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[1] 章锦才[2] 张蕴惠[3] 

机构地区：[1]南京医科大学口腔医院口腔内科,江苏南京210029 [2]广东省口腔医院口腔内科,广东广州510280 [3]四川大学华西口腔医院牙周病科,四川成都610041

出　　处：《华西口腔医学杂志》2005年第4期335-337,共3页West China Journal of Stomatology

基　　金：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)

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。Objective Human soluble tumor necrosis factor receptor（sTNFR） can interfere with the biological functions of interleukin- 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（ ＋ ） DNA were digested with Kpn I and Xho I . After purification, the two fragments were ligated by TakaRa DNA Ligation Kit （Ver 2.0）. This recombinant DNA was then transformed into E. Coli Competent Cells JM109 and positive clones were selected on the LB agarese plate containing ampicillin （80μg/ul）. Results Six single clones were indentified by double digestion with kpn I and xho I and two fragments with the size of 5.4 kb and 1.0 kb were produced as expected. Conehusion The sTNFR gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3.1 （ ＋ ） by recombination technology in vitro.

关 键 词：人可溶性肿瘤坏死因子α受体 真核载体 质粒 

分 类 号：R780.2[医药卫生—口腔医学]