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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
引用本文:陈庆梅,吴国君,吴洁,李泰明,曹荣月,刘景晶.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[J].药物生物技术,2007,14(4).
作者姓名:陈庆梅  吴国君  吴洁  李泰明  曹荣月  刘景晶
作者单位:中国药科大学,生命科学与技术学院,江苏,南京210009
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。

关 键 词:热休克蛋白65(Hsp65)  融合蛋白  纯化  降解  稳定性

Purification and Stability Research of Mycobacterial Heat Shock Protein 65 and Its Fusion Protein Hsp65-6 P277
CHEN Qing-mei,WU Guo-jun,WU Jie,LI Tai-ming,CAO Rong-yue,LIU Jing-jing.Purification and Stability Research of Mycobacterial Heat Shock Protein 65 and Its Fusion Protein Hsp65-6 P277[J].Pharmaceutical Biotechnology,2007,14(4).
Authors:CHEN Qing-mei  WU Guo-jun  WU Jie  LI Tai-ming  CAO Rong-yue  LIU Jing-jing
Abstract:Heat shock protein 65(Hsp65) of Mycobacterium bovis 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 1mmol/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.
Keywords:Heat shock protein 65 (Hsp65)  Fusion protein  Purification  Degradation  Stability
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