心脏转录因子Nkx2.5真核表达质粒的构建

目的构建人类心脏特殊转录因子Nkx2.5的真核表达质粒,为进一步探讨Nkx2.5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pCMV6-XL5-Nkx2.5为模板,并设计上下游引物扩增Nkx2.5编码区序列,将真核表达载体pCMV-HA和Nkx2.5的PCR产物分别在双酶切后用T4连接酶连接,构建重组质粒pCMV-HA-Nkx2.5,转化至大肠杆菌,提取质粒后经PCR、双酶切及测序鉴定。结果成功设计引物扩增出Nkx2.5基因编码区约1 000 bp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基因序列。结论成功构建了含有Nkx2.5的真核表达重组质粒,为后续研究奠定基础。

Construction of Eukaryotic expression Recombinant Plasmid pCMV-HA-Nkx2.5

Objective To construct the recombinant plasmid pCMV-HA-Nkx2.5 which is capable of expressing in mammalian cells for further study.Methods Upstream and downstream primers were previously designed and plasmid pCMV6-XL5-Nkx2.5 was used as template to amplify the coding area of Nkx2.5 with PCR.Double enzyme digestion was conducted for pCMV-HA and Nkx2.5.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 Primers were effectively designed to amplify the coding area of Nkx2.5 for approximate 1 000 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 gene is successfully constructed and identified.