人CD45基因克隆及其在Hela细胞中的表达  

Cloning of human CD45 gene and its expression in Hela cells

作　　者：李捷[1] 许天昱 吴璐琳 张丽芸[1] 卢晓[1] 左大明[1] 陈政良[1] 

机构地区：[1]南方医科大学基础医学院免疫学教研室,广东广州510515

出　　处：《南方医科大学学报》2015年第11期1575-1578,1585,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30972679)~~

摘　　要：目的克隆人CD45 c DNA并导入Hela细胞中表达,建立研究CD45功能的细胞模型。方法采用RT-PCR方法从人外周血单个核细胞中扩增CD45基因PTPRC的c DNA,将其克隆至p MD-18T载体。构建重组真核表达载体Pc DNA3.1-3xflag-CD45,经HindⅢ和XhoⅠ双酶切及测序验证。将其转染至Hela细胞,以流式细胞术(FCM)和免疫印迹(WB)分析CD45在Hela细胞中的表达情况,碱性磷酸酶试剂盒检测CD45的活性。结果分离到长约3900 bp的人PTPRC c DNA片段,将其插入p MD-18T载体获得了c DNA克隆。酶切和测序结果证实重组表达载体Pc DNA3.1-3xflag-CD45构建成功,FCM和WB分析表明CD45能在Hela细胞中有效表达,且表达的重组CD45蛋白具有生物学活性。结论成功获得人PTPRC c DNA克隆并在Hela细胞中有效表达,为进一步研究CD45功能奠定了基础。Objective To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. Methods The intact c DNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells（PBMCs） of a healthy donor was cloned into p MD-18 T vector. The CD45 c DNA fragment amplified from the p MD-18T-CD45 by PCR was inserted to the coding region of the Pc DNA3.1-3xflag vector, and the resultant recombinant expression vector Pc DNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. Results The c DNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into p MD-18 T vector. The recombinant expression vector Pc DNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity.Conclusion The c DNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

关 键 词：CD45 基因克隆 真核表达 HELA细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q78[医药卫生—基础医学]