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目的:构建携tagln基因的真核表达载体,建立稳定转染该质粒的人结肠癌细胞株RKO并鉴定.方法:通过Gateway克隆技术,使用pOTB7-TAGLN-mut与pDONR221进行BP重组反应产生入门克隆,再与pcDNA6.2/EmGFP-BsdV5-DEST空载体进行LR重组反应,生成目的质粒pcDNA6.2/EmGFP-Bsd/V5-TAGLN-mut通过测序验证目的质粒的插入序列.将携带tagln的真核表达载体和对照质粒稳定转染至人结肠癌细胞株RKO.通过实时荧光定量PCR和免疫印迹检测tagln的mRNA和蛋白表达水平.通过细胞侵袭实验了解transgelin在结肠癌细胞RKO中的作用.结果:携带tagln的真核表达载体测序分析显示插入序列及位点正确;实时荧光定量PCR及免疫印迹结果显示,稳定转染重组目的质粒的细胞株(RKO-TAGLN细胞)中tagln的表达水平与转染对照质粒的细胞株(RKO-CTRL细胞)及野生型RKO细胞相比明显上调,差异具有统计学意义(mRNA相对表达水平分别为45.58±12.79、1.32±0.43和1,P<0.01;蛋白质灰度定量值为1.69±0.04、0.29±0.05和0.29±0.04,P<0.01).细胞侵袭实验提示,RKO-TAGLN细胞较RKO-CTRL细胞的侵袭能力提高(161.76%±61.18%,P<0.01).结论:成功构建携tagln基因的真核表达载体并建立过表达transgelin的稳定细胞株和对照细胞株,为研究transgelin在结肠癌中的作用奠定基础.
OBJECTIVE: To construct a eukaryotic expression vector carrying tagln gene and establish a human colon cancer cell line RKO stably transfected with the plasmid and identify it.Methods: The cloned recombinant plasmid pTB7-TAGLN-mut and pDONR221 The recombinant plasmid pcDNA6.2 / EmGFP-Bsd / V5-TAGLN-mut was inserted into pcDNA6.2 / EmGFP-BsdV5-DEST empty vector to generate the desired plasmid pcDNA6.2 / EmGFP-Bsd / V5-TAGLN- The expression vector and control plasmid were stably transfected into human colon cancer cell line RKO.The mRNA and protein expression of tagln were detected by real-time fluorescence quantitative PCR and Western blotting.Transfection assay was used to investigate the role of transgelin in colon cancer cell RKO.Results: The results of real-time fluorescence quantitative PCR and Western blot showed that the expression of tagln in RKO-TAGLN cells stably transfected with the recombinant plasmid was similar to the transfection Compared with the wild type RKO cells, the control plasmid (RKO-CTRL cells) was significantly up-regulated, the difference was statistically significant (mRNA relative expression levels were 45.58 ± 12.79,1. 32 ± 0.43 and 1, P <0.01, and the gray value of protein was 1.69 ± 0.04, 0.29 ± 0.05 and 0.29 ± 0.04, respectively, P <0.01) .The cell invasion assay indicated that RKO-TAGLN cells were more invasive than RKO-CTRL cells (161.76% ± 61.18%, P <0.01) .Conclusion: The eukaryotic expression vector carrying tagln gene was successfully constructed and stable and overexpressed transgelin cell lines and control cell lines were established, which laid the foundation for the study on the role of transgelin in colon cancer .