心脏转录因子Nkx2-5、GATA-4真核表达质粒的构建  

Construction of Eukaryotic expression Recombinant Plasmid PCDNA3.1-HA-Nkx2-5 and pCDNA3.1-flag-GATA-4

作　　者：赖平[1] 罗耀玲[2] 张敏鸿[2] 杨建琼[2] 邱悦群[3] 

机构地区：[1]赣南医学院,江西赣州341000 [2]赣南医学院第一附属医院临床科研中心,江西赣州341000 [3]赣南医学院第一附属医院心内科,江西赣州341000

出　　处：《赣南医学院学报》2017年第1期42-45,共4页JOURNAL OF GANNAN MEDICAL UNIVERSITY

基　　金：江西省科技支撑计划项目(编号:20141BBG70069)

摘　　要：目的:构建人类心脏特异性转录因子Nkx2-5、GATA-4的真核表达质粒,探讨Nkx2-5、GATA-4在心脏发育及先天性心脏病发病过程中的作用奠定基础。方法:以质粒人脑c DNA为模板,并设计上下游引物扩增Nkx2-5、GATA-4编码区序列。将真核表达载体pCDNA 3.1-HA、pCDNA 3.1-flag分别和Nkx2-5、GATA-4的PCR产物分别在双酶切后用T4连接酶连接,构建重组质粒pCDNA 3.1-HA-Nkx2-5、pCDNA 3.1-flag-GATA-4,转化至大肠杆菌,质粒抽提后经PCR、双酶切及测序鉴定。结果:成功设计引物扩增出Nkx2-5、GATA-4基因编码区约1 kbp及1.3 kbp的目的片段,PCR和酶切鉴定结果显示,构建的重组真核表达质粒中含有正确的Nkx2-5、GATA-4基因序列。结论:成功构建了含有Nkx2.5、GATA4的真核表达重组质粒,为后续研究奠定基础。Objective： To construct the recombinant plasmid PCDNA3.1-HA-Nkx2-5 and pCDNA3. I-flag-GATA-4 which are capable of expressing in mammalian cells for further study. Methods： Upstream and downstream primers were previ- ously designed and plasmid human brain eDNA was used as template to amplify the coding area of Nkx2-5 and GATA-4 with PCR. Double enzyme digestion was conducted for PCDNA3.1-HA/pCDNA3.1-flag and Nkx2-5/GATA-4. Both frag- ments were connected by using T4 ligase and transferred to DHSa. Then plasmid was extracted and detected by PCR and double enzyme digestion and sequencing. Results： Primers were effectively designed to amplify the coding area of Nkx2-5 and GATA-4 for approximate 1 000 bp and 1 300 bp. The results of PCR and enzyme digestion showed that the recombi- nant expression plasmid had correct codogenic gene fragment. Conclusion： The recombinant expression plasmid of Nkx2- 5 and GATA-4 gene is successfully constructed and identified.

关 键 词：转录因子 NKX2-5 GATA-4 重组质粒 真核表达载体 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]