胱抑素C基因真核表达载体的构建及在转染的人主动脉平滑肌细胞中的表达

Construction and Expression of Eukaryotic Expression Vector Containing Gene Cystatin C

目的克隆人胱抑素C基因及构建胱抑素C基因真核表达载体并研究其在人血管平滑肌细胞中的表达。方法培养人脐静脉内皮细胞系,并从中提取总RNA,采用逆转录聚合酶链反应扩增胱抑素C基因,构建其真核表达载体,双酶切及聚合酶链反应鉴定阳性克隆,转染人血管平滑肌细胞,逆转录聚合酶链反应及Western blot法检测其表达情况。结果成功克隆人胱抑素C基因及构建其真核表达载体,转染后成功高表达。结论克隆人胱抑素C基因及构建其真核表达载体成功,转染人血管平滑肌细胞获得其高表达。

Aim To clone human Cystatin C gene and construct its eukaryotic expression vector to observe the expression in the human vascular smooth muscle cells（ VSMC ）. Methods Cystatin C gene was obtained from human endothelial cells by reverse transefiption-polymerase chain reaction （RT-PCR） .Molecular cloning technique was used to construct this kind of eukaryotic expression vector, pCDNA3.1-Cystatin C was identified by restriction enzyme and PCR. Using liposome- mediated transfection, the eukaryotic expression vector pCDNA3.1-Cystatin C was transfected into human VSMC and proved by RT-PCR and Western blot. Results The eukaryotic expression vector pCDNA3.1-Cystatin C was constructed correctly and was highly expressed in human VSMC. Conclusion Cystatin C was cloned successfidly, its eukaryotic expression vector was constructed correctly and was expressed highly in human VSMC.