TAGLN真核表达载体pcDNA6.2/EmGFP-Bsd/V5-TAG-LN-mut及稳定转染细胞株的构建

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.

Construction of a eukaryotic expression vector carrying tagln and establishment of a cell line stably expressing tagln

AIM:To construct a eukaryotic expression plasmid carrying tagln(the gene encoding transgelin) and establish the human colon carcinoma cell line RKO stably expressing tagln.METHODS:Using the Gateway Technology,a BP recombination reaction was performed using a construct carrying the tagln gene(pOTB7-TAGLN-mut) and a donor vector(pDONR221) to create an entry clone(pDONR221-TAGLN-mut).An LR recombination reaction was then performed between the entry clone and the destination vector(pcDNA6.2/EmGFP-Bsd/V5-DEST) to generate a recombinant plasmid(pcDNA6.2/EmGFP-Bsd/V5-TAGLN-mut).The recombinant plasmid was confirmed by sequencing.Lipofectamine-mediated transfection was performed in RKO cells and stable transfectants were selected.The stable expression of tagln in RKO cells was validated by real-time RT-PCR and Western blot.Matrigel invasion assay was performed with these stable cell lines.RESULTS:Sequencing analysis showed that tagln was successfully inserted into the pcDNA6.2/EmGFP-Bsd/V5-DEST plasmid.Real-time RT-PCR and Western blotting indicated that the expression of tagln increased remarkably in RKO cells transfected with the pcDNA6.2/EmGFP-Bsd/V5-TAGLN-mut plasmid(RKO-TAGLN cells) as compared to those transfected with the control vector(RKO-CTRL cells) and non-transfected RKO cells(relative mRNA expression levels:45.58 ± 12.79,1.32 ± 0.43 vs 1,both P < 0.01;protein expression levels:1.69 ± 0.04,0.29 ± 0.05 vs 0.29 ± 0.04,both P < 0.01).Overexpression of tagln increased cell invasion by 161.76% ± 61.18% in RKO cells(P < 0.01).CONCLUSION:A eukaryotic expression plasmid carrying tagln has been successfully generated and a RKO cell line stably expressing tagln has been established.These lay a foundation for further research of the role of transgelin in human colon carcinoma.