人肿瘤坏死因子相关凋亡诱导配体在大肠杆菌中的克隆、表达及纯化

[Cloning, expression and purification of human TNF-related apoptosis-inducing ligand in Escherichia coli].

作者信息

Yao Gen-hong, Luan Jian-feng, Ye Dong, Lei Qian-hong, Zhu Pei-yuan, Jin Jie, Hou Ya-yi

机构信息

Department of Transfusion, Jinling Hospital, Nanjing 210002, China.

出版信息

Wei Sheng Yan Jiu. 2006 Nov;35(6):697-700.

PMID:17290744

Abstract

OBJECTIVE

To clone human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, 114-281) cDNA and develop an inducible system for expression in E. coli.

METHODS

The human TRAIL (114-281) cDNA was amplified with the total RNA from the human peripheral blood monocytes by RT-PCR. The cDNA was inserted into pMD T vector. The selected integrants were confirmed by PCR screen, digestion with restriction enzymes and DNA sequencing. Then, the insert of human TRAIL cDNA was subcloned into prokaryotic expression vector pET28a. The construction of prokaryotic expression vector was proofed correct by RT-PCR and digestive identification. The recombinant protein was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside) . The sTRAIL inclusion bodies were refolded by dilution method. Refolded protein was purified with column chromatography.

RESULTS

Agarose gel electrophoresis of product of RT-PCR revealed a band around 500bp, which was expected. The positive integrants were confirmed by PCR screen and digestion with restriction enzymes. The same band as RT-PCR product was showed by PCR screen and digestion with restriction enzymes. Then the selected integrants were confirmed by DNA sequencing. The sequence was checked in GenBank. The construction of prokaryotic expression vector pET 28a was proofed correct by RT-PCR and digestive identification. TRAIL protein was successfully induced by IPTG in E. coli BL21. The results also showed that the protein was expressed as inclusion bodies. After the sTRAIL inclusion bodies were solubilized and refolded, the protein expressed was purified with one band, which was about 20kD, analyzed by SDS-PAGE.

CONCLUSION

These results suggested that the hsTRAIL was cloned, expressed and purified in the present study. Significant quantities of TRAIL produced by this method should allow further studies in determining the physiological significance and function of TRAIL in the future.

摘要

目的

克隆人肿瘤坏死因子相关凋亡诱导配体（TRAIL，114 - 281）cDNA，并构建一个可在大肠杆菌中诱导表达的系统。

方法

采用逆转录聚合酶链反应（RT-PCR）从人外周血单核细胞的总RNA中扩增出人TRAIL（114 - 281）cDNA。将该cDNA插入pMD T载体。通过PCR筛选、限制性内切酶消化及DNA测序对筛选出的整合体进行鉴定。然后，将人TRAIL cDNA插入原核表达载体pET28a。通过RT-PCR和酶切鉴定证明原核表达载体构建正确。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组蛋白表达。采用稀释法对可溶性TRAIL包涵体进行复性。用柱层析法纯化复性后的蛋白。

结果

RT-PCR产物的琼脂糖凝胶电泳显示出一条约500bp的条带，符合预期。通过PCR筛选和限制性内切酶消化鉴定出阳性整合体。PCR筛选和限制性内切酶消化显示出与RT-PCR产物相同的条带。然后通过DNA测序对筛选出的整合体进行确认。将该序列在GenBank中进行比对。通过RT-PCR和酶切鉴定证明原核表达载体pET 28a构建正确。IPTG成功诱导大肠杆菌BL21表达TRAIL蛋白。结果还表明该蛋白以包涵体形式表达。可溶性TRAIL包涵体溶解并复性后，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，纯化后的蛋白呈现一条约20kD的条带。