CCNL1真核表达载体的构建及其在MDA-MB-231细胞中表达的鉴定

目的:构建pcDNA3.1(+)-CCNL1、pVAX Ⅰ -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EcoR Ⅰ、HindⅢ酶切CCNL1基因,将其分别与pcDNA3.1(+)、pVAX Ⅰ和pEGFP-C1...

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

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.