野生型p27和核定位信号缺失型p27真核表达载体的构建及表达  

Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27

作　　者：焦鑫艳 张英[3] 盛秋 张妙 杨泽健 高小倩 赵谦[5] 王博[1,2] 刘培军 JIAO Xinyan;ZHANG Ying;SHENG Qiu;ZHANG Miao;YANG Zejian;GAO Xiaoqian;ZHAO Qian;WANG Bo;LIU Peijun(Center for Translational Medicine,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Key Laboratory for Tumor Precision Medicine of Shaanxi Province,Xi’an 710061;Department of Gastroenterology,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Hospital of Northwestern Polytechnical University,Xi’an 710072;Department of Otolaryngology‒Head and Neck Surgery,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China)

机构地区：[1]西安交通大学第一附属医院转化医学中心,陕西西安710061 [2]陕西省肿瘤精准医学重点实验室,陕西西安710061 [3]西安交通大学第一附属医院消化内科,陕西西安710061 [4]西北工业大学医院,陕西西安710072 [5]西安交通大学第一附属医院耳鼻喉头颈外科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2022年第4期516-521,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81872272);陕西省重点研发计划资助项目(No.2015SF015,2019SF-147);西安交通大学第一附属医院基金资助项目(No.2019ZYTS-13)。

摘　　要：目的构建人野生型p27(p27WT)和核定位信号缺失型p27(p27△NLS)的真核表达载体,并在HEK293T细胞中进行表达,为细胞中p27核浆定位的变化及其不同的生物学功能研究提供细胞基础。方法提取人乳腺癌细胞系MCF7总RNA,反转录cDNA后,分别进行p27全长和非NLS区片段的PCR扩增,获得p27WT全长(CDKN1B,NM_004064.5)和p27△NLS编码区序列,分别将其克隆至真核表达载体pCMV-Blank,经菌液PCR、双酶切和测序鉴定。电穿孔法转染HEK293T细胞,48 h时分别提取胞核蛋白和胞质蛋白,Western blotting检测p27在细胞质和细胞核中的蛋白表达。结果测序结果显示,真核表达载体pCMV-Blank中插入的p27WT序列和p27△NLS序列均与NM_004064.5序列相一致。pCMV-p27WT转染HEK293T细胞后,细胞总p27蛋白表达显著增加,核浆分离检测发现其在细胞质和细胞核中均表达;而pCMV-p27△NLS转染HEK293T细胞的总p27蛋白表达也显著增加,核浆分离检测发现其主要在细胞质中表达。结论成功构建人p27WT和p27△NLS的真核表达载体,并在HEK293T细胞中成功表达,为p27蛋白在肿瘤细胞周期中的功能研究提供了细胞基础。Objective To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences,and the express them in HEK293T cells,which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus.Methods Total RNA was prepared from human breast cancer MCF7 cells,and cDNA was obtained by reverse transcription-polymerase chain reaction(RTPCR).After amplification of the p27 CDs and non-NLS fragments by PCR,full length p27WT(CDKN1B,NM_004064.5)and p27△NLS coding regions were obtained.PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank.After identification with bacterial PCR,double restriction enzyme digestion and sequencing,they were defined officially as pCMV-p27WT and pCMV-p27△NLS,respectively.The recombinant plasmids were transfected into HEK293T cells by electroporation.After 48 h,the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting.Results The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5.After transfection with pCMV-p27WT,total p27protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells.After transfection with pCMV-p27△NLS,p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells.Conclusion The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells.This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.

关 键 词：P27 CDKN1B 核定位信号序列 真核表达 

分 类 号：R393[医药卫生—基础医学]