CAJ | 학술논문

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。
목적：우화종류배사인자상관조망유도배체（ TRAIL ）단백표체조건，탐색포함체복성화순화방법。방법：용IPTG유도pGEX-6P-1/TRAIL재대장간균BL21（DE3）중표체，표체산물경희석복성화투석복성법사GST-rTRAIL단백회복천연구상；병운용Glutathione-Superflow Resin친화층석주순화GST-rTRAIL목적단백，Western blotting분석목적단백。결과：GST-rTRAIL재대장간균BL21（ DE3）중표체약40 kDa적단백조대，여예기치상부，해단백이포함체형식존재；0．2 mmol· L-1 IPTG 37℃유도8 h시，전균단백표체량최고；복성후적단백용액경Glutathione-Superflow Resin친화층석순화획득순적목적단백，Western blotting현시능피서항GST적표첨항체식별。결론：본실험결과현시IPTG농도위0．2 mmol· L-1，37℃유도8 h，GST-rTRAIL전균단백적표체량최고；GST-rTRAIL단백포함체경희석복성、투석복성화Glutathione-Superflow Resin친화층석주분리순화획득목적단백，위진일보연구기생물학공능급림상응용전정기출。
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 .