人Prp8蛋白基因片段的克隆  

Cloning of Sequence of Human Prp8 Protein

作　　者：洪敬欣[1] 步天栩[1] 史雪彬[1] 邵洁[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11期801-803,I0001,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Prp8基因全长真核表达质粒。方法:从HeLa细胞中提取总体RNA,两步法合成cDNA,利用逆转录聚合酶链反应(RT-PCR)法,扩增出人Prp8基因的四段序列,首先克隆至pTZ57R/T载体,再定向克隆至真核表达载体pcDNA3.1(+),构建pcDNA3.1(+)-Prp8重组质粒。结果:RT-PCR法获得Prp8基因的4段序列,长度分别为2025、1090、1966、1963bp,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(+)真核表达载体连接、转化、酶切鉴定及序列分析后,证实pcDNA3.1(+)-Prp8重组质粒构建成功。结论:成功克隆了人Prp8的编码基因,并构建了表达载体pcDNA3.1(+)-Prp8。Objective： To construct the eukaryotic expression plasmid about the whole Prp8 sequence. Methods： The total RNA was extracted from Hela cell and the cDNA was synthesized by two steps,and the four fragments of human Prp8 were amplified by RT-PCR. The four fragments were cloned to the pTZ57R/T vector,subcloned to eukaryotic expression vector pcDNA3.1（＋）and constructed the pcDNA3.1（＋）-Prp8 constitutive plasmid. Results： The four fragments of Prp8 produced by RT-PCR were 2 025 bp,1 090 bp,1 966 bp and 1 963 bp in size respectively,they were ligated with the pTZ57R/T vector,and formed the whole sequence by selecting suitable enzyme-digested sites and subcloned into the pcDNA3.1（＋）. The constitutive plasmid was successful confirmed by sequencing. Conclusion： The cloning of Prp8 gene and the construction of the eukaryotic expression vector pcDNA3.1（＋）-Prp8 are achieved.

关 键 词：抗体 单克隆 基因表达 遗传载体 质粒 真核细胞 逆转录聚合酶链反应 人类 

分 类 号：R382.31[医药卫生—医学寄生虫学]