细胞角蛋白8真核表达载体的构建、表达及生物信息学分析  

Bioinformatics analysis on CK8 full length coding sequence and eukaryotic expression in SMMC7721 cells

作　　者：寻萌[1] 赵四海[2] 曹春霞 薛欣[1] 邵明明[4] 李薇[1] 徐琨[1] 楚雍烈[1] 

机构地区：[1]西安交通大学医学院免疫与病原生物学系,陕西西安710061 [2]西安交通大学医学院实验动物中心,陕西西安710061 [3]Department of Medicine,University of Florida [4]西安医学院医学技术系,陕西西安710021

出　　处：《细胞与分子免疫学杂志》2011年第2期123-127,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30470089);国家高技术研究发展计划(863)资助项目(2005AA214160)

摘　　要：目的:构建人细胞角蛋白8(CK8)全长CDS序列真核表达载体,并转染人肝癌细胞SMMC7721,为研究CK8的生物学功能提供细胞模型。方法:RT-PCR扩增CK8全长CDS,克隆入pMD18-Tsimplevector质粒,鉴定正确后,亚克隆入质粒pEGFP-C1,构建真核表达载体pEGFP-CK8。脂质体介导转染SMMC7721细胞,荧光显微镜、realtimePCR和Westernblot检测CK8的表达。生物信息学软件分析CK8理化特性、信号肽及功能位点等。结果:PCR、酶切和测序结果均证实重组质粒含有人CK8全长CDS序列,转染实验表明CK8在SMMC7721细胞中发生了高表达。结论:成功地构建了人CK8全长CDS真核表达载体,并使其在SMMC7721细胞中获得高表达,为研究CK8的生物学功能奠定了实验基础。AIM： To construct an eukaryotic expression vector containing the coding region of human full length cytokeratin 8 gene and to detect its expression in SMMC7721 cells. METHODS： CK8 cDNA was amplified by RT-PCR and cloned to pMD18-T simple vector. After confirming the sequence, the cDNA was inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. The recombinant plasmid was transfected into SMMC7721 cells with LipofectamineTM2000 and the expression was detected by fluorescence microscope, real time PCR and Western blot. The physical-chemical properties, signal peptide and functional motifs were predicted by the bioinformatics software. RESULTS： PCR, restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the coding region of full length CK8 gene. Observation under fluorescence microscope and the results of real time PCR and western blot indicated CK8 was over-expressed in SMMC7721 cells. CONCLUSION： The eukaryotic expression vector containing the CK8 gene was successfully constructed and expressed, which provides a basis for the study for biological function of CKS.

关 键 词：细胞角蛋白8 全长编码序列 真核表达 生物信息学分析 

分 类 号：R34[医药卫生—基础医学]