MDM2/4-p53双靶点抑制蛋白原核表达载体的构建与蛋白制备  

The construction of prokaryotic expression vector and preparation of MDM2 /4- p53 dualtarget inhibitory protein

作　　者：董丹凤[1] 王举波[2] 吴胤瑛[1] 耿倩倩[1] 李恩孝[1] 李毅[1] 

机构地区：[1]西安交通大学第一附属医院肿瘤内科,陕西西安7100612 [2]西安交通大学第二附属医院神经外科,陕西西安710004

出　　处：《现代肿瘤医学》2016年第6期865-870,共6页Journal of Modern Oncology

基　　金：博士点基金新教师类项目(编号:20130201120079);陕西省攻关项目(编号:S2013SF3846)

摘　　要：目的:采用分子克隆技术和大肠杆菌原核表达系统制备具备生物学活性MDM2/4-p53双靶点抑制蛋白。方法:首先应用PCR合成大肠杆菌硫氧还蛋白(thioredoxin,Trx)A的c DNA序列,将其插入到大肠杆菌原核表达载体p ET40b中,将人工合成的人免疫缺陷病毒(human immuno-deficiency virus,HIV-1)反式激活蛋白(transactivator,TAT)的蛋白转导结构域(protein transduction domain,PTD)和MDM2/4双抑制肽p DI(peptide double inhibition)的c DNA序列分别插入到Trx序列的C端和核心位点,从而构建出可表达MDM2/4-p53双靶点抑制蛋白的原核表达载体p ET-TAT-Trx A-p DI。然后将p ET-TAT-Trx A-p DI原核表达载体转化大肠杆菌表达菌种BL21感受态细胞,应用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导蛋白表达,筛选最优表达的单克隆菌种,分析重组蛋白的表达部位,并了解不同IPTG浓度(0.1mmol/L、0.4mmol/L、0.7mmol/L、1.0mmol/L)、温度(16℃、25℃、37℃)、表达诱导时间(1h、2h、3h、4h、5h、6h、7h)对MDM2/4-p53双靶点抑制蛋白表达的影响,以获得最优的蛋白表达条件。最终利用MDM2/4-p53双靶点抑制蛋白携带的His标签,采用能够特异性结合His标签的蛋白纯化柱进行蛋白纯化,并应用梯度透析法和氧化还原法进行MDM2/4-p53双靶点抑制蛋白的体外折叠。结果:成功构建可以表达MDM2/4-p53双靶点抑制蛋白的原核表达载体p ET-TAT-Trx A-p DI,并实现MDM2/4-p53双靶点抑制蛋白在大肠杆菌表达菌种BL21(DE3)中的高效表达,表达量占总蛋白表达量的60%以上,并且主要存在于包涵体中。蛋白表达条件优化结果发现,温度对重组蛋白表达无明显影响,而应用0.4mmol/L的IPTG浓度和3h的诱导时间即可获得重组蛋白的高效表达。经过蛋白纯化后获得纯度99.99%的重组蛋白,将纯化的MDM2/MDM4双靶点抑制蛋白溶液用梯度透析复性和氧化还原复性法最终成功获得包涵体蛋白的体外正确�Objective： To prepare MDM2 /4- p53 dual- target inhibitory protein with activity by molecular cloning and E. coli prokaryotic expression technology. Methods： Thioredoxin（ Trx） A was amplificated via PCR and inserted into the prokaryotic expression vector p ET40 b. The protein transduction domain（ PTD） and the small molecule MDM2 /4- p53 inhibitory peptide p DI（ peptide double inhibition） were synthesized according to the reported sequence. Then the prokaryotic expression vector,which could express MDM2 /4- p53 dual- target inhibitory protein,was constructed by separately inserting PTD and p DI into the C- terminal and active site of Trx A. To obtain high purity and biological MDM2 /4- p53 dual- target inhibitory protein,the expression of recombinant protein was induced by IPTG in E. coli BL21 cells,the optimal expression of recombinant protein expression was analyzed,and the optimal expression condition was selected in different IPTG concentration（ 0. 1 tendency,the tendency for 0. 4L and 0. 4L tendency tendency for L,1L）,temperature（ 16℃,25℃ and 37℃）,induction time（ 1h,2h,3h,4h,5h,6h,7h）. Then it was purified via His tag protein purification system and refolded via dialysis and redox system. Results： The prokaryotic expression vector p ET- TAT- Trx A- p DI expressing MDM2 /4- p53 dual- target inhibitory protein was successfully constructed and transformed into E. coli BL21 cells,finally more than 60% MDM2 /4- p53 dual- target inhibitory protein was obtained. The optimal condition of protein expression was 0. 4mmol / L IPTG concentration and 3hours induction time. Then the MDM2 /4- p53 dual- target inhibitory protein was purified via His tag protein purification system and refolded via dialysis and redox system. The purity and the refolding rate of the recombinant protein after separately were over 99% and 80% respectively. Conclusion： The MDM2 /4- p53 dual- target inhibitory protein with activity was successfully prepared by molecular cloning and E. coli prokaryotic expressi

关 键 词：鼠双微体2 鼠双微体4 P53 硫氧还蛋白 蛋白转导域 

分 类 号：R73-3[医药卫生—肿瘤]