化脓性链球菌脂蛋白FtsB的基因克隆、表达、纯化与功能研究  

Cloning,expression,purification and functional study of lipoprotein FtsB from Streptococcus pyogenes

作　　者：李慧[1] 吕洁[1] 王红翠[1] 王南杰[1] 李楠[1] 詹胜[1] 阳小燕[1] 孙雪松[1] 

机构地区：[1]暨南大学生命与健康工程研究院,广东广州510632

出　　处：《微生物学通报》2011年第12期1786-1792,共7页Microbiology China

基　　金：国家自然科学基金项目(No.31000373);广东省自然科学基金项目(No.10451063201005247);暨南大学创新与培育项目(No.21611201)

摘　　要：体外克隆化脓性链球菌5005的ftsb基因,表达并纯化FtsB蛋白,并对其铁色素结合特性进行初步研究。首先通过PCR方法从基因组中扩增ftsb基因,将其连接到pGEX4T-1载体上,转入大肠杆菌DH5α中大量扩增,将测序正确的重组载体转化到表达宿主大肠杆菌BL21进行表达,优化诱导表达条件。利用亲和层析方法纯化表达产物,并对FtsB的铁色素结合特性进行研究,同时通过DEPC封闭组氨酸实验对其结合位点进行初步研究。成功构建原核表达载体pGEX-ftsb,获得分子量约33 kD的野生型FtsB蛋白,产率为30 mg/L。紫外-可见光谱研究表明FtsB和铁色素结合后在450 nm处出现紫外吸收峰,DEPC封闭组氨酸实验证明FtsB中组氨酸不参与铁色素的结合。对FtsB蛋白进行铁色素结合特性和结合位点进行研究,为进一步研究细菌中的铁色素转运机理及开发疫苗候选物和药靶奠定一定的理论基础。This study aims to cloneftsb gene from Streptococcus pyogenes 5005, to express and purify FtsB, and to characterize its ferrichrome binding properties in vitro. DNA fragments encodingfisb was obtained by PCR and inserted into plasmid pGEX4T-1, followed by transformation into Escherichia coli DH5α. The constructed recombinant plasmid was transformed into Escherichia coli BL21 to express FtsB. The conditions used to induce expression were optimized. The protein was purified using affinity chromatography. The ferrichrome binding property was initially characterized, and the binding site was investigated by blocking histidine residues, with DEPC. The 33 kD FtsB was successfully ex- pressed and purified, and the yield was 30 mg/L. The UV-visible spectra showed the FtsB had a visible absorption peak at 425 nm with ferrichrome and that histidine didn＇t participate in ferrichrome binding. This approach reports the ferrichrome binding properties and binding site of FtsB, providing theoretical foundations for the further study of ferrichrome transport mechanism in bacteria and the development of drug target and vaccine candidate.

关 键 词：化脓性链球菌 FtsB蛋白 铁色素 DEPC 

分 类 号：R378[医药卫生—病原生物学]