人TRAIL细胞外段基因在大肠杆菌中的表达及其包涵体的复性

目的　在大肠杆菌中表达人肿瘤坏死因子相关凋亡诱导配体细胞外段 (sTRAIL)基因 ,并研究其包涵体的复性。方法　应用RT- PCR法从正常人外周血单核细胞中获得sTRAIL的编码基因 ,将其克隆到原核表达载体pBV2 2 0 ,于大肠杆菌DH5α中通过温控诱导表达 ;将包涵体进行变性和复性处理后 ,采用离子交换层析纯化表达产物 ,并经Dotblot及MTT试验鉴定。结果　通过温度诱导 ,目的基因主要以包涵体形式高效表达 ,包涵体蛋白经梯度透析法复性可获得具有抗肿瘤活性的重组人sTRAIL。结论　已获得大量纯化重组人sTRAIL ,为开发新型抗肿瘤制剂奠定基础。

Expression of Extracellular Domain Gene of Human TRAIL in E. coli and Re naturalization of Inclusion Body

Objective To express the extracellular domain gen e of human tumor necrosis factor-related apoptosis inducing ligand (sTRAIL) in E.coli and study the re-naturalization of inclusion body.Methods Amplify the gene encoding sTRAIL from normal human peripheral blood monon uclear cells by RT-PCR,clone into prokaryotic expression vector pBV220 and tran sform to E.coli for expression under induction of temperature.The expressed product existing in a form of inclusion body was de-naturalized,re-naturalized and purified by ion exchange chromatography,then identified by dot blot and MTT test.Results sTRAIL was highly expressed in a form of incl usion body and showed anti-tumor activity after re-naturalization by gradient dialysis.Conclusion A large quantity of purified recombinant hu man sTRAIL was obtained.It laid a foundation of developing novel anti-tumor dru gs.