人Kiss-1真核表达荧光载体的构建

Construction of eukaryotic expression fluorescent vector Kiss-1 in human

[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 plas- mid. [Methods]Kiss-1 gene was taken from EC109 cell by primers designed from gene bank,and cloned into pIRES2-EGFP vector to construct human Kiss-1 eukaryotic expression fluorescent plasmid and send to Se- quencing. The plasmid was transfected into EC-109 cell line with Lipofectamine 2000. The expression level of the RNA and protein from the pIRES2-EGFP-Kiss-1 transfeeted group and control plRES2-EGFP trans- fected group were collected and gauged. [Results] The PCR, enzyme digestion and sequencing results showed that the recombinant pIRES2-EGFP-Kiss-1 fluorescent plasmid fully achieved our intended design. The mRNA Kiss-1 and protein expression levels in transfected group were significantly higher than those in the control group. [Conclusion]In the study, we successfully construct the recombinant pIRES2-EGFP- Kiss-1 plasmid and EC109 cell that transient expression Kiss-1 gene. This will lay the foundation for estab- lishing the Kiss-1 high expression positive cell clones subsequently,and provide experimental basis for the effect and mechanism of Kiss-1 gene study in the proliferation, invasion and metastasis ability of human esophageal cancer cell line.