TRAIL分子胞外区基因工程蛋白的制备与鉴定

The cloning and expression of human outer-membrane region of TRAIL in E. Coli

目的 制备人TRAIL配体胞外区基因工程蛋白。方法 应用RT PCR技术从激活的人外周血淋巴细胞总RNA中扩增TRAIL配体胞外区cDNA ,克隆入PCR2 .1 载体 ,测序验证后用基因重组法构建了TRAIL配体胞外区和组氨酸盒的原核表达质粒pQE hTRAIL。将重组质粒转入大肠杆菌M15中表达 ,经 1mmol LIPTG在 37℃条件下诱导 4h ,亲和层析柱纯化。用SDS -PAGE电泳和Westernblot鉴定表达产物。结果 所表达融合蛋白为人TRAIL配体胞外区分子 ,分子量 30kD。表达量约为 2mg ml。结论 人TRAIL配体胞外区在大肠杆菌中得到良好表达 ,为深入研究TRAIL配体分子在肿瘤与自身免疫病中的可能应用提供了材料。

Objective To obtain recombinant human outer-membrane region of TRAIL molecule.Methods RT-PCR was applied to amplify cDNA of outer-membrane region of TRAIL molecule from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR 2.1 vector. After sequencing, the cDNA of outer-membrane region of TRAIL molecule was inserted into pQE vector and expressed in E. Coli M15. The protein was purified through affinity chromatography column with ligand of 6 X His tag and identified by SDS-PAGE and Western Blotting.Results The protein expressed was purify with one band which was 30 KD analyzed by SDS-PAGE. The expressing protein was outer-membrane region of TRAIL molecule which identified by Western-blot.Conclusion The fusion human outer-membrane region of TRAIL molecule was obtained. It lays the foundation for the further studying.