人CCR7基因真核表达载体构建及表达

目的构建人CCR7基因真核表达质粒，使其在真核细胞中稳定表达，为其临床应用奠定基础。方法以RT—PCR法从临床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导人肺腺癌A549细胞中，加入G418对细胞进行筛选获得稳定表达CCR7的细胞，并用流式细胞仪对重组质粒的表达进行鉴定。结果PCR、酶切及测序结果证明重组质粒pEGFP—CCR7构建正确，荧光显微镜及流式细胞术在稳定转染A549细胞中检测到人CCR7的表达。结论人CCR7基因真核表达质粒已成功构建并稳定转染肺癌A549细胞株；本研究为临床应用提供了实验依据。

Construction and expression of human CCR7 gene eukaryotic cell expression vector

Objective] To construct a eukaryotie expression vector of human CCR7 gene and transfeet stably it into A549 cells,so to set foundation for its clinical application. Methods] CCR7 coding domain was amplified from a patient with lung adenoeareinoma tissue by RT-PCR, direetionally cloned into pEGFP-N1 to construct PEGFP-CCR7 plasmid. The recombinant vectors were transfeeted into A549 cell lines by DOTAP liposome and screened by G418 selective medium. The expression of CCR7 gene was verified by flow eytometry. Results] Correct construction of pEGFP-CCR7 was identified by methods of restriction enzyme analysis,PCR amplieation and nueleotide-sequeneing method. Green fluorescence was emited from transfeeted cells under fluorescent micro- scope,flow eytometry showed the expression of CCR7 protein of the transfeeted A549 cells. Conclusion] The pEGFP-CCR7 eukaryotie expression plasmid is constructed successfully and CCR7 is expressed Stably in lung adenoeareinoma A549 cells. The research sets the experimental basis for its clinical application.