心脏转录因子GATA结合蛋白4真核表达质粒的构建

Construction of eukaryotic expression recombinant plasmid pCMV-Myc-GATA4

背景:有研究表明心脏转录因子GATA结合蛋白4(GATA4)与先天性心脏病的发生有密切的关系,但其机制不明,而目前尚未查及GATA4真核表达质粒构建类的报道。目的:构建人类心脏特殊转录因子GATA4的真核表达质粒。方法:对重组质粒pUC57-GATA4进行酶切获得GATA4基因编码区序列,将真核表达载体pCMV-Myc和GATA4基因在双酶切后用T4连接酶连接,构建重组质粒pCMV-Myc-GATA4,转化至大肠杆菌,提取质粒后经聚合酶链反应、双酶切及测序鉴定。结果与结论:实验成功酶切重组质粒获得GATA4基因编码区约1.3kb的目的片段,聚合酶链反应和酶切鉴定以及基因测序结果显示,构建的重组真核表达质粒中含有正确的GATA4基因序列,证实成功构建了含有GATA4基因的真核表达重组质粒。

BACKGROUND：Studies showed that heart transcription factor GATA bind protein 4（GATA4） are closely related to congenital heart disease.However,there are few reports about the construction of GATA4 expression plasmid and how it effects the development and proliferation of myocardial cell.OBJECTIVE：To construct the recombinant plasmid pCMV-Myc-GATA4.METHODS：Recombinant plasmid pUC57-GATA4 was digested by EcoRⅠand Kpn I to obtain the target gene GATA4.Double enzyme digestion was conducted for pCMV-Myc and GATA4.Both fragments were connected by using T4 ligase and transferred to DH5α.Then plasmid was extracted and detected by PCR and double enzyme digestion and sequencing.RESULTS AND CONCLUSION：The coding area of GATA4 for approximate 1.3 kb was obtained.The results of PCR,enzyme digestion and gene sequencing showed that the recombinant expression plasmid had correct codogenic gene fragment.The recombinant expression plasmid of GATA4 gene is successfully constructed and identified.