小鼠热休克蛋白gp96羧基端片段的克隆、表达及纯化

热休克蛋白(Heat shock protein)gp96(Grp94)是近年来新发现的一类糖蛋白，除了分子伴侣的功能外，现有越来越多的献报道了它在先天性免疫和获得性免疫中的重要作用。gp96可以促进抗原呈递细胞的成熟以及细胞因子的分泌。热休克蛋白抗原肽复合体可以引起特异性的细胞毒T淋巴细胞效应，应用这个特点可以设计抗病毒及抗肿瘤药物。但是gp96全长分子量大，蛋白在大肠杆菌中表达量低，不稳定，难纯化。组织提取的gp96又受组织来源和样品量的限制。对gp96的结构和功能的研究带来困难。克隆并表达了小鼠热休克蛋白gp96的羧基端560．751aa约四分之一长的功能片段，该段包含gp96的一个肽结合区和二聚化位点。将该功能片段在大肠杆菌中进行融合表达，纯化后将融合的片段切掉，并对目的片段进行了分析，结果表明该段可能是形成二聚体密切相关的片段，为进一步研究其结构和功能打下基础。

Cloning,Expression and Purification of the C-terminal Section of Murine Heat Shock Protein gp96

Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti virus and anti tumor vaccines. However the expression level of whole length gp96 was relatively low in E.coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment( 560aa 751aa )of murine gp96 into the pGEX 6p 1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.