黄杆菌肝素酶I基因克隆及表达.docVIP

黄杆菌肝素酶I基因克隆及表达摘要：目的 克隆并表达黄杆菌肝素酶I（HepI）基因。方法通过PCR从黄杆菌基因组DNA中扩增黄杆菌HepI 基因，验证后插入到表达质粒pET-22b 中构建表达载体，重组质粒转化大肠杆菌BL21(DE3)进行蛋白质表达，并用Azure A法测定酶活性。结果PCR 扩增得到序列正确的HepI基因，并将该基因成功插入到pET-22b 表达载体中。重组质粒转入E.coli BL21(DE3)后表达的蛋白质经SDS分析，与目的蛋白质的相对分子质量基本一致，其活性约为50 U/LA600。结论克隆并成功表达了黄杆菌HepI基因。 关键词：肝素酶I；克隆；基因表达；酶活性 中图分类号：Q557文献标识码：A文章编号：1672-979X(2008)07-0005-03 Cloning and Expression of Flavobacterium heparinum Heparinase I Gene FU Wen-bin, XIONG Ke-qiang, KAN Meng-yuan, ZHAO Jian*, LI Su-xia, JI Sheng-li, YUAN Qin-sheng (1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; 2. School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China) Abstract：Objective To clone and express the heparinase I gene from Flavobacterium heparinum. Methods The heparinase I gene was amplified with PCR from the genomic DNA of Flavobacterium heparinum, and it was linked into the expression plasmid pET-22b after sequencing, then, the recombinant plasmid was expressed in E.coli BL21 (DE3) and the enzyme activity of recombinant heparinase I was verified by Azure A assay. Results The recombinant pET-22b with the PCR product of heparinase I which was confirmed by DNA sequencing was successfully constructed. The expression product of recombinant plasmid pET-22b in E.coli BL21 (DE3) was tested by SDSelectrophoresis. The enzyme activity of recombinant heparinase I was 50 U/LA600. Conclusion The heparinase I gene from Flavobacterium heparinum can be cloned and expressed successfully. Key words：heparinase I; clone; genetic expression; enzyme activity 肝素酶是目前发现的自然界中唯一能够特异性裂解肝素大分子中糖苷键的酶类。最初由肝素黄杆菌（F. heparinum）中分离得到，后又陆续在其它微生物和动物组织中发现。该酶可作为分析肝素多糖分子及其降解产物序列的有用工具[1]。国外已有用肝素酶制备抗血栓药物低分子肝素（LMWH）的报道。目前已从肝素黄杆菌中发现3种肝素酶（heparinase，Hep）：HepI、HepII和HepIII。3种肝素酶各有其不同的底物特异性，HepI作用位点为2~3个硫酸化的二糖GlcNS(6S)-IdoUA(2S)连接的糖苷键（因该部位硫酸化程度较高，又称S-domain）；HepIII作用位点为GlcNS/NAC-GlcUA连接的N-乙酰化寡糖区域（又称NAC-domain）；HpaII具有广泛的选择性，兼有HepI和HepIII活性，