携带Egr-1启动子和Endostatin基因的腺病毒穿梭质粒构建及鉴定

Construction and identification of recombinant adenoviral shuttle vector with Egr-1 promoter and Endostatin gene

目的构建含辐射敏感启动子Egr-1和人内皮抑素(endostatin)基因的腺病毒穿梭质粒pshuttle-Egr1-Endostatin。方法利用RT-PCR方法从人胎肝中扩增出人endostatin cDNA序列,从T-Egr1质粒中扩增出Egr-1基因序列,并将它们连接到pMD19T质粒进行测序,利用基因重组技术构建含有辐射诱导启动子Egr-1的腺病毒穿梭质粒pshuttle-Egr1-Endostatin。结果经测序证实,获得的人endostatin基因和Egr-1基因与GenBank公布的完全一致,表明克隆人endostatin基因和Egr-1启动子成功,并经鉴定证实含有辐射诱导启动子Egr-1的腺病毒穿梭质粒pshuttle-Egr1-Endostatin构建正确。结论本实验成功克隆了人endostatin基因和Egr-1启动子并构建了含辐射敏感启动子的腺病毒穿梭质粒pshuttle-Egr1-Endostatin。

Objective To construct a recombinant adenoviral shuttle vector pshuttle-Egr1-Endostatin which contains endostafin and Egr-1 which acts as the radiation-sensitive promoter and can induce its downstream gene expression. Methods The endostatin gene fragment was amplificated from the fetal fiver by RT-PCR.The Egr-1 gene was acquired from plasmld T-Egr1 .Then the two genes were ligated to pMD19T and sequenced them.Finally,using the gene recombinant technique,the recombinant plasmid pshuttle-Egr1- Endostatin with radiatlon-inducible promoter Egr-1 was constructed. Results The sequence of both acquired endostatin and Egr-1 genes were in concordance with published that on Genebank confirmed by sequencing,indicating that the two genes were cloned successfully. Moreover, the recombinant ph, unid pshuttle-Egrl-Endostatin was constructed correctly by PCR and restrictive digestion identification.Conclusion The endostatin gene and the Egr-1 promoter were cloned and the recombinant plasmid pshuttle-Egr1-Endostatin with radiation-sensitive promoter was constructed suceessfully in our research.