CCNL1真核表达载体的构建及其在MDA-MB-231细胞中表达的鉴定

Construction of CCNL1 eukaryotic expression vector and identification of expression of CCNL1 in MDA-MB-231 cells

目的:构建pcDNA3.1(+)-CCNL1、pVAXⅠ-CCNL1和pEGFP-C1-CCNL1三组重组载体,鉴定其在MDA-MB-231细胞中的表达情况。方法:用PCR方法扩增得到CCNL-1基因,然后用EcoRⅠ、HindⅢ酶切CCNL1基因,将其分别与pcDNA3.1(+)、pVAXⅠ和pEGFP-C1载体连接,构建pcDNA3.1(+)-CCNL1、pVAXⅠ-CCNL1和pEGFP-C1-CCNL1三组重组载体,然后用Fugene HD转染试剂将构建的真核表达质粒转染入MDA-MB-231细胞,48h后,用荧光定量PCR和Western blotting方法检测CCNL1在MDA-MB-231细胞中的差异表达。结果:转染MDA-MB-231细胞48h后,在荧光显微镜下,pEGFP-C1-CCNL1重组载体能观测到绿色荧光,而pcDNA3.1(+)-CCNL1、pVAXⅠ-CCNL1重组载体未观测到;在MDA-MB-231细胞中CCNL1的mRNA和蛋白质表达水平由高到低依次为pcDNA3.1(+)-CCNL1、pVAXⅠ-CCNL1和pEGFP-C1-CCNL1。结论:成功构建pcDNA3.1(+)-CCNL1、pVAXI-CCNL1和pEGFP-C1-CCN1三组重组载体,且pcDNA3.1(+)-CCNL1在MDA-MB-231细胞中高表达。

Objective To construct the CCNL1 eukaryotic expression vector pcfDNA3.1（＋）-CCNL1,pVAXⅠ-CCNL1 and pEGFP-C1-CCNL1 and identify the expressions of CCNL1 genes in MDA-MB-231 cells.Methods The eukaryotic expression plasmid pcDNA3.1（＋）-CCNL1,pVAX1-CCNL1 and pEGFP-C1-CCNL1 were transfected into the MDA-MB-231 cells by Fugene HD Transfection Reagent.After 48 h transfection,the differential expressions of pcDNA3.1（＋）-CCNL1,pVAX1-CCNL1 and pEGFP-C1-CCNL1 in MDA-MB-231 cells were detected by real time PCR and Western blotting.Results After 48 h transfection,there was green fluorescence only in pEGFP-C1-CCNL1 by fluorescent microscope,not in pcDNA3.1（＋）-CCNL1 and pVAX1-CCNL1;the expressions of mRNA and protein from high to low were pcDNA3.1（＋）-CCNL1,pVAX1-CCNL1 and pEGFP-C1-CCNL1 recombinant vector in MDA-MB-231 cells.Conclusion The recombinant vectors pcDNA3.1（＋）-CCNL1,pVAX1-CCNL1 and pEGFP-CCNL1 are constructed succesfully.The expression of pcDNA3.1（＋）-CCNL1 in MDA-MB-231 cells is higher.