热休克蛋白65及其融合蛋白Hsp65-6×P277的分离纯化和稳定性研究(英文)  

Purification and Stability Research of Mycobacterial Heat Shock Protein 65 and Its Fusion Protein Hsp65-6 P277

作　　者：陈庆梅[1] 吴国君[1] 吴洁[1] 李泰明[1] 曹荣月[1] 刘景晶[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2007年第4期249-254,共6页Pharmaceutical Biotechnology

基　　金：This work was supported by China National Natural Science Fund Committee(Grant No.30500458).

摘　　要：来源于牛分枝杆菌的热休克蛋白65(Hsp65)在没有佐剂的情况下可作为载体分子将抗原表位递呈给免疫系统。热休克蛋白具有蛋白酶的催化活性,容易发生自溶而降解,这制约了基于Hsp65疫苗的研究。该研究中,建立了纯化Hsp65及其融合蛋白Hsp65-6×P277(P277为来源于人热休克蛋白60的肽段,线性重复6次)的方法。Hsp65与Hsp65-6×P277以可溶形式表达于大肠杆菌。在低温及添加EDTA的条件下经细胞裂解、硫酸铵沉淀及阴离子交换树脂等纯化方法,可得到电泳纯的目的蛋白。用纯化的融合蛋白Hsp65-6×P277免疫小鼠,可激发机体产生强烈的免疫应答,该融合蛋白有希望作为免佐剂的糖尿病疫苗加以进一步研究开发。Heat shock protein 65 （Hsp65） of Mycobacterium boris could be used as a suitable and safe carrier molecule for presenting epitopes to the immune system in vivo in the absence of adjuvants. One of the problems associated with the development of Hsp65-based vaccines is that heat shock proteins display proteolytic activity and are easily degraded by autoproteolysis. A method was reported here to purify Hsp65 and the fusion protein Hsp65-6 P277 with 6 tandem repeat P277 sequences derived from human Hsp60. Hsp65 and Hsp65-6 P277 were expressed as soluble proteins in Escherichia coli. The proteins were prepared by means of cell disruption, ammonium sulfate precipitation, and anion exchange column chromatography at 1 mmol/L EDTA and low temperature. Hsp65 and Hsp65-6 P277 could be purified to near homogeneity by minimized self-degradation. Immunization of mice with Hsp65-6 P277 could elicit strong immune response, and the fusion protein Hsp65-6 P277 might be further developed to be a vaccine against diabetes in the absence of adjuvants.

关 键 词：热休克蛋白65(Hsp65) 融合蛋白 纯化 降解 稳定性 

分 类 号：R378.911[医药卫生—病原生物学]