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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 eukaryotic expression plasmid of human CCR7 gene and obtain H157 cell stably expressing CCR7 protein. Methods The coding region of CCR7 was amplified from human lung adenocarcinoma by reverse transcription polymerase chain reaction (RT-PCR) and cloned into vector pEGFP-N1 to construct plasmid pEGFP-CCR7. Liposome-mediated gene transfection Recombinant plasmid DNA was introduced into lung adenocarcinoma H157 cells. G418 cells were screened to obtain cells stably expressing CCR7. The expression of CCR7 was identified by fluorescence microscopy, RT-PCR and flow cytometry. Results PCR, restriction enzyme digestion and sequencing proved that the recombinant plasmid pEGFP-CCR7 was constructed correctly. The expression of human CCR7 was detected by fluorescence microscope, RT-PCR and flow cytometry in stable transfected H157 cells. Conclusion The eukaryotic expression plasmid of human CCR7 gene was successfully constructed and H157 cell line stably expressing human CCR7 was obtained.