GST-gAD在大肠杆菌中的优化表达及其生物活性测定

Optimizing Expression in E.coli and Bioactivity Assay of Recombinant GST-gAD

目的探讨可溶性重组脂联素球状结构域融合蛋白(GST-gAD)在大肠杆菌中的优化表达及其抑制人乳腺癌细胞MDA-MB-231增殖的生物学活性。方法用含有GST-gAD基因的原核表达载体pGEX-KG-gAD转化E.coli J M109,同理空载质粒pGEX-KG作为内对照同时转化E.coli J M109;将影响融合蛋白表达的4个因素,温度、IPTG浓度、细菌密度OD600和诱导时间进行正交设计,确定其优化表达条件,用亲和层析法纯化GST-gAD;用MTT法检测GST-gAD对MDA-MB-231增殖的影响。结果GST-gAD最佳表达条件为温度32℃,IPTG的浓度1.0mmol/L,诱导前细菌密度OD6001.0,诱导表达时间1.5h。GST-gAD浓度高于0.5μmol/L时对MDA-MB-231细胞的生长具有明显的抑制作用。结论通过正交设计确定了可溶性GST-gAD融合蛋白在大肠杆菌中表达的优化条件并且当重组融合蛋白在大于0.5μmol/L时对MDA-MB-231细胞的生长具有明显的抑制作用。

Objective To explore the best expressing condition of the recombinan human globular domain of human adiponection（gAD） and study its bioactivity. Methods The plasmid pGEX- KG-gAD and the empty plasmid pGEX-KG, as control, were transformated into E. eoli host JM109. The best expressing condition of GST-gAD was explored through orthogonal design which took growth temperature, IPTG concentration, culture density,incubation time as indices. The expressed products（GST-gAD） proteins were purified from supernatant bacterial lysates by affinity chromatography using reduced glutathione resin. The effect of GST-gAD on proliferation of MDA-MB-231 cells were measured by MTT essay. Results The expression level was the highest in the conditions of OD600 value 1.0,1.0 mmol/L IPTG,under 32 ℃ and 1.5 hours of induction time. The growth ability of MDA-MB-231 cells were obviously Inhibited when concentration of GST-gAD was higher than 0.5/,mol/L. Conclusion The best expressing condition of the recombinant GST-gAD in E. coli was determined through orthogonal design and GST-gAD show natural biological activities.