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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。
OBJECTIVE: To establish a prokaryotic expression vector for efficient expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein and to obtain a highly biologically active TRAIL protein. Methods Total RNA was extracted from peripheral blood of healthy volunteers. The extracellular region of TRAIL gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET30a. The positive clones were identified by double enzyme digestion, PCR and sequencing. Recombinant vector was transformed into competent cell BL21 to optimize the protein expression conditions; affinity chromatography purified recombinant protein; SDS-PAGE protein electrophoresis and Western blot identification. Recombinant proteins and TRAIL protein standards Huh-7 cells, CCK8, flow cytometry to detect the role of protein. Results DNA sequencing confirmed the successful construction of recombinant plasmid pET30a / TRAIL, SDS-PAGE protein electrophoresis, Western blot showed that pET30a / TRAIL induced soluble His-rTRAIL. CCK8, flow cytometry results showed that, compared with the standard TRAIL protein, His-rTRAIL Huh-7 cells have a good pro-apoptotic effect. Conclusion The prokaryotic expression vector pET30a / TRAIL, which efficiently expresses the soluble TRAIL protein, has been constructed successfully. His-rTRAIL has high biological function and provides a new experimental basis for the biological treatment of tumors.