人CXC型趋化因子受体CXCR6真核表达载体的构建及生物学功能研究

目的:克隆和构建带人CXC型趋化因子受体6(CXCR6)基因的真核表达载体pcDNA3.1(+).CXCR6,分析瞬时转染CXCR6基因对人乳腺癌细胞MDA-MB-231迁移能力的影响.方法:Trizol一步法抽提人外周血单个核细胞(PBMC)总RNA,RT-PCR扩增CXCR6全长基因,克隆入T载体,测序,双酶切连入真核表达载体peDNA3.1(+),酶切鉴定.将构建好的pcDNA3.1(+)-CXCR6瞬时转染.MDA-MB-231细胞,RT-PCR和Western blotting鉴定重组质粒的表达.利用微孔隔离小室检测转入人CXCR6基因的MDA-MB-231细胞的迁移能力.结果:扩增出人CXCR6基因全长,测序结果和GenBank记载完全一致,成功克隆入真核表达载体pcDNA3.1(+).瞬时转染pcDNA3.1(+)-CXCR6显著增强MDA.MB-23l细胞的迁移能力.结论:成功构建带有人CXCR6基因的真核表达载体pcDNA3.1(+)-CXCR6,为CXCR6在肿瘤学中的研究奠定基础.

Construction of eukaryotic expression vector of human chemokine CXC motif receptor 6 and characterization of its biological function

Aim:To construct the eukaryotic expression vector for human CXCR6 and to study the biological function of CXCR6 in cancer metastasis.Methods:The total RNA was isolated from human peripheral blood mononuclear cell (PBMC) with Trizol. CXCR6 gene was amplified by RT-PCR,and then inserted into pGEM-T vectors.DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1(+) identified by the double digestion.The recombinant vector was transiently transfected...