人CD40分子基因转染人脐静脉内皮细胞ECV-304

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (12): 1474-1477.

人CD40分子基因转染人脐静脉内皮细胞ECV-304

王维蓉¹, 林蓉¹, 杨玉琮², 甘伟杰¹, 刘俊田¹, 吕社民³

1. 西安交通大学医学院药理教研室, 陕西, 西安, 710061;
2. 西安交通大学医学院第一医院实验医学中心, 陕西, 西安, 710061;
3. 西安交通大学医学院生物化学教研室, 陕西, 西安, 710061

出版日期:2005-12-20 发布日期:2005-12-20
基金资助:
收稿日期:2005-5-16。
基金项目:国家自然科学基金(30371759)
作者简介:王维蓉(1979- ),女,在读硕士研究生,电话:029-85274383,E-mail:wangweirong1222@163.com.
通讯作者:林蓉(1963- ),女,西安交通大学医学院药理系,副教授,电话:029-82656215,E-mail:linrong@mail.xjtu.edu.cn.

Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells

WANG Wei-rong¹, LIN Rong¹, YANG Yu-cong², GAN Wei-jie¹, LIU Jun-tian¹, LÜ She-min³

1. 西安交通大学医学院药理教研室, 陕西, 西安, 710061;
2. 西安交通大学医学院第一医院实验医学中心, 陕西, 西安, 710061;
3. 西安交通大学医学院生物化学教研室, 陕西, 西安, 710061

Online:2005-12-20 Published:2005-12-20

摘要/Abstract

摘要： 目的构建人CD40的真核表达载体,建立持续﹑稳定高表达CD40的人脐静脉内皮细胞ECV-304。方法将含有CD40的克隆载体pUCD40酶切后,再克隆到真核表达载体pCDNA3.1中,构建重组质粒pCDNA3.1(+)/CD40,并对重组质粒进行酶切鉴定。然后用脂质体法将重组质粒转染ECV-304,G418筛选转染的细胞。RT-PCR﹑Western-blotting和流式细胞仪定性定量检测转染后的ECV-304的CD40的表达情况。结果经酶切鉴定,重组体中已插入目的基因片段CD40。RT-PCR和Western-blotting证实,重组质粒转染的ECV-304中有CD40基因的表达,流式细胞仪检测转染后的ECV-304的CD40的表达率为95%。结论成功构建了真核表达载体pCDNA3.1(+)/CD40,并建立了高表达CD40的ECV-304,为筛选抗动脉粥样硬化药物奠定了基础。

Abstract: Objective To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. Methods The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. Results Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. Conclusion The rcombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

中图分类号:

王维蓉¹, 林蓉¹, 杨玉琮², 甘伟杰¹, 刘俊田¹, 吕社民³. 人CD40分子基因转染人脐静脉内皮细胞ECV-304[J]. 南方医科大学学报, 2005, 25(12): 1474-1477.

WANG Wei-rong¹, LIN Rong¹, YANG Yu-cong², GAN Wei-jie¹, LIU Jun-tian¹, LÜ She-min³. Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells[J]. Journal of Southern Medical University, 2005, 25(12): 1474-1477.

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