大鼠脑红蛋白(NGB)的原核表达、抗体制备及其细胞分布

脑红蛋白 (NGB)是新发现的与脑内氧供应密切相关的分子 .为了检测细胞内脑红蛋白的表达、亚细胞分布从而对该分子进行深入的功能研究 ,成功地将大鼠脑红蛋白基因编码区构建于原核表达载体pGEX 4T 2 ,转化大肠杆菌BL2 1(DE3) ,获得融合表达产物 .对含有融合蛋白的包含体进行溶解和复性 ,用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化 ,通过免疫家兔获得了兔源性抗NGB多克隆抗体 .采用Western印迹分析技术 ,用该抗体检测NGB基因的真核表达产物 ,证明该抗体有较好的针对NGB蛋白的专一性 ,可用于对NGB的结构和功能研究 .同时 ,用该抗体进行免疫组化分析发现 ,正常成年大鼠神经系统中有较多的NGB免疫反应阳性细胞分布 ,提示NGB是与神经系统功能密切相关的重要分子

Prokaryotic Expression, Antibody Preparation and Cellular Distribution of Rat Neuroglobin

Neuroglobin(NGB), a globin recently identified in the nervous system, is a monomer similar to myoglobin with a high oxygen affinity. In order to study the expression, sub cellular distribution and the function of NGB protein, the coding region of rat neuroglobin(NGB) gene was successfully sub cloned into the prokaryotic expression vector pGEX 4T 2. The vector was then introduced into E. coli BL21(DE3) cells for highly efficient expression. A GST fusion protein of 41 kD was detected in the inclusion body. After refolded and purified, this protein was used to immunize rabbits to produce polyclonal antibodies. The specificity of the anti NGB antibody was successfully proved by Western blotting analysis on both of the prokaryotic and eukaryotic expressed products of NGB protein. Moreover, the NGB immunoreactive cells were found to be widely distributed in adult rat brain including cerebral cortex, hippocampus, cerebellum, etc. All of the results demonstrated that the polyclonal antibody for NGB in this study could be further used for analyzing the function of NGB protein.