老年小鼠脑和血清NAMPT变化及重组人的NAMPT的制备和鉴定
发布时间：2019-03-01 16:15
【摘要】：尼克酰胺磷酸核糖转移酶(NAMPT)是合成NAD的关键酶。已有大量文献表明,NAMPT与许多衰老相关疾病密切相关,如肿瘤、糖尿病等。其中NAMPT大量表达于脑中的神经元,且在脑缺血过程中起保护作用。然而有许多问题仍不明确,如NAMPT在脑中的具体分布、NAMPT在老年过程中的表达变化,以及NAMPT表达和活性的变化是否与动物的神经行为相关等。在本研究中,我们发现小鼠在老年过程中,NAMPT的表达发生特异性的改变,其中皮层和海马的NAMPT水平显著下降而血清的NAMPT水平则显著增加；老年小鼠的小胶质细胞出现NAMPT表达,而年轻小鼠的小胶质细胞却不表达NAMPT;老年小鼠海马和小脑的NAD水平显著下降,而皮层和纹状体的NAD水平则没有显著改变。行为学试验显示,老年小鼠在开放场中的运动量、中心区的探索以及恐惧记忆水平都显著下降；皮层NAMPT水平与小鼠运动量正相关,而小脑和血清的NAMPT水平与小鼠运动量负相关,脑和血清的NAMPT与小鼠在开放场中心区的探索及恐惧记忆没有明显相关。因此,我们的研究展示了小鼠在衰老过程中脑和血清NAMPT表达、分布及活性的变化特征,提示NAMPT可能与动物老年过程中运动能力下降的现象有关。 目的：制备和纯化重组人NAMPT和NAMPT (H247A)蛋白,并对其体外酶活性进行检测。 方法：以pcDNA3.1-hnampt为模板,通过PCR扩增获得两端分别为BamHⅠ和NdeⅠ酶切位点的hnampt片断,将该片断与pET-11a(+)表达型载体连接,通过点突变获得pET-11a(+)-hnampt (H247A),以测序鉴定构建质粒。将野生型和突变型分别转化至BL21star大肠杆菌,以IPTG诱导蛋白表达,以镍柱和分子筛纯化目标蛋白,以SDS凝胶电泳和质谱鉴定目标蛋白,以核磁共振的方法检测两个重组蛋白在体外酶活性。 结果：测序结果表明,pET-11a(+)-hnampt(野生型)及pET-11a(+)-hnampt (H247A)(突变型)表达载体构建成功,突变位点正确。两种蛋白均在BL21star大肠杆菌获得表达,裂解后为可溶性蛋白,通过镍柱、分子筛获得纯化蛋白,SDS凝胶电泳和质谱鉴定证明重组蛋白为分子量约56 KD的NAMPT。核磁共振体外酶活性检测发现野生型NAMPT具有酶活性,而NAMPT (H247A)的酶活性降低了约5-10倍。 结论：成功获得了有体外酶活性的人NAMPT蛋白和酶活性较低的人NAMPT (H247A)蛋白,为NAMPT蛋白作用研究提供了基础。
[Abstract]:Nicotinamide phosphate ribosyltransferase (NAMPT) is the key enzyme in the synthesis of NAD. A large number of literatures have shown that NAMPT is closely related to many aging-related diseases, such as cancer, diabetes, and so on. NAMPT is expressed in a large number of neurons in the brain, and plays a protective role in the course of cerebral ischemia. However, many problems remain unclear, such as the specific distribution of NAMPT in the brain, the change of NAMPT expression in the elderly, and whether the change of NAMPT expression and activity is related to the neurobehavior of animals, and so on. In this study, we found that the expression of NAMPT in aged mice changed specifically, in which the level of NAMPT in cortex and hippocampus decreased significantly, while the level of NAMPT in serum increased significantly. The expression of NAMPT was observed in the microglia of the aged mice, while the level of NAD in the hippocampus and cerebellum of the aged mice was significantly lower than that of the young mice, while the NAD level of the cortex and striatum did not change significantly. The expression of NAD in the hippocampus and cerebellum of the aged mice was not significantly changed. Behavioral tests showed that the amount of exercise in the open field, the exploration of the central area and the level of fear memory were significantly decreased in the aged mice. The level of NAMPT in cortex was positively correlated with the amount of exercise in mice, while the level of NAMPT in cerebellum and serum was negatively correlated with the amount of exercise in mice. There was no significant correlation between NAMPT in brain and serum and the exploration of the central area of the open field and fear memory in mice. Therefore, our study showed the changes of the expression, distribution and activity of NAMPT in brain and serum of mice during aging, suggesting that NAMPT may be related to the decline of motor ability during the aging of animals. Aim: to prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzyme activities in vitro. Methods: using pcDNA3.1-hnampt as template, the hnampt fragment with BamH 鈪，
本文编号：2432586
[Abstract]:Nicotinamide phosphate ribosyltransferase (NAMPT) is the key enzyme in the synthesis of NAD. A large number of literatures have shown that NAMPT is closely related to many aging-related diseases, such as cancer, diabetes, and so on. NAMPT is expressed in a large number of neurons in the brain, and plays a protective role in the course of cerebral ischemia. However, many problems remain unclear, such as the specific distribution of NAMPT in the brain, the change of NAMPT expression in the elderly, and whether the change of NAMPT expression and activity is related to the neurobehavior of animals, and so on. In this study, we found that the expression of NAMPT in aged mice changed specifically, in which the level of NAMPT in cortex and hippocampus decreased significantly, while the level of NAMPT in serum increased significantly. The expression of NAMPT was observed in the microglia of the aged mice, while the level of NAD in the hippocampus and cerebellum of the aged mice was significantly lower than that of the young mice, while the NAD level of the cortex and striatum did not change significantly. The expression of NAD in the hippocampus and cerebellum of the aged mice was not significantly changed. Behavioral tests showed that the amount of exercise in the open field, the exploration of the central area and the level of fear memory were significantly decreased in the aged mice. The level of NAMPT in cortex was positively correlated with the amount of exercise in mice, while the level of NAMPT in cerebellum and serum was negatively correlated with the amount of exercise in mice. There was no significant correlation between NAMPT in brain and serum and the exploration of the central area of the open field and fear memory in mice. Therefore, our study showed the changes of the expression, distribution and activity of NAMPT in brain and serum of mice during aging, suggesting that NAMPT may be related to the decline of motor ability during the aging of animals. Aim: to prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzyme activities in vitro. Methods: using pcDNA3.1-hnampt as template, the hnampt fragment with BamH 鈪，
本文编号：2432586
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