化脓链球菌中金属结合蛋白Dpr的表达与纯化.pdfVIP

中国科技论文在线 化脓链球菌中金属结合蛋白 Dpr 的表达与 纯化# 王南杰，李楠，徐倩，阳小燕，孙雪松** 5 （暨南大学生命与健康工程研究院，广州 510632 ） 摘要：化脓链球菌是 目前已知的引起细菌性炎症的主要病原体之一。 Dpr 是化脓链球菌细 胞内与金属铁的结合转运相关。本文通过 PCR 扩增化脓链球菌 M5005 的Dpr 基因片段，通 过双酶切，用 T4 连接酶连接至同种酶双酶切处理后 的含有 GST 标签 的质粒载体 PGEX-4T-1/GST 中，转化至大肠杆菌 BL-21 后，用IPTG 诱导重组蛋白的表达。得到的重 10 组蛋白通过 GST 亲和层析柱纯化以后，用外切酶-凝血酶切除 GST 标签，得到纯化的Dpr 蛋白。通过 ICP-MS 测得 Dpr 与金属Fe 的结合比例是 1:1，与Mn ，Zn 的结合比较微弱。 关键词：化脓链球菌；Dpr ；基因克隆；融合蛋白；ICP-MS 中图分类号：Q519 15 Expression and purification of iron-binding protein Dpr of Streptococcus pyogenes WANG Nanjie, LI Nan, XU Qian, YANG Xiaoyan, SUN Xuesong (Institute of Life and Health Engineering, Jinan University, GuangZhou 510632) Abstract: Streptococcus pyogenes is one of the major pathogens of bacterial pneumonia . Dpr 20 have been reported related to c iron transport in Streptococcus pyogenes. Dpr gene fragment was amplified by polymerase chain reaction (PCR) from Streptococcus pyogenes M5005 and cloned into the expression vector PGEX-4T-1/GST containing GST tag. Dpr gene and vetor was connected with T4 DNA ligase after digestion with EcoRI and SalI.Then the recombinant plasmid was transformed into Escherichia coli BL-21. The expression of the recombinant protein was 25 induced with IPTG. The obtained recombinant protein later was purified by GST affinity column, digested with the thrombin to remove GST tag and obtain pure Dpr