摘要
目的:构建人多药耐药基因(MDR1)的真核表达载体,转染CHO细胞,研究MDR1基因在真核细胞中的表达及功能。方法:将MDR1全长基因克隆到真核表达载体pcDNA3.1(+),利用阳离子脂质体转染CHO细胞,RT-PCR检测MDR1转染阳性细胞,并用G418筛选稳定表达MDR1基因的细胞,通过测定细胞对Rh123的外排作用以及对阿霉素的耐受性确定MDR1基因在正常细胞中的功能。结果:酶切鉴定和测序结果证明构建的重组表达载体pcDNA-MDR1序列正确。RT-PCR分析以及荧光显微镜观察结果表明:该重组表达载体已在CHO细胞中有效转录并稳定表达。Rh123外排和MTT药敏实验结果显示:转染pcDNA-MDR1的CHO细胞对Rh123外排作用显著高于未转染的CHO细胞,并且转染后的CHO细胞对阿霉素的耐药性也有显著提高。结论:成功构建了稳定表达MDR1并具有多药耐药功能的细胞株,有望成为多药耐药性药物研究的细胞模型。
AIM： To study the construction of eukaryotic expressing vectors containing human MDR1 gene and their expression in CHO as cells as well as the function of MDR 1. METHODS： The MDR1 gene fragment was cloned into pcDNA3.1 （＋） eukaryotic expressing vector and transfected into CHO cell through positive ion liposome method. The transcription and expression of MDR1 in the transfected cells were assayed by RT-PCR 48 hours after transfection. The cells which constantly expressed MDR1 gene were screened by G418. The function of MDR1 in transfected cells was assessed by determining the stagnation ratio when treated with Rh123 and/or the inhibition ratio when treated with doxorubicin. RESULTS： The eukaryotic expressing vector containing human MDR1 gene was successfully constructed. The transcription and expression of MDR1 in the transfected cells were verified by RT-PCR. The function of MDR1 in transfected cells was confirmed through determining the stagnation ratio treated with Rh123 and the inhibition ratio treated with doxorubicin. CONCLUSION： The exogenous MDR1 gene could be normally expressed in CHO cells, and the product could exert the functions.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2005年第6期594-598,共5页
Journal of China Pharmaceutical University
基金
国家重点基础研究发展计划("九七三"计划)资助项目(No.G1998051116)~~
