人可溶性TRAIL原核表达载体的构建和蛋白表达、纯化及活性鉴定
Construction of prokaryotic expression vector of human soluble TNF-related apoptosis inducing ligand and studies of the expression, purification and activity identification of recombinant protein
尤红娟 1刘雯 1赵金金 1徐志辉 1党景东 1刘转转 1郑葵阳 1汤仁仙1
作者信息
- 1. 徐州医学院 病原生物学与免疫学教研室 江苏徐州221004
- 折叠
摘要
目的 建立高效表达可溶性肿瘤坏死因子相关凋亡诱导配体(TRAIL)蛋白的原核表达载体,获得高生物学活性的TRAIL蛋白.方法 取健康人外周血提取总RNA,RT-PCR扩增TRAIL基因的胞外区片段,克隆入原核表达载体pET30a中,经双酶切、PCR及测序鉴定阳性克隆.重组载体转化感受态细胞BL21,优化蛋白表达条件；亲和层析法纯化重组蛋白；SDS-PAGE蛋白电泳、Western blot鉴定.重组蛋白与TRAIL蛋白标准品分别作用Huh-7细胞,CCK8、流式细胞术检测蛋白生物学作用.结果 DNA测序结果证实成功构建重组质粒pET30a/TRAIL,SDS-PAGE蛋白电泳、Western blot显示pET30a/TRAIL诱导的为可溶性的His-rTRAIL.CCK8、流式细胞术检测结果显示,与TRAIL蛋白标准品相比,His-rTRAIL对Huh-7细胞具有良好的促凋亡作用.结论 成功构建高效表达可溶性TRAIL蛋白的原核表达载体pET30a/TRAIL,表达的可溶性蛋白His-rTRAIL具有高度的生物学作用,为肿瘤的生物学治疗提供新的实验依据.
Abstract
Objective To construct the TNF-related apoptosis inducing ligand ( TRAIL) gene prokaryotic expression vector expressing soluble protein with high activity. Methods Genomic RNA was extracted from the peripheral blood of health adults. The extracellular domain region of TRAIL gene was amplified with RT-PCR and cloned into the prokaryotic expression vector pET30a. The recombinant was confirmed by EcoR I/ Xho I digestion, PCR, and DNA sequencing. Meanwhile, the recombinant was induced into E. coli BL21. Recombinant protein was analyzed by SDS-PAGE and Western blot. Huh-7 cells were stimulated by recombinant protein and standard protein respectively to compare their bioactivity by CCK8 and FCM. Result pET30a/TRAIL was successfully constructed and soluable His-rTRAIL could be expressed and identified by SDS-PAGE and Western blot analysis. The results of CCK8 and FCM showed that His-rTRAIL could induce apoptosis of Hch-7 cells. Conclusion A prokaryotic expression vector pET30a/TRAIL was constructed successfully, which expressed soluble protein with high bioactivity. This provided a new idea for tumor therapy.
关键词
肿瘤坏死因子相关凋亡诱导配体/克隆/原核表达/凋亡Key words
TRAIL/cloning/prokaryotic expression/apoptosis引用本文复制引用
基金项目
江苏省高校自然科学基金(10KJD310002)
出版年
2012
NETL
NSTL
维普
万方数据