人p53不同突变体的构建、表达及其对亚砷酸盐诱导细胞凋亡的影响  

Construction and expression of different mutants of human p53 and their effects on arsenite-induced cell apoptosis

作　　者：刘爱华[1] 龚小卫[2] 魏洁[2] 明小燕[2] 王达安[2] 邓鹏[2] 罗深秋[3] 姜勇[2] 

机构地区：[1]南方医科大学南方医院呼吸科,广东广州510515 [2]南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东广州510515 [3]南方医科大学细胞生物学教研室,广东广州510515

出　　处：《南方医科大学学报》2008年第5期671-674,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30572151);国家自然科学基金委员会-广东省人民政府自然科学联合基金(U0632004);广东省自然科学基金(05004730)~~

摘　　要：目的构建人p53的不同突变体,并在真核细胞中表达,研究这些突变体对应激刺激诱导细胞凋亡的影响。方法采用PCR方法扩增人p53 cDNA序列,使用常规酶切、连接方法将其重组至pcDNA3/HA载体中,对阳性克隆进行酶切和PCR鉴定。将阳性重组体的第15位和第46位丝氨酸突变为丙氨酸,并转染NIH3T3细胞,观察其在NIH3T3细胞内的表达情况。转染不同突变体的NIH3T3细胞用Annexin V-FITC和碘化丙啶双标后,利用流式细胞术检测亚砷酸盐刺激前后的凋亡情况。结果构建的pcDNA3/HA-p53(WT)、pcDNA3/HA-p53(S15A)和pcDNA3/HA-p53(S46A)是正确的,且能在NIH3T3细胞内有效表达。流式细胞术检测表明,p53(WT)的表达使亚砷酸盐诱导的细胞凋亡增加,p53(S15A)的表达使细胞凋亡减少,而p53(S46A)没有明显影响。结论p53的第15位丝氨酸在其介导亚砷酸盐诱导的细胞凋亡中具有重要作用。Objective To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis. Methods Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Set^15 and Ser^15 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry. Results The recombinant plasmids of HA-tagged wild-type p53, HA-p53（WT）, and its mutants, HA-p53（S15A） and HA-p53（S46A）, were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53 （WT）-transfected cells induced by arsenite increased and that of p53 （S15A）-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53 （S46A）-transfected cells. Conclusion The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.

关 键 词：P53 定点突变 载体构建 基因表达 细胞凋亡 亚砷酸盐 

分 类 号：Q78[生物学—分子生物学]