反义DNA甲基化酶3b基因片断真核表达载体的构建及鉴定  

Construction and indentification of eukaryotic expressionvector for antisense DNA methylferase3b gene fragment

作　　者：左石[1] 罗剑[1] 刘民锋[1] 徐立宁[1] 董泾青[1] 郭伟[1] 邹声泉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院

出　　处：《山东医药》2005年第26期7-9,共3页Shandong Medical Journal

基　　金：国家高科技研究发展计划(863计划)

摘　　要：目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA 3.1(+)的多克隆位点,构建反义DNM T 3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNM T 3b基因片断真核表达载体可为进一步研究DNM T 3b基因功能提供实验工具。Objective： To construct the eukaryotic expression vector for antisense DNA metbylferase 3b （DNMT3b）gene and to provide a tool for studying the DNMT3b gene function. Methods.. PCR primers were designed according to the coding sequence of DNMT3b gene and Xba Ⅰ and Kpn Ⅰ recognition sequence were added to the 5′end of the sense and antisense primer respectively. The 485bp specific PCR fragment was obtained from the cDNA of bile duct cancer cell line QBC-939 by RT-PCR,tbe purified PCR fragment was then inserted reversedly into the multiple cloning site of eukaryotic expression vector pcDNA3. 1 （＋）. The constructed recombinant DNA was identified by PCR confirmation,Xba Ⅰ and Kpn Ⅰ double endonuclease digestion and DNA sequencing. Results： Tbe 467bp specific DNA fragment was obtained by PCR,Xba Ⅰ and Kpn Ⅰ double digestion produced 471bp and 5.4kb DNA fragments which represented the insert DNA fragment aod the vector respectively. The sequencing result confirmed that the sequence of inserted fragment was correct. Conclusion：Eukaryotic expression vector of antisense DNMT3b gene fragment can be constructed successfully by using gene cloning technique.

关 键 词：DNMT3B 反义技术 表达载体 基因克隆 DNA甲基化 

分 类 号：R394-33[医药卫生—医学遗传学]