒pQE30多克隆位点中.筛选的重组质粒经酶切和DNA序列测定证实正确后,转染感受态大肠杆菌,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并通过10%聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹法进行鉴定.结果 琼脂糖凝胶电泳发现扩增的小鼠组织因子cDNA序列相对分子量大小和预期值相一致,重组质粒酶切结果和预期值相符,DNA序列测定显示质粒的目的基因阅读框架准确无误.用IPTG诱导可以有效诱导重组蛋白质在大肠杆菌中表达,表达的重组蛋白质相对分子量与预期值相一致.结论 成功构建了小鼠组织因子的原核表达载体,并在大肠杆菌中诱导了有效表达,为将组织因子应用于治疗和功能研究奠定了基础.

Prokaryotic Vector Construction and Expression of Murine Tissue Factor

Objective To construct a prokaryotic vector encoding murine tissue factor and to express the recombinant tissue factor protein.Methods The cDNAs of murine tissue factor were amplified from total RNAs of murine livers.The corresponding prokaryotic expression vectors were constructed by restrictive endonuclease digestion and ligation,and then the recombinant vectors pQE-mTF were proofed by restricting enzymatic digestion and DNA sequencing.The confirmed vectors were transfected into E.coli JM109 and recombinant protein expression was induced by Isopropyl-β-D-thiogalactoside (IPTG).Results The recombinant expression vectors of murein tissue factor(pQE-mTF)were constructed successfully.The recombinant murine tissue factor protein was expressed stably in E.coli JM109.Conclusion Recombinant murine tissue factor lays a solid foundation for applying tissue factors to treatment and further functional study.