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。
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.
出处
《第一军医大学学报》
CAS
CSCD
北大核心
2005年第12期1474-1477,共4页
Journal of First Military Medical University
基金
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.
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.
