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[目的]构建并鉴定人Kiss-1真核表达荧光质粒。[方法]按照Genebank中人Kiss-1序列设计引物,以EC109细胞cDNA为模板,扩增基因片段与载体pIRES2-EGFP连接,得到人Kiss-1真核表达荧光载体并测序鉴定,通过脂质体体外转染至EC-109细胞,根据转染情况分为2组:空载体组(转染pIRES2-EGFP空载体)、转染组(转染pIRES2-EGFP-Kiss-1重组质粒)。裂解细胞分别提取RNA和蛋白样品用于检测Kiss-1表达水平。[结果]构建的pIRES2-EGFP-Kiss-1荧光重组质粒,经PCR、酶切、测序结果完全达到预期设计,转染组细胞中Kiss-1mRNA和蛋白表达水平均显著高于空载体组。[结论]成功重组质粒pIRES2-EGFP-Kiss-1和瞬时表达Kiss-1基因的食管癌细胞,为后续建立Kiss-1高表达阳性细胞克隆奠定了基础,为研究Kiss-1对人食管癌细胞增殖、侵袭和转移能力提供了实验基础。
[Objective] To construct and identify human Kiss-1 eukaryotic expression fluorescent plasmid. [Method] According to the Kiss-1 sequence in Genebank, primers were designed and EC109 cell cDNA was used as a template. The gene fragment was amplified and ligated with vector pIRES2-EGFP to obtain the human Kiss-1 eukaryotic expression vector and identified by liposome The cells were transfected into EC-109 cells and divided into 2 groups according to the transfection conditions: empty vector group (empty vector transfected with pIRES2-EGFP) and transfected group (transfected with recombinant plasmid pIRES2-EGFP-Kiss-1). Lysed cells were extracted RNA and protein samples were used to detect Kiss-1 expression levels. [Results] The recombinant plasmid pIRES2-EGFP-Kiss-1 was constructed by PCR, digestion and sequencing. The result of sequencing confirmed that the expression level of Kiss-1 mRNA and protein in transfected cells was significantly higher than that in empty vector group. [Conclusion] The successful construction of recombinant plasmid pIRES2-EGFP-Kiss-1 and kiss-1 gene-overexpressing esophageal cancer cells will provide the basis for the subsequent establishment of Kiss-1 overexpression positive cell clones. To study the effect of Kiss-1 on esophageal cancer cells Proliferation, invasion and metastasis provide experimental basis.