肿瘤坏死因子相关凋亡诱导配体蛋白表达及条件优化-东南大学学报医学版

肿瘤坏死因子相关凋亡诱导配体蛋白表达及条件优化

单位：1. 东南大学医学院附属徐州医院泌尿外科, 江苏徐州 221009;
2. 徐州医学院病原生物学与免疫学教研室, 江苏徐州 221004

分类号：Q78

出版年·卷·期（页码）：2013·32·第四期（394-398）

摘要：

目的: 优化肿瘤坏死因子相关凋亡诱导配体(TRAIL)蛋白表达条件,探索包涵体复性和纯化方法。方法: 用IPTG诱导pGEX-6P-1/TRAIL在大肠杆菌BL21(DE3)中表达,表达产物经稀释复性和透析复性法使GST-rTRAIL蛋白恢复天然构象;并运用Glutathione-Superflow Resin亲和层析柱纯化GST-rTRAIL目的蛋白,Western blotting分析目的蛋白。结果: GST-rTRAIL在大肠杆菌BL21(DE3)中表达约40 kDa的蛋白条带,与预期值相符,该蛋白以包涵体形式存在;0.2 mmol·L^-1 IPTG 37℃诱导8 h时,全菌蛋白表达量最高;复性后的蛋白溶液经Glutathione-Superflow Resin亲和层析纯化获得纯的目的蛋白,Western blotting显示能被鼠抗GST的标签抗体识别。结论: 本实验结果显示IPTG浓度为0.2 mmol·L^-1,37℃诱导8 h,GST-rTRAIL全菌蛋白的表达量最高;GST-rTRAIL蛋白包涵体经稀释复性、透析复性和Glutathione-Superflow Resin亲和层析柱分离纯化获得目的蛋白,为进一步研究其生物学功能及临床应用奠定基础。

Objective: To optimize the expressed condition of human tumor necrosis factor-related apoptosis inducing ligand(TRAIL) and search for the method of renaturalization and purification of inclusion body. Methods: pGEX-6P-1/TRAIL was induced by IPTG in E.coli BL21(DE3)and analyzed by SDS-PAGE. The protein existing in a form of inclusion body was denaturalized and then renaturalized by dilution and dialysis. The protein solution was purified by Glutathione-Superflow Resin affinity chromatography, then identified by Western blotting.Results: The molecular weight of TRAIL expressed in E.coli BL21(DE3)was approximately 40 kDa. GST-rTRAIL was highly expressed in a form of inclusion body. Total bacterial protein induced by 0.2 mmol·L^-1 IPTG for 8 h in 37℃ was the optimum condition for the expression. The purified GST-rTRAIL was obtained by Glutathione-Superflow Resin affinity chromatography. Western blotting revealed that it could be recognized by mouse monoclonal antibody to GST. Conclusion: In the research,it is revealed that the expression of total bacterial protein is the optimum condition when it is induced by 0.2 mmol·L^-1 IPTG for 8 h in 37℃. Inclusion body is refolded by dilution and dialysis, purified by Glutathione-Superflow Resin affinity chromatography. It will lay a foundation of developing biological function and clinical application.

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