心脏转录因子Nkx2-5、GATA-4真核表达质粒的构建

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cDNA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 pCDNA3.1-flag-GATA-4 which are capable of expressing in mammalian cells for further study.Methods: Upstream and downstream primers were previously designed and plasmid human brain cDNA 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 fragments were connected by using T4 ligase and transferred to DH5a.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 recombinant expression plasmid had correct codogenic gene fragment.Conclusion: The recombinant expression plasmid of Nkx2-5 and GATA-4 gene is suecessfully constructed and identified.