【作者】 王静；

【导师】 罗志军； 尹东；

【作者基本信息】 南昌大学 ， 生物化学和分子生物学， 2008， 硕士

【摘要】 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:①用CaCl2法制备感受态细菌JM109,质粒pGEX-KG-gAD转化大肠杆菌JM109,空载质粒pGEX-KG作为内对照同时转化JM109,筛选建株;②以正交设计软件正交设计助手Ⅱ选择L9(34)正交设计表在三个水平,四个因素(IPTG浓度,诱导时间,诱导温度,细菌密度OD600)下确定优化表达的最佳组合条件;③亲和层析法纯化GST-gAD;④用MTT法检测融合蛋白GST-gAD对人乳腺癌细胞MDA-MB-231增殖的影响。结果:①重组质粒pGEX-KG-gAD成功转化大肠杆菌JM109;②对GST-gAD诱导表达影响最大的因素为温度,最佳诱导表达组合条件为温度32℃,IPTG的浓度1.0 mmol/L,诱导前细菌的生长密度OD600 1.0,诱导表达时间1.5h,此条件下其表达量占菌体总蛋白的6 %左右;③获得纯化的分子量为42KD的GST-gAD;④GST-gAD浓度高于0.5μmol/L时对人乳腺癌细胞MDA-MB-231的增殖具有明显的抑制作用结论:①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;②GST-gAD融合蛋白在原核表达系统中可以有效表达,并且具有较好的生物学活性。更多还原

【Abstract】 Objective: To explore the best expressing condition of the recombinant GST-gAD( globular domain of human adiponection,gAD) in E.coli and study its bioactivity in inhibiting the proliferation of human breast cancer cell MDA-MB-231.Methods:①Competent bacterium JM109 was prepared with CaCl2. The plasmid pGEX-KG-gAD and the empty plasmid pGEX-KG, as control, were transformed into E.coli host JM109;②The best expressing condition of GST-gAD was explored through orthogonal design which took L 9(34),including: growth temperature, IPTG concentration,culture density,incubation time as indices by orthogonal design assistantⅡ;③GST- gAD and GST were purified by GSH-agarose;④The effect of GST—gAD on proliferation of MDA-MB-231 cells were measured by MTT essay.Results:①The pGEX-KG-gAD recombinant vector was transformed successfully into E.coli host JM109;②The expression level was highest in the conditions of OD600 value 1.0,1.0mmol/L IPTG, 32℃and 1.5 hours of induction time. The amount of the fusion protein was about 6% of the total bacterial protein;③The molecular weight of GST-gAD was 42KD;④The GST-gAD can obviously inhibit the proliferation of MDA-MB-231 when its concentration was more than 0.5μmol/L. Conclusion:①The best expressing condition of the recombinant GST-gAD in Ecoli was determined through orthogonal design;②The GST-gAD fusion protein was efficiently expressed in E.coli and showed natural biological activities.更多还原