含HSV－tk基因的人大肠癌特异性逆转录病毒载体的构建

Construction of retroviral vector containing HSV-tk gene for colorectal carcinoma tissue-specific gene therapy

目的：为使自杀基因在大肠癌细胞中特异性表达，以达到靶向基因治疗的目的，首先构建以ＣＥＡ基因转录调控序列驱动的重组逆转录病毒载体。方法：将目的基因片段ＨＳＶ－ｔｋ定向克隆入ｐＵＣ１１８质粒载体中，以相应的内切酶取出合适的酶切片段后，与来自ｐＣＥＡ４２４／２ＣＡＴ质粒的ＣＥＡ５＇转录调控序列相连接，克隆入逆转录病毒载体中。结果：经鉴定及筛选，构建成重组逆转录病毒载体Ｇ１ＣＥＡｔｋＮａ。结论：Ｇ１ＣＥＡｔｋＮａ的成功构建，为进一步研究大肠癌组织特异性基因治疗奠定了基础。

Objective : In order to make suicide gene express in colorectal carcinoma cells for targeted killing carcinoma cells, we constructed the chimeric CEA/tk gene contained in a retroviral vector. Methods: A 1. 3 kb EcoR I /BamH I fragment of the HSV-tk gene was isolated from pLNtk, and inserted into pUC118, and then the Hind Ⅲ /EcoR I tk fragment was isolated from it. A 420 bp Hind Ⅲ /BamH I fragment in pCEA424/2CAT was isolated and subcloned into pUC118 to harvest Hind Ⅲ /EcoR I CEATRS according to the same procedure above. These two fragments were ligated with the retroviral vector G1 Na which had been lineared and dephosphorylated by using T4 DNA ligase. Results: Using Sal I to identify a positive clone B5,G1CEAtkNa was constructed. Conclusion: Successful construction of G1CEAtkNa provides a basis for colorectal carcinoma specific prodrug gene therapy.