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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。
Objective To investigate the pro-apoptotic effect of p53 P53c on SW480 mutant p53 cell line. Methods P53c was transfected into tumor cells using the protein transduction domain (TAT) of human immunodeficiency virus TAT protein and the stability of P53c in tissues was controlled by oxygen-dependent degradation region (ODD). The TAT-ODD-p53c (TOPc), TAT-p53c (TPc) and p53c genes were prepared by PCR and expressed in E. coli after they were ligated with the pGEX4T vector. The results showed that glutathione S-transferase (GST) P53c (GST-TOPc), GST-TAT-P53c (GST-TPc) and GST-P53c. The effect of TAT fusion protein on SW480 cell membrane was detected by immunohistochemistry. The effect of fusion protein on SW480 cell viability was detected by MTT assay. The apoptosis of SW480 cells was detected by flow cytometry. Results The pGEX series expression vector was successfully constructed and expressed in E. coli. Western blotting results showed that the recombinant protein could specifically bind to GST monoclonal antibody. Immunohistochemistry showed that both GST-TPc, GST-TOPcm and GST-TOPc could enter into SW480 cells. GST-TPc could significantly reduce the activity of SW480 cells and lead to the apoptosis of SW480 cells. ODD-containing fusion proteins have a mild pro-apoptotic effect on tumor cells under normoxic conditions. Conclusion TAT can mediate its fusion protein across the cell membrane. GST-TPc can induce p53 mutant tumor cell apoptosis.