老年小鼠脑和血清NAMPT变化及重组人的NAMPT的制备和鉴定
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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。
Nicotinomide phosphoribosyltransferase (NAMPT) is a key enzyme for NAD biosynthesis. NAMPT is involved in age-related disorders, such as cancers, diabetes. In brain, NAMPT is expressed in neurons and plays a protective role against ischemia. However, the detailed expression of NAMPT in brain, its changes during aging, and its correlation with behavioral performance are not clear. In this study, we found a region-specific change of NAMPT expression during aging-NAMPT level dropped in cortex and hippocampus whereas increased in serum; we also found that NAMPT was expressed in microglia of aged mice but not in young mice, and NAD level decreased in hippocampus and cerebellum while remained constant in cortex and striatum. In behavioral tests, aged mice showed attenuated motor activity, central exploration and memory. The expression of NAMPT in cortex was positively correlated with motor activity, while that in cerebellum and serum was negatively correlated. Thus, we have demonstrated an age-related expression, distribution and activity pattern of NAMPT in brain and serum, which could be responsible for the declined motor activity during aging.
Objective:To prepare and purify recombinant human NAMPT and NAMPT (H247A) protein, and detection their enzymatic activity.
Methods:Using pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) muatant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT(H247A) were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities were assessed by solution NMR.
Results:The DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased relative to wildtype NAMPT.
Conclusion:The recombinant hNAMPT and hNAMPT (H247A) were successful prepared and purified. The H247A mutation dramatically decreased the enzymatic activity of NAMPT. This work provides a basis for further research on the NAMPT protein.
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。
Nicotinomide phosphoribosyltransferase (NAMPT) is a key enzyme for NAD biosynthesis. NAMPT is involved in age-related disorders, such as cancers, diabetes. In brain, NAMPT is expressed in neurons and plays a protective role against ischemia. However, the detailed expression of NAMPT in brain, its changes during aging, and its correlation with behavioral performance are not clear. In this study, we found a region-specific change of NAMPT expression during aging-NAMPT level dropped in cortex and hippocampus whereas increased in serum; we also found that NAMPT was expressed in microglia of aged mice but not in young mice, and NAD level decreased in hippocampus and cerebellum while remained constant in cortex and striatum. In behavioral tests, aged mice showed attenuated motor activity, central exploration and memory. The expression of NAMPT in cortex was positively correlated with motor activity, while that in cerebellum and serum was negatively correlated. Thus, we have demonstrated an age-related expression, distribution and activity pattern of NAMPT in brain and serum, which could be responsible for the declined motor activity during aging.
Objective:To prepare and purify recombinant human NAMPT and NAMPT (H247A) protein, and detection their enzymatic activity.
Methods:Using pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) muatant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT(H247A) were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities were assessed by solution NMR.
Results:The DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased relative to wildtype NAMPT.
Conclusion:The recombinant hNAMPT and hNAMPT (H247A) were successful prepared and purified. The H247A mutation dramatically decreased the enzymatic activity of NAMPT. This work provides a basis for further research on the NAMPT protein.
引文
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