人类U5·116 ku蛋白基因片段的克隆  

Cloning of Full-length Human U5·116 ku Gene

作　　者：洪敬欣[1] 邵洁[1] 史雪彬[1] 姚智[1] 杨洁[1] 

机构地区：[1]天津医科大学免疫学教研室,300070

出　　处：《天津医药》2007年第6期401-404,共4页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(项目编号:30670441;30300070);天津市科委应用基础研究重点项目(项目编号:043802811);国家教育部新世纪人才支持计划(项目编号:NCET-04-0245);高等学校博士学科点专项基金(项目编号:20040062003)

摘　　要：目的:构建人类U5·116 ku基因全长真核表达质粒。方法:从HeLa细胞中提取总体RNA,一步法合成单链cDNA,利用逆转录聚合酶链反应(RT-PCR)法,扩增出人类U5·116 ku基因两段连续的序列,首先分别克隆至pTZ57R/T载体,两段序列连接成全长后再定向克隆至真核表达载体pcDNA3.1(-),构建pcDNA3.1(-)-U5·116 ku重组质粒。结果:RT-PCR法获得U5·116 ku基因两段连续的序列,长度分别为1672bp和1246bp,分别与pTZ57R/T载体连接后,选择合适的酶切位点再连接成全长序列,然后将全长序列和pcDNA3.1(-)真核表达载体进行连接、转化、酶切鉴定及序列分析后,证实pcDNA3.1(-)-U5·116 ku重组质粒构建成功。结论:成功克隆了人类U5·116 ku的编码基因,并构建了其真核表达质粒pcDNA3.1(-)-U5·116 ku。To construct recombinant eukaryotic expression plasmid about the whole U5.116 ku sequences. Methods： Total RNA was extracted from Hela cell. The eDNA was synthesized by a one-step process. The two consecutive fragments of human U5· 116 ku were amplified by reverse transcriptase polymerase chain reaction （RT-PCR）. The two fragments were cloned to the pTZ57R/T vector and then subcloned to eukaryotic expression vector pcDNA 3.1 （-）, constructed the pcDNA3.1（-）-U5 · 116 ku constitutive plasmid.Rosults： The two fragments of U5.116 ku produced by RT-PCR were 1 672 bp and 1 246 bp. They were ligated with the pTZ57R/T vector respectively and formed the whole sequence by selecting suitable enzyme-digested sites and then subcloned into the pcDNA3.1 （-）. By sequencing, the constitutive plasmid was correct. Conclusion： The cloning of U5·116 ku gene and the construction of the eukaryotic expression vector pcDNA3.1 （-）-U5 · 116 ku has been achieved.

关 键 词：HELA细胞 基因表达 遗传载体 质粒 克隆 分子 逆转录聚合酶链反应 人类 U5·116ku蛋白 

分 类 号：R730.5[医药卫生—肿瘤]