發(fā)布時間：2019-03-01 16:15

【摘要】：尼克酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)是合成NAD的關(guān)鍵酶。已有大量文獻表明,NAMPT與許多衰老相關(guān)疾病密切相關(guān),如腫瘤、糖尿病等。其中NAMPT大量表達于腦中的神經(jīng)元,且在腦缺血過程中起保護作用。然而有許多問題仍不明確,如NAMPT在腦中的具體分布、NAMPT在老年過程中的表達變化,以及NAMPT表達和活性的變化是否與動物的神經(jīng)行為相關(guān)等。在本研究中,我們發(fā)現(xiàn)小鼠在老年過程中,NAMPT的表達發(fā)生特異性的改變,其中皮層和海馬的NAMPT水平顯著下降而血清的NAMPT水平則顯著增加；老年小鼠的小膠質(zhì)細(xì)胞出現(xiàn)NAMPT表達,而年輕小鼠的小膠質(zhì)細(xì)胞卻不表達NAMPT;老年小鼠海馬和小腦的NAD水平顯著下降,而皮層和紋狀體的NAD水平則沒有顯著改變。行為學(xué)試驗顯示,老年小鼠在開放場中的運動量、中心區(qū)的探索以及恐懼記憶水平都顯著下降；皮層NAMPT水平與小鼠運動量正相關(guān),而小腦和血清的NAMPT水平與小鼠運動量負(fù)相關(guān),腦和血清的NAMPT與小鼠在開放場中心區(qū)的探索及恐懼記憶沒有明顯相關(guān)。因此,我們的研究展示了小鼠在衰老過程中腦和血清NAMPT表達、分布及活性的變化特征,提示NAMPT可能與動物老年過程中運動能力下降的現(xiàn)象有關(guān)。 目的：制備和純化重組人NAMPT和NAMPT (H247A)蛋白,并對其體外酶活性進行檢測。 方法：以pcDNA3.1-hnampt為模板,通過PCR擴增獲得兩端分別為BamHⅠ和NdeⅠ酶切位點的hnampt片斷,將該片斷與pET-11a(+)表達型載體連接,通過點突變獲得pET-11a(+)-hnampt (H247A),以測序鑒定構(gòu)建質(zhì)粒。將野生型和突變型分別轉(zhuǎn)化至BL21star大腸桿菌,以IPTG誘導(dǎo)蛋白表達,以鎳柱和分子篩純化目標(biāo)蛋白,以SDS凝膠電泳和質(zhì)譜鑒定目標(biāo)蛋白,以核磁共振的方法檢測兩個重組蛋白在體外酶活性。 結(jié)果：測序結(jié)果表明,pET-11a(+)-hnampt(野生型)及pET-11a(+)-hnampt (H247A)(突變型)表達載體構(gòu)建成功,突變位點正確。兩種蛋白均在BL21star大腸桿菌獲得表達,裂解后為可溶性蛋白,通過鎳柱、分子篩獲得純化蛋白,SDS凝膠電泳和質(zhì)譜鑒定證明重組蛋白為分子量約56 KD的NAMPT。核磁共振體外酶活性檢測發(fā)現(xiàn)野生型NAMPT具有酶活性,而NAMPT (H247A)的酶活性降低了約5-10倍。 結(jié)論：成功獲得了有體外酶活性的人NAMPT蛋白和酶活性較低的人NAMPT (H247A)蛋白,為NAMPT蛋白作用研究提供了基礎(chǔ)。
[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，

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