人CCR7基因真核表达载体构建及表达

Construction and expression of human CCR7 gene eukaryotic cell expression vector

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导入肺腺癌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。

[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.