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-1IPTG 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 BI21 （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- IFFG 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.
出处
《东南大学学报（医学版）》
CAS
2013年第4期394-398,共5页
Journal of Southeast University(Medical Science Edition)
基金
江苏省分子和功能影像重点实验室开放基金资助项目(PYZX2011001)
徐州市科技计划项目(XM12B038)
