pcDNA3.1(+)/caveolin-1真核表达质粒的构建及其在MCF-7乳腺癌细胞株中的表达

Construction of pcDNA3.1(+)/caveolin-1 eukaryotic expression plasmid and its expression in MCF-7 breast cancer cell lines

目的明确正常组织和人乳腺癌细胞株MCF-7中caveolin-1的表达情况,构建pcDNA3.1(+)/caveolin-1表达质粒,并鉴定其表达。方法 RT-PCR法检测9种正常人体组织及人乳腺癌细胞株MCF-7中caveolin-1基因的扩增情况;Western blotting法检测MCF-7细胞中caveolin-1蛋白的表达情况。设计克隆引物和表达引物,RT-PCR法扩增caveolin-1基因,用EcoRⅠ+XbaⅠ内切酶消化回收PCR产物和质粒pcDNA3.1(+),连接后构建pcDNA3.1(+)/caveolin-1重组表达质粒。挑选pcDNA3.1(+)/caveolin-1转染感受态细菌后的单菌落进行PCR鉴定,阳性菌落培养后提取质粒行酶切鉴定及测序鉴定。瞬时转染MCF-7细胞后Western blotting检测caveolin-1蛋白的表达情况。结果①9种正常组织中均有caveolin-1基因扩增,MCF-7细胞中无caveolin-1基因扩增且无蛋白表达;②pcD-NA3.1(+)/caveolin-1重组表达质粒转染MCF-7细胞后caveolin-1蛋白表达良好。结论成功构建了pcDNA3.1(+)/caveolin-1重组表达质粒,瞬时转染乳腺癌MCF-7细胞后caveolin-1表达稳定。

Objective To identify the expression of caveolin-1 in human breast cancer cell line MCF-7 and normal tissues, construct pcDNA3.1 （＋）/caveolin-1 expression plasmid and detect its expression. Methods The gene amplification of caveolin-1 in 9 kinds of normal tissues and MCF-7 was detected by RT-PCR; the protein expression of caveolin-1 in MCF-7 cells was detected by Western blotting. Caveolin-1 gene was amplified by RT-PCR with its special primer; PCR products and pcDNA3.1 （＋） plasmid were digested and recycled by EcoR I ＋ Xba I endonuclease, and then pcDNA3. 1 （＋）/caveolin-1 recombinant expression plasmid was constructed. The single colonies of pcDNA3.1 （＋）/caveolin-1 were selected and transfected into competent bacteria for PCR identification. Positive colonies were selected and cultured, and restriction endonuclease and sequence identification were carried out. Western blotting was used to detect the protein expression of caveolin-1 in transient transfected MCF-7 cells. Results ① There were gene amplifications of caveolin-1 in all the 9 kinds of normal tissues, but none in MCF-7 cells, so was protein expression. ② When pcDNA3. 1 （＋）/caveolin-1 recombinant expression plasmids were transfected into MCF-7 cells, the caveolin-1 protein was expressed well. Conclusion The pcDNA3. 1 （＋）/ caveolin-1 recombinant expression plasmid was successfully constructed, and the caveolin-1 protein was expressed stably in transient transfected MCF-7 cells.