CD基因构建及鉴定

Construction and identification of cytosine deaminase gene

目的利用分子生物学技术,构建胞嘧啶脱胺酶(CD)自杀基因并测定其结构,证实与GENBANK发表的序列具有同源性。为该自杀基因的进一步研究提供良好的实验基础。方法以大肠杆菌JM109基因组为模板,利用聚合酶链式反应(PCR)法,扩增出约为1280 bp CD基因,定向克隆到载体PEGM-T,构建克隆载体。两端分别引入限制性内切酶BamH I、Not I酶切位点,经PCR及酶切鉴定初步证实为重组体后,送上海生物工程技术服务有限公司测序认证。结果经PCR、酶切鉴定和测序鉴定完成CD基因的构建,同源性高达99.30%,完全具备表达该目的基因的要求。结论成功扩增出CD基因,为继续研究打下基础。

Objective To construct cytosine deaminase suicide gene and to estimate its structure. Methods The cytosine deaminase gone fragment was amplified with PCR from the genome DNA of Escherichia coli JM109. The PCR product was digested by the restriction endonucleases BamH I and Not I. The constructed cytosine deaminase gene fragment in the recombinant plasmid was confirmed by PCR and enzyme-digestive method, and the sequence was measured. Results we successfully got the cytosine deaminase gone with the homology of 99.3%. Conclusion The successful construction of cytosine deaminase gene will lay the foundation for further study.