pcDNA3.1/CXCR7真核细胞表达载体的构建及其在内皮细胞中的表达鉴定  

Construction of pcDNA3.1/CXCR7 Eukaryotic Expression Plasmid and its Expression in Endothelial Cells

作　　者：戴小珍[1,2] 王兰[1] 李红[1] 何浪[1] 张坤[1] 田志杰[1] 

机构地区：[1]成都医学院生物医学系,四川成都610500 [2]重庆大学生物流变科学与技术教育部重点实验室,重庆400044

出　　处：《现代生物医学进展》2013年第19期3661-3664,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81200917);四川省教育厅资助科研项目(11ZA205);重庆大学"生物流变科学与技术"教育部重点实验室访问学者基金(CQKLBST-2012-003)

摘　　要：目的:CXCR7是基质衍生因子1(stroma derived factor-1,SDF-1)的新受体,且该受体在血管新生部位的内皮细胞中表达上调,故本研究拟构建CXCR7的真核表达载体pcDNA3.1/CXCR7,并检测其在人脐静脉内皮细胞中的表达。方法:采用RT-PCR法从人肝癌细胞HepG2的cDNA中扩增出约1100 bp的CXCR7基因片段。采用KpnI、XbaI将目的基因和载体pcDNA3.1进行双酶切,将酶切产物加入T4 DNA连接酶16℃连接过夜。将连接产物转化到感受态大肠杆菌中。挑取阳性克隆、提质粒,用双酶切、质粒DNA PCR扩增及DNA序列分析鉴定正确后,采用阳离子脂质体LipofectamineTM2000将其转染人脐静脉内皮细胞(HU-VEC),通过western-blot检测目的基因在内皮细胞中的表达。结果:阳性克隆经双酶切法鉴定含有CXCR7基因片段,质粒DNAPCR扩增出与CXCR7同等大小的基因片段,基因测序结果与GenBank中序列相同。转染HUVEC后,细胞中CXCR7的表达水平显著上升。结论:成功构建了CXCR7的真核表达载体,可在内皮细胞中正常表达并。为进一步研究其作用机制奠定了基础。Objective： CXCR7 is a novel receptor of stroma derived factor-1 （SDF-1）, and the expression of CXCR7 is elevated in endothelial cells ofneovascular sites. Thus in this study, we construct pcDNA3.1/CXCR7 expression plasmid and detect its expression in HUVEC. Methods： CXCR7 gene was amplified by RT-PCR with the cDNA from human hepatic carcinoma HepG2 cell lines; PCR products and pcDNA3.1 plasmid were digested and recycled by KpnI ＋ XbaI endonuclease, the digested products were connected by T4 DNA lig- ase and then transformed into competent bacteria. The positive clones were selected, from which plasmid DNA was abstracted and identi- fied by restriction endonuclease, sequence identification and sequencing. The pcDNA3.1/CXCR7 recombinant expression plasmid was transfected into HUVECs by LipofectamineTM 2000. And then western blotting was used to detect the protein expression of CXCR7 in transient transfected HUVECs. Results： The size of PCR products and double digested section were 1100 bp, which was in accordance with the expected results; sequence analysis of inserted fragment revealed the same sequence as GenBank report. When pcDNA3.1/CXCR7 recombinant expression plasmids were transfected into HUVECs, the expression of CXCR7 was up-regulated. Conclusion： The pcDNA3. 1 /CXCR7 recombinant expression plasmid was successfully constructed. It could up-regulate the expression of CXCR7 in HUVECs, which will establish a foundation for further investigating the mechanisms of CXCR7.

关 键 词：趋化因子受体7 真核表达载体 内皮细胞 

分 类 号：Q75[生物学—分子生物学] Q78