CCR7基因载体的构建及其在肺腺癌H157细胞的稳定表达

Construction of human CCR7 gene vector and its stable expression in lung adenocarcinoma H157 cells

目的构建人CCR7基因真核表达质粒,获得稳定表达CCR7蛋白的H157细胞。方法应用逆转录PCR法(RT-PCR)自人肺腺癌标本中扩增出CCR7编码区序列,定向克隆至载体pEGFP-N1中构建质粒pEGFP-CCR7,采用脂质体介导的基因转染技术将重组质粒DNA导入肺腺癌H157细胞中,加入G418对细胞进行筛选,获得稳定表达CCR7的细胞,并用荧光显微镜、RT-PCR和流式细胞术对CCR7的表达进行鉴定。结果 PCR、酶切及测序结果证明,重组质粒pEGFP-CCR7构建正确,荧光显微镜、RT-PCR及流式细胞术在稳定转染H157细胞中检测到人CCR7的表达。结论成功构建人CCR7基因真核表达质粒并获得稳定表达人CCR7的H157细胞株。

Objective To construct expression vectors of human CCR7 gene and to obtain H157 cells that can express CCR7 stably.Methods CCR7 coding domain was amplified from lung adenocarcinoma patient using RT-PCR,directionally cloned into pEGFP-N1 plasmid.The recombinant vectors were transfected into H157 cell lines by DOTAP liposome and screened by G418 selective medium.The expression of CCR7 was verified by RT-PCR and flow cytometry.Results Correct construction of pEGFP-CCR7 was identified by methods of restriction enzyme analysis,PCR amplication and nucleotide-sequencing.CCR7 was found to be expressed in the transfected H157 cells with fluorescent microscopy,RT-PCR and flow cytometry.Conclusions The CCR7-expressing plasmid has been constructed successfully and CCR7 is expressed stably in human adenocarcinoma H157 cells.