论文部分内容阅读
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。
Ftsb gene of Streptococcus pyogenes 5005 was cloned in vitro, FtsB protein was expressed and purified, and its iron-binding properties were studied. Firstly, the ftsb gene was amplified from the genome by PCR and ligated into pGEX4T-1 vector. The ftsb gene was transformed into E. coli DH5α for amplification. The correct recombinant vector was transformed into E. coli BL21 for expression and optimization Expression conditions. The expressed products were purified by affinity chromatography, and the iron-binding properties of FtsB were studied. Meanwhile, the binding sites were studied by DEPC-blocked histidine. The prokaryotic expression vector pGEX-ftsb was successfully constructed and a wild-type FtsB protein with a molecular weight of about 33 kD was obtained with a yield of 30 mg / L. Ultraviolet-visible spectroscopy studies show that FtsB and iron pigments at 450 nm after UV absorption peak appears, DEPC blocked histidine experiments showed that FtsB histidine does not participate in iron pigment binding. FtsB protein ferritin binding properties and binding sites for further study of iron transport mechanism in bacteria and the development of vaccine candidates and drug targets to lay a theoretical foundation.