人Cdc25C基因克隆及其原核表达载体的构建与表达  

Cloning and prokaryotic expression of human Cdc25C

作　　者：卓少元[1] 陈承晓[2] 钟卫干[1] 农蔚霞[2] 黄天明[2] 马步国[2] 莫发荣[2] 

机构地区：[1]广西中医药大学生物化学与分子生物学教研室,广西壮族自治区南宁市530001 [2]广西医科大学组织学与胚胎学教研室,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2014年第15期2140-2144,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.81160264;肝脏保护与再生调节北京市重点实验室基金资助项目;No.2011-2~~

摘　　要：目的:构建人Cdc25C基因的克隆载体和原核表达载体,并诱导其在大肠杆菌中表达.方法:从人肝癌细胞株Bel-7404中提取总R N A,经RT-P C R法扩增人C d c25C c D N A后,将其正确插入克隆载体pMD18-T和表达载体pET-32a(+),并转化至BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中,分别采用0.25 mmol/L IPTG和ArtMediaTM Protein Expression自动诱导表达培养基诱导表达,并对纯化的融合蛋白进行考马斯亮蓝染色和质谱分析鉴定.结果:成功扩增了Cdc25C基因,并获得p M D18-T-C d c25C克隆载体和p E T-32a(+)Cdc25C表达载体;重组质粒在BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中均诱导表达出TRx-His-Cdc25C融合蛋白;考马斯亮蓝染色和蛋白质谱分析结果显示基因重组蛋白与目的蛋白相符.结论:获得肿瘤相关抗原Cdc25C重组蛋白,为后续研究奠定基础.AIM： To clone the human Cdc25C gene and construct a recombinant prokaryotic system to express this protein. METHODS： Total RNA were isolated from human hepatocellular carcinoma Bel-7404 cells and reverse transcribed, and the resulting cDNA was used as a template to amplify the human Cdc25Cgene by RT-PCR. The amplified PCR product was cloned into pMD18-T and pET-32a （＋） vectors and sequenced. Next, pET-32a（＋）-Cdc25C was transformed into chemically competent E. coli strains, including BL21 （DE3）, BL21 （DE3） pLysS and Transetta （DE3）, to express the protein after induction with 0.25 mmol/L IPTG and ArtMediaTM protein expression, respectively. The fusion protein was identified by Coomassie staining and mass spectrometry analysis. RESULTS： The Cdc25C gene and pMD18-T- Cdc25C and pET-32a（＋）-Cdc25C vectors were obtained successfully. Three strains of E. coli which harbored the recombinant plasmid could express the TRx-His-Cdc25C fusion protein. The expressed protein was identical to the Cdc25C protein as revealed by Coomassie staining and mass spectrometry. CONCLUSION： The recombinant protein of tumor-associated antigen Cdc25C has been successfully obtained.

关 键 词：肿瘤相关抗原 人Cdc25C 原核表达 

分 类 号：R735.7[医药卫生—肿瘤]