血管生成抑制因子arresten基因原核表达载体的构建及其在大肠杆菌中的表达

目的:构建血管生成抑制因子arresten基因的原核表达载体, 并在大肠杆菌中进行表达。方法:利用聚合酶链式反应(PCR), 由我们构建的重组质粒pGEM-Arr中扩增出arresten基因;采用基因重组技术, 将该基因定向克隆于原核表达载体pRSET中, 转化大肠杆菌BL21(DE3), 用IPTG诱导表达, 并对表达产物行SDS-PAGE分析。结果:酶切鉴定和DNA测序证实arresten基因正确地插入表达载体中。重组arresten在大肠杆菌中获得高效表达, 其分子量约为26kD, 表达量约占菌体总蛋白量的30%。结论:Arresten基因原核表达载体的成功构建和重组arresten蛋白在大肠杆菌中的高效表达, 为进一步研究其生物学功能奠定了基础。

Construction of prokaryotic expression vector of human angiogenesis inhibitor arresten and its expression in E.coli

AIM: To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli. METHODS: Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis. RESULTS: Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION: The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.