ZO-1基因的慢病毒载体构建

Construction of Lentiviral Vector Carrying ZO-1 Gene

目的:相邻两心肌纤维的连接处称心肌闰盘,它在心肌的机电活动中扮演重要角色。目前有关闰盘相关基因Cx43的研究已被广泛关注,而在不同心肌病理状态下,其变化及功能都与另一闰盘相关基因ZO-1有极为密切的联系。目前有关ZO-1的研究比较少,此次欲将猪ZO-1基因克隆到慢病毒表达载体并进行鉴定,为进一步研究其功能奠定基础。方法:利用聚合酶链反应(PCR)扩增猪ZO-1基因,并克隆到慢病毒表达载体,通过转化、抽提质粒、双酶切和测序鉴定构建的Lenti-EF1α-EGFP-TRE-ZO-1重组载体。结果:猪ZO-1基因片段重组到慢病毒载体;PCR和双酶切鉴定,电泳结果显示均能得到与理论大小相符的片段;经测序证实成功构建Lenti-EF1α-EGFP-TRE-ZO-1慢病毒表达载体。结论:通过一系列实验证实Lenti-EF1α-EGFP-TRE-ZO-1慢病毒表达载体构建成功,可利用其感染相关细胞或注入动物体内,为后续的体外及在体研究奠定基础,明确其在不同心肌病理状态下所发挥的独特作用。

Objective： The joint of adjacent heart muscle fibers is called myocardial intercalated disc, which is important for myocardial electrical activity. It has been widely concerned about the study of intercalated disc related gene Cx43. Under different pathological status, it is related to another gene ZO-1. However, the research of ZO-1 is scanty, so we cloned the ZO-1 gene of porcine into the lentiviral vector and identificate the recombinant plasmid, which will lay the foundation for further research. Methods： The porcine ZO-1 gene was amplified by PCR, and cloned into lentiviral expression vector. The reconstructed Lenti- EFlct-EGFP-TRE-ZO-1 plasmid was identified by transformation, plasmid extraction, double enzyme digestion and DNA sequencing analysis. Results： The porcine ZO-1 DNA sequences were successfully inserted into the lentiviral vector.The reconstructed plasmid was identified correctly by PCR and double enzyme digestion. The recombinant Lenti-EFlcx-EGFP-TRE-ZO-1 vector was confirmed by DNA sequencing. Conclusion： Lenti-EFloL-EGFP-TRE-ZO-1 lentiviral vector has been successfully constructed, so we can use it to infect related cells or animals, which will be useful for further study in vitro and in vivo, to understand its function in different pathological status.