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

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

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。

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. [