人血管生成抑制因子canstatin原核表达载体的构建及表达

Construction of prokaryotic expression vector for Chinese canstatin and its expression

目的 :构建国人血管生成抑制因子canstatin的原核表达载体并在大肠杆菌BL2 1中表达canstatin重组蛋白。方法 :用Trizol试剂提取人肝脏组织总RNA ,通过RT -PCR扩增canstatin的cDNA ,克隆到pMD1 8-T载体中并进行序列分析。将canstatincDNA定向克隆于原核表达载体pET30a(+)中 ,而后在大肠杆菌BL2 1中经IPTG诱导表达。结果 :canstatincDNA克隆入质粒pET30a(+)获得了重组表达载体pET30a(+) /Cans,canstatin的cDNA长度为 6 84bp ,编码 2 2 7个氨基酸。IPTG诱导原核表达载体pET30a(+) /Cans在大肠杆菌BL2 1中的表达量约占菌体总蛋白量的 35 %。结论 :原核表达载体pET30a(+) /hCans在大肠杆菌BL2

Aim:To construct prokaryotic expression vector for human canstatin and to express recombinant canstatin in E.coli BL21. Methods: Total RNA was extracted from fresh liver tissue by Trizol Reagent, and human canstatin cDNA was amplified by RT-PCR, then cloned into vector pMD18-T for sequencing. Prokaryotic expression vector pET30a(+)/hCans was constructed and expressed in E.coli BL21 with induction of IPTG. Resaults: In this study, Chinese canstatin cDNA cloned was 684bp in length encoding 227 amino acids and prokaryotic expression vector pET30a(+)/hCans was expressed in E.coli BL21 with amount of 35% of the total bacterial protein after being induced by IPTG for 4 h. Conclution: Constructed pET30a(+)/hCans is efficiently expressed in E.coli BL21. It is required for the further research on its anti-angiogenic activity and potential application for cancer therapy.