人可溶性白介素-1受体真核表达载体pcDNA3/sIL-1R的构建
Construction of Eukaryote Expression Vector Carrying Human Soluble Interleukin-1 Receptor Gene
作者：李鹏,徐艳,张蕴惠,章锦才,王大章
Author：LIPeng*,XUYan,ZHANG Yunhui,et al.
收稿日期：2003-04-25 年卷（期）页码：2003,21(02):140-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：人可溶性白细胞介素-1受体,真核表达载体,质粒pcDNA 3·1(+),
Key words：human soluble interleukin-1 receptor,eukaryote expression vector,plasmid pcDNA 3·1(+),
基金项目：
本课题为国家自然科学基金(编号39170781)和浙江省教育厅科研基金(编号20010283)资助项目
中文摘要
目的 本研究旨在构建人可溶性白介素-1受体(sIL-1R)真核表达载体pcDNA3/sIL-1R,为研究骨关节病的基因治疗奠定实验基础。方法 将人sIL-1R基因的RT-PCR纯化产物及质粒pcDNA3·1(+) DNA经KpnI和 XhoI双酶切、胶回收纯化酶切片段后,体外连接酶切片段,使其定向重组,再将重组DNA转化E.coliCompetent Cells JM109,经复苏后,在含Ampr的LB固体培养基上筛选出阳性克隆。结果 挑取的LB固体培养基上的6个单菌落经酶切及测序鉴定,证实均为阳性克隆,即sIL-1R与pcDNA3·1(+)体外重组成功。结论 采用体外重组技术,成功地将人sIL-1R cDNA插入了真核表达载体pcDNA3·1(+)中。
英文摘要
Objective The purpose of this study was to construct a eukaryote expression vector carrying human sIL-1R gene.Methods Both sIL-1R gene and plasmid pcDNA3·1(+) DNAwere digestedwithKpnIandXhoI. After purification, the two fragments obtained were ligated by usingTakaRa DNALigation Kit. This recombinant DNAwas then transformed intoE.coli Competent Cells JM109 and positive cloneswere selected on the LB agarose plate containingAmpicillin (80μg/ml).Results Six single clones were identified by double digestionwithKpnIandXhoI, and two fragmentswith the size of 5·4 kb and 1·0 kbwere produced as expected.Conclusion The sIL-1R gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3·1(+) by the recombination techniquein vitro.
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