黄杆菌肝素酶Ⅰ基因的克隆与表达.pdfVIP

食品与药品 Food and Drug 2008年第1O卷第O7期 5 · 论 著 黄杆菌肝素酶I基因的克隆与表达 傅文彬，，熊可强 ，阚梦元 ，赵 健h，李素霞 ，姬胜利。，袁勤生 (1．华东理工大学生物反应器工程国家重点实验室，上海 200237；2．山东大学药学院，山东 济南 250012) 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一22b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一22b表达载体 中。重组质粒转入E．coli BL21(DE3)后表达的蛋白质经SDS—PAGE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／LA…。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 f1．State Key Laboratory ofBioreactor Engineering，East China University ofScience and Technology,Shang— hai 200237，China；2．School ofPharmaceutical 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一22b after sequencing，then，the recombinant plasmid was expressed in E．coli BL21 (DE3)and the enzyme activity of recombinant heparinase 1 was verified by Azure A assay．Results The recombi— nant pET一22b with the PCR product of heparinase 1 which was confirmed by DNA sequencing was successfully constructed．The expression product of recombinant plasmid pET一22b in E．coli BL2 1(DE3)was tested by SDS— PAGE electrophoresis． e enzyme activity of recombinant heparinase 1 was 50 U几A ．Conclusion e 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 为2-3个硫酸化的二糖GlcNS(6S)一IdoUA(2S)连接的 解肝素大分子中糖苷键的酶类。最初由肝素黄杆菌 糖苷键 (因该部位硫酸化程度较高，又称s— (F．he