人可溶性肿瘤坏死因子α受体真核表达载体pcDNA3.1(+)/sTNFR的构建
Construction of the Eukaryote Expression Vector of Human Soluble Tumor Necrosis Factor Receptor
作者：徐艳,章锦才,张蕴惠
Author：XUYan1, ZHANG Jin-cai2, ZHANG Yun-hui3
收稿日期：2005-08-25 年卷（期）页码：2005,23(04):335-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：人可溶性肿瘤坏死因子α受体,真核载体,质粒,
Key words：human soluble tumor necrosis factor receptor,eukaryote vector,plasmid,
基金项目：
国家科技部“九五”攻关资助项目;江苏省自然科学基金创新人才资助项目(BK2003422);南京医科大学科技创新基金资助项目 (CX2002004)
中文摘要
目的 构建人可溶性肿瘤坏死因子α受体(sTNFR)真核表达载体pcDNA3·1(+)/sTNFR,为研究人sTNFR 在哺乳动物细胞中的合成与表达提供条件。方法 采用体外重组技术,将sTNFR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,体外连接这两个酶切片段进行定向重组,再将重组DNA转化感受态细胞E.ColiCompetent Cells JM109。复苏后,在含氨苄青霉素的LB固体培养基上筛选阳性克隆,进行酶切及测序鉴定。结果 挑取的LB固体培养基上的6个单菌落经证实均为阳性克隆,即sTNFR与 pcDNA3·1(+)体外重组成功。结论 将sTNFR cDNA成功地插入了真核表达载体pcDNA3·1(+)中,构建了质粒 pcDNA3·1(+)/sTNFR。
英文摘要
Objective Human soluble tumor necrosis factor receptor(sTNFR) can interfere with the biological functions of in- terleukin-1, which may be appropriate to the treatment of periodontitis. The eukaryote expression vector of the human sTNFR gene must be constructed prior to conducting transgene therapy of periodontitis.Methods Both sTNFR gene and plasmid pcDNA 3·1(+) DNAwere digested withKpnⅠandXhoⅠ. After purification, the two fragmentswere ligated byTakaRa DNALigation Kit (Ver 2·0). This recombinant DNAwas then transformed intoE.ColiCompetent Cells JM109 and positive cloneswere select- ed on the LB agarose plate containing ampicillin (80μg/ul).Results Six single cloneswere indentified by double digestionwith kpnⅠandxhoⅠand two fragments with the size of 5·4 kb and 1·0 kb were produced as expected.Conclusion The sTNFR gene was successfully inserted into the eukaryote expression vector plasmid pcDNA3·1(+) by recombination technologyin vitro
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