GST-TAT-P53c融合蛋白的表达及对p53基因突变型肿瘤细胞的促凋亡作用  

Expression of GST-TAT-P53c fusion protein and its promotive action on apoptosis of tumor cells with mutational p53 gene

作　　者：吴少平[1] 王玉霞[1] 武军华[1] 贾培媛[1] 王晨宇[1] 李前[1] 孙曼霁[1] 

机构地区：[1]军事医学科学院毒物药物研究所

出　　处：《中国药理学与毒理学杂志》2007年第1期28-33,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目(35072207)~~

摘　　要：目的研究P53蛋白C端(P53c)对p53基因突变型肿瘤细胞SW480的促凋亡作用。方法用人类免疫缺陷病毒TAT蛋白的蛋白转导域(TAT)将P53c运载进入肿瘤细胞,用氧依赖性降解区域(ODD)控制P53c在组织中的稳定性。通过PCR方法制备TAT-ODD-p53c(TOPc),TAT-p53c(TPc)及p53c基因,与pGEX4T载体连接后在大肠杆菌中表达谷胱甘肽-S-转移酶(GST)-TAT-ODD-P53c(GST-TOPc),GST-TAT-P53c(GST-TPc)及GST-P53c等融合蛋白。免疫组化方法检测TAT蛋白运载融合蛋白穿过SW480细胞膜的作用,MTT法检测融合蛋白对SW480细胞活力的影响,流式细胞仪检测致SW480细胞凋亡作用。结果成功构建pGEX系列表达载体,并在大肠杆菌中可溶性表达。Western印迹结果表明,重组蛋白可以和GST单克隆抗体特异性结合。免疫组化显示GST-TPc,GST-TOPcm及GST-TOPc均能进入SW480细胞,GST-TPc能明显降低SW480细胞活性,导致SW480细胞凋亡。含ODD的融合蛋白在常氧环境中对肿瘤细胞有轻度促凋亡作用。结论TAT可以介导其融合蛋白跨越细胞膜。GST-TPc可引起p53突变型肿瘤细胞凋亡。AIM To investigate the apoptosis- promotive action of the P53 protein C-terminus （P53c） agsinst tumors with p53 gene mutation. METHODS Protein transduction domain of human immunodeficiency virus TAT protein （TAT） was used to deliver P53c into tumor cells, and the oxygen-dependent degradation domain （ODD） was used to control the stability of P53c in tissue. TAT-p53c （TPc） gene was constructed by PCR, and cloned into the pGEX4T vector. The fusion proteins of glutathi-one-S- transferase （ GST ）-TAT-ODD-P53c （ GST-TOPc ） and GST-TAT-P53c （ GST- TPc ）, etc, were expressed in BL21 （ DE3 ） and purified by affinity column. Immunohistochemistry analysis was used to determine the transduction of TAT fusion protein into the SW480 cells. The viability of SW480 cells treated with the fusion proteins were measured by the MTT method, and the flow cytometry was employed to analyze the apoptosis of SW480 cells caused by the fusion proteins. RESULTS A series of pGEX vectors were constructed, and the soluble proteins were expressed in E. coli. The result of Western blot showed that the fusion pro- teins well immunoreacted with the mouse anti- Schistosoma japonica GST monoclonal antibody. Immunohistochemistry analysis showed that GST-TPc, GST-TOPcm and GST-TOPc could all permeate into the SW480 cells. GST-TPc obviously decreased the viability of SW480 cells and led to the cell apoptosis in vitro. The fusion proteins bearing the ODD domain exhibits mild apoptosis-promoting action on the tumor cells under the aerobic condition. CONCLUSION TAT mediates the fusion proteins accross the cell membrane. GST-TPc induces the apoptosis of the P53 mutated-tumor cells.

关 键 词：蛋白质P53 蛋白转导域 氧依赖性降解域 细胞凋亡 

分 类 号：R730.5[医药卫生—肿瘤]