细菌透明质酸酶基因工程菌的构建及高效表达  

Construction and High Expression of Hyaluronidase Gene from Streptococcus zooepidemicus in Escherichia coli

作　　者：钊倩倩[1,2] 张秀华[1,2] 刘飞[1,2] 李珍爱[1,2] 朱希强[1,2,3] 凌沛学 

机构地区：[1]山东省生物药物研究院山东省生物药物重点实验室,山东济南250101 [2]山东省药学科学院山东省多糖类药物工程实验室,山东济南250101 [3]山东福瑞达医药集团公司,山东济南250101

出　　处：《食品与药品》2014年第5期305-308,共4页Food and Drug

基　　金：济南市重大专项(编号:201403009)

摘　　要：目的将兽疫链球菌透明质酸酶hyl基因在大肠杆菌原核表达系统中高效分泌表达。方法通过PCR方法扩增得兽疫链球菌hyl基因,Xho I和Nco I双酶切后,连入表达载体pET26b(+),并转化入大肠杆菌,经低温异丙基-β-D-硫代半乳糖苷或乳糖诱导表达,DNS法检测胞外透明质酸酶活性。结果成功构建含兽疫链球菌透明质酸酶基因hyl的大肠杆菌基因工程菌株,经IPTG诱导并添加甘氨酸后胞外透明质酸酶活性高达5.3×104 U/mL。结论实现了透明质酸酶在原核系统中的表达,为工业化生产奠定了基础,具有很好的市场前景。Objective To obtain high level secretory expression of hyaluronidase hyl gene from Streptococcus zooepidemicus in Escherichia coll. Methods The Streptococcus zooepidemicus hyaluronidase hyl gene was amplified by polymerase chain reaction（PCR）, connected to pET26b（＋） vector after digested by Xho I and Nco I, and transformed into Escherichia coli BL21 strain. The recombinant plasmid pET26-hyl was expressed in Escherichia coli BL21, which was induced by isopropyl fl-D-l-thiogalactopyranoside （IPTG） or lactose at a low temperature. The extracellular hyaluronidase activity was detected by 3,5-dinitrosalicylic acid（DNS） method. Results The Streptococcus zooepidemicus hyaluronidase hyl gene was successfully expressed in Escherichia coli at a high level. The crude enzyme activity detected by DNS method was up to 5.3 ~ 104 U/mL after induction by IPTG and glycine. Conclusion An extracellular prokaryotic expression system can be constructed in Escherichia coli to express hyaluronidase, which lays the foundation for industrial production and has good market prospects.

关 键 词：兽疫链球菌 透明质酸酶 大肠杆菌 基因工程 分泌表达 

分 类 号：Q78[生物学—分子生物学]