突变型PUMA质粒的构建及表达  

Construction of expression plasmid of the mutant PUMA and its expression in Hela cells

作　　者：黄伟[1] 万福生[2] 

机构地区：[1]南昌大学生物化学与分子生物学教研室,南昌330006 [2]南昌大学实验动物科学部,南昌330006

出　　处：《安徽医科大学学报》2013年第4期341-345,共5页Acta Universitatis Medicinalis Anhui

基　　金：江西省自然科学基金(编号:2010JZY0237)

摘　　要：目的构建促凋亡基因p53上调凋亡调制物(PU-MA)的真核表达载体和针对其磷酸化位点定点突变的真核表达载体,为进一步研究其功能奠定基础。方法通过PCR方法从pCEP4-(HA)2-PUMA质粒中扩增PUMA基因,并克隆到pIRES2-EGFP载体上,构建PUMA真核表达载体pIRES2-EGFP-(HA)2-PUMA,利用定点突变技术构建pIRES2-EGFP-(HA)2-PUMA-T28G,行PCR及测序鉴定;脂质体分别将两种质粒转染Hela细胞,同时设未转染的阴性对照组及转染空载体组(4个实验组);PI染色观察PUMA对细胞的促凋亡作用;流式细胞仪检测细胞凋亡率;RT-PCR检测PUMA的表达;Western blot检测突变型PUMA蛋白和标签蛋白的表达。结果经PCR及测序结果表明,正确构建野生型、突变型PUMA重组质粒,PUMA基因第10位氨基酸的第28～30位碱基由丝氨酸(TCC)突变为丙氨酸(GCC),其他碱基均无突变;野生型、突变型PUMA重组质粒转染Hela细胞荧光显微镜观察到绿色荧光,而PI染色观察到野生型、突变型PUMA对Hela细胞的促凋亡作用;流式细胞术检测结果显示突变型和野生型PUMA转染组凋亡率高于阴性对照组和空载体转染组;Western blot和RT-PCR检测出目的基因的高表达。结论成功构建了PUMA基因及其定点突变载体,为深入研究PUMA基因的抑癌功能及其翻译后调节奠定基础。Objective To construct the eukaryotic expression vector of a pro-apoptotic gene PUMA （P53-upregulat- ed modifier of apoptosis） and the site-directed mutagenesis vector for the further functional study. Methods PUMA was amplified by PCR from plasmid of pCEP4-（ HA）E-PUMA and subsequently cloned into the eukaryotic vector pIRES2-EGFP to generate pIRES2-EGFP- （ HA ） E-PUMA. pIRES2-EGFP- （ HA ） 2-PUMA-T28 G was constructed through site-directed mutagenesis. Sequence of the inserted gene was identified by Plasmid PCR and analyzed by sequencing subsequently. Hela cells were divided into 4 groups：Wild type PUMA transfection group, mutant PUMA transfection group, the empty vector control group, and the control group. Characteristic morphological changes of ap- optosis were observed by PI staining. The cellular apoptosis rates were examined by FCM. The expression of PUMA was detected by Western blot and RT-PCR. Results DNA sequencing showed that TCC ,which encoded the $10 of PUMA,was changed to GCC ;there were no mutation sites in other codons of PUMA in pIRESE-EGFP-（HA） E-PU- MA-T28G recombinant plasmid. High expression of EGFP was detected by fluorescence microscope and PI dyeing displayed that HeLa cells had a series of apoptosis morphologic changes after transfecting Wild type and mutant PU- MA into HeLa cells. The rate of apoptosis by FCM was significantly higher in the experimental group compared with the empty vector control group and the control group. Western blot and RT-PCR displayed the high expression of PUMA gene. Conclusion The eukaryotic expression vectors for PUMA and its mutant can be successfully construc- ted, which may pave the way for further study on tumor suppressor function of PUMA and its post-translational regu- lation mechanism.

关 键 词：p53上调凋亡调制物 PIRES2-EGFP 定点突变 真核表达载体 

分 类 号：R737.33[医药卫生—肿瘤] R329.2[医药卫生—临床医学]