Xu Yan, Zhang Jin-Cai, Zhang Yun-Hui
Dept. of Oral Medicine, College of Stomatology, Nanjing Medical University, Nanjing 210029, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2005 Aug;23(4):335-7.
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.
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 agarose plate containing ampicillin (80 microg/ul).
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.
The sTNFR gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3.1 (+) by recombination technology in vitro.
人可溶性肿瘤坏死因子受体（sTNFR）可干扰白细胞介素-1的生物学功能，这可能适用于牙周炎的治疗。在进行牙周炎转基因治疗之前，必须构建人sTNFR基因的真核表达载体。
用Kpn I和Xho I对sTNFR基因和质粒pcDNA 3.1(+) DNA进行酶切。纯化后，用宝生物DNA连接试剂盒（Ver 2.0）将两个片段连接起来。然后将这种重组DNA转化到大肠杆菌感受态细胞JM109中，并在含有氨苄青霉素（80微克/微升）的LB琼脂平板上筛选阳性克隆。
通过用Kpn I和Xho I双酶切鉴定出6个单克隆，预期产生了大小为5.4 kb和1.0 kb的两个片段。
通过体外重组技术成功地将sTNFR基因插入到真核表达载体质粒pcDNA 3.1(+)中。
