人CD52基因的克隆及稳定转染细胞系的建立  

Cloning of Human CD52 Gene and Establishment of Cell Line for Stable Transfection

作　　者：刘友[1,2] 屈浩 石琳 刘红芹 黄丽华 张宇光[1,3] 朱卫彬 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所血液病医院,天津300020 [2]内蒙古科技大学包头医学院,内蒙古包头014010 [3]协和干细胞基因工程有限公司,天津300384

出　　处：《中国生物制品学杂志》2010年第5期471-474,共4页Chinese Journal of Biologicals

基　　金：天津市科技创新专项资金项目(08FDZDSH03000)

摘　　要：目的克隆人CD52基因,构建真核表达载体,并在CHO细胞中稳定表达。方法提取人Hut-78细胞总RNA,采用RT-PCR法扩增CD52基因,定向克隆至真核表达载体pcDNA3.1(+),构建重组表达质粒pcDNA3.1(+)/CD52,通过脂质体法转染CHO细胞,建立稳定转染的细胞系CHO-CD52。采用RT-PCR、免疫荧光组化技术及流式细胞术检测目的基因和蛋白的表达。结果 RT-PCR扩增得到186bp的DNA片段。重组表达质粒pcDNA3.1(+)/CD52经PCR、双酶切及测序证明构建正确。CHO-CD52细胞经RT-PCR分析,可见186bp的目的基因条带;经免疫荧光检测,可见绿色荧光分布;经流式细胞术分析,阳性细胞百分率及荧光平均值分别为98.18和193.56。结论已成功克隆了人CD52基因,并建立了稳定转染的CHO细胞系,为CD52单克隆抗体的制备及抗体药物的开发奠定了基础。Objective To clone human CD52 gene and construct a eukaryotic expression vector for stable expression in CHO cells.Methods The total RNA of human Hut-78 cells was extracted for amplification of CD52 gene by RT-PCR.The amplified gene was cloned into eukaryotic expression vector pcDNA3.1（＋）,and the constructed recombinant plasmid pcDNA3.1（＋）/CD52 was transfected to CHO cells in mediation of liposome to establish a CHO-CD52 cell line for stable transfection.The transcription of target gene was determined by RT-PCR,and the expression of target protein by immunofluorescent histochemical assay and flow cytometry.Results The DNA fragment at a length of 186 bp was amplified by RT-PCR.PCR,restriction analysis and sequencing proved that recombinant plasmid pcDNA3.1（＋）/CD52 was constructed correctly.The target gene band at a length of 186 bp was detected in CHO-CD52 cells by RT-PCR.Green fluorescence was observed in CHO-CD52 cells by immunofluorescent histochemical assay.Flow cytometry showed that the percentage of positive cells and mean fluorescence intensity of CHO-CD52 cells were 98.18 and 193.56 respectively.Conclusion Human CD52 gene was successfully cloned,and the CHO cell line for stable transfection was established,which laid a foundation of preparation of CD52 monoclonal antibody and development of antibody drugs.

关 键 词：CD52基因 真核表达 转染 CHO细胞 

分 类 号：Q789[生物学—分子生物学] R392-33[医药卫生—免疫学]