| 靶向克隆法构建NY-ESO-1基因的原核表达载体 |
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| 引用本文： | 俞远东,徐少勇,王斌,王家宁,黄永章.靶向克隆法构建NY-ESO-1基因的原核表达载体[J].武汉大学学报(医学版),2009,30(2). |
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| 作者姓名： | 俞远东 徐少勇 王斌 王家宁 黄永章 |
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| 作者单位： | 1. 郧阳医学院附属人民医院肿瘤科消化内科,湖北,十堰,442000
2. 郧阳医学院附属人民医院肿瘤科临床医学研究所,湖北,十堰,442000 |
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| 基金项目： | 湖北省科技厅资助项目(编号:2004AA304B08) |
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| 摘 要： | 目的:利用PCR产物靶向克隆法构建人癌-睾丸抗原NY-ESO-1的原核表达载体。方法:从人新鲜的食管癌组织中提取总RNA,通过RT-PCR技术扩增NY-ES0-1基因3′端的340bp片段,采用靶向克隆法将目的基因插入到原核表达载体pET-15b中,得到重组表达质粒pET-15b-NY-ESO-1,经过筛选,挑选出阳性克隆进行XhoⅠ和BamHⅠ双酶切图谱分析、PCR检测和扩增产物核苷酸序列分析等,鉴定所构建的原核表达载体。结果:扩增得到NY-ESO-1基因片段,经测序证实与GenBank公布的序列一致,重组的质粒经过PCR,酶切鉴定获得了一个大小为340bp的特征性片段,证明重组质粒中已成功的插入了目的基因NY-ESO-1。结论:靶向克隆法可快速、高效地构建人NY-ESO-1基因的原核表达载体,为制备pET-15b-NY-ESO-1融合蛋白奠定了基础。
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| 关 键 词： | NY-ESO-1 靶向克隆 原核表达 |
Construction of NY-ESO-1 Gene Prokaryotic Expression Vector by LP Recco PCR Cloning |
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| YU Yuandong,XU Shaoyong,WANG Bin,WANG Jianing,HUANG Yongzhang.Construction of NY-ESO-1 Gene Prokaryotic Expression Vector by LP Recco PCR Cloning[J].Medical Journal of Wuhan University,2009,30(2). |
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| Authors: | YU Yuandong XU Shaoyong WANG Bin WANG Jianing HUANG Yongzhang |
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| Institution: | YU Yu,ong1,XU Shaoyong1,WANG Bin1,WANG Jianing2,HUANG Yongzhang21Dept.of Gastroenterology,2Institute of Clinical Medicine,People's Hospital,Yunyang Medical College,Shiyan 442000,China |
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| Abstract: | Objective: To construct the prokaryotic expression vector of human cancer-testis gene NY-ESO-1 by the method of LP Recco PCR Cloning.Methods: Total RNA was extracted from esophageal carcinoma tissue,the primer containing special enzyme was designed,NY-ESO-1 fragment was amplified by RT-PCR,and then was cloned into the expression vector pET-15b by LP Recco PCR Cloning.The recombinants plasmid pET-15b-NY-ESO-1 were identified by restriction endonucleases digest analysis and sequence analysis.Results: The sequ... |
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| Keywords: | NY-ESO-1 Gene LP Recco PCR Cloning Prokaryotic Expression |
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