本文選題：多巴胺　切入點(diǎn)：多巴胺醌　出處：《華東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:帕金森病作為一種常見的的神經(jīng)退行性疾病,其發(fā)病原因尚不清楚。我們認(rèn)為神經(jīng)元細(xì)胞內(nèi)游離的多巴胺能夠氧化產(chǎn)生多巴胺醌,多巴胺醌能夠結(jié)合并修飾蛋白,使蛋白功能喪失,導(dǎo)致細(xì)胞死亡。本實(shí)驗(yàn)用多巴胺對體外純化的LDHB和ENO1酶蛋白進(jìn)行處理,來研究多巴胺氧化產(chǎn)生的多巴胺醌對酶蛋白活性的影響,進(jìn)而探究多巴胺醌毒性與帕金森病之間的關(guān)系。方法:體外擴(kuò)增LDHB和ENO1酶蛋白基因序列,將兩種蛋白基因序列分別連接進(jìn)入表達(dá)載體pProEX-HTb。將重組表達(dá)質(zhì)粒轉(zhuǎn)化入BL21表達(dá)感受態(tài)菌中,利用IPTG(Isopropy1β-D-Thiogalactoside)誘導(dǎo)蛋白表達(dá)。超聲破碎菌體提取蛋白,利用Ni-NTA親和層析法對兩種蛋白進(jìn)行純化。加入相應(yīng)的酶底物,通過吸光度變化來檢測酶生物活性。在不同條件下用多巴胺對酶蛋白進(jìn)行處理,之后對樣品的酶活性進(jìn)行測定。將經(jīng)過處理的樣品進(jìn)行變性聚丙烯酰胺凝膠電泳,利用考馬斯染色法和NBT/甘氨酸鹽顯色法對蛋白樣品進(jìn)行分析。最后通過蛋白免疫印跡實(shí)驗(yàn),分析LDHB蛋白樣品中的醌結(jié)合蛋白。結(jié)果:體外擴(kuò)增的LDHB和ENO1蛋白基因序列測序無錯(cuò)配。誘導(dǎo)表達(dá)純化的兩種蛋白具有生物催化活性。用相同濃度的多巴胺對LDHB和ENO1蛋白處理不同時(shí)間之后,隨著處理時(shí)間的增加,LDHB和ENO1蛋白酶活性的損失都增加。經(jīng)過不同DA濃度處理相同時(shí)間后,隨著DA處理濃度增加,兩種蛋白酶活性損失增加,蛋白酶活性下降;若在處理樣品中預(yù)加入谷胱甘肽(GSH),能夠阻止酶活性的損失,使蛋白保持活性。酪氨酸酶催化多巴胺氧化生成多巴胺醌,但不會(huì)生成活性氧等產(chǎn)物,生成的多巴胺醌降低了蛋白酶催化活性,同樣預(yù)加入谷胱甘肽后,蛋白活性損失消失�？捡R斯染色結(jié)果顯示經(jīng)過多巴胺處理的蛋白樣品中生成多聚物,多聚物的量與處理時(shí)間以及處理濃度成正比;NBT/甘氨酸鹽染色結(jié)果表明處理樣品中的多聚物,是由于多巴胺醌修飾產(chǎn)生的蛋白積聚。而谷胱甘肽能夠阻止蛋白樣品中多聚物形成。最后對LDHB蛋白進(jìn)行Western Blots實(shí)驗(yàn),結(jié)果表明處理的樣品中的多聚物是醌結(jié)合的LDHB蛋白。結(jié)論:體外表達(dá)純化的LDHB和ENO1蛋白具有一定的生物催化活性。純化的LDHB和ENO1蛋白經(jīng)過多巴胺處理,多巴胺氧化生成的多巴胺醌能夠結(jié)合并修飾蛋白,使蛋白聚集形成多聚物,降低了蛋白酶活性。隨著處理時(shí)間增加,處理的多巴胺濃度增加,這種影響越嚴(yán)重。
[Abstract]:Objective: as a common neurodegenerative disease, the pathogenesis of Parkinson's disease is unclear. We believe that dopamine free in neurons can oxidize dopamine quinone, and dopamine quinone binds and modifies proteins. In this study, we used dopamine to treat purified LDHB and ENO1 proteins in vitro to study the effect of dopamine quinone on the activity of enzyme protein. To explore the relationship between dopamine quinone toxicity and Parkinson's disease. Methods: LDHB and ENO1 protein gene sequences were amplified in vitro. The two protein gene sequences were ligated into the expression vector pProEX-HTb.Recombinant expression plasmid was transformed into BL21 expression receptive bacteria, and protein expression was induced by IPTG(Isopropy1 尾 -D-Thiogalactoside. Two kinds of proteins were purified by Ni-NTA affinity chromatography. The enzyme bioactivity was detected by absorbance change by adding the corresponding enzyme substrate. The enzyme protein was treated with dopamine under different conditions. After that, the enzyme activity of the samples was determined. The treated samples were analyzed by denaturing polyacrylamide gel electrophoresis, and the protein samples were analyzed by Cormas staining and NBT/ glycine salt colorimetry. Analysis of quinone binding proteins in LDHB protein samples. Results: there was no mismatch in sequencing of LDHB and ENO1 gene sequences amplified in vitro. The two proteins expressed by induction and purification had biocatalytic activities. The same concentration of dopamine was used to treat LDHB and ENO1. After ENO1 protein was treated for different time, The loss of both LDHB and ENO1 protease activity increased with the increase of treatment time. After different DA concentrations for the same time, with the increase of DA concentration, the loss of the two protease activities increased and the protease activity decreased. If glutathione glutathione glutathione GSH is added in the treated sample, it can prevent the loss of enzyme activity and keep the protein active. Tyrosinase catalyzes the oxidation of dopamine to dopamine quinone, but does not produce the products such as reactive oxygen species. The resulting dopamine quinone reduced protease catalytic activity, and the loss of protein activity disappeared after the addition of glutathione. Coomas staining showed that a polymer was formed in the dopamine-treated protein sample. The results of NBT / glycine salt staining showed that the amount of polymer was proportional to the treatment time and concentration. Glutathione prevents the formation of polymers in protein samples. Finally, the LDHB protein is tested by Western Blots. The results showed that the polymers in the treated samples were quinone-bound LDHB proteins. Conclusion: the purified LDHB and ENO1 proteins have certain biocatalytic activity. The purified LDHB and ENO1 proteins are treated with dopamine. The dopamine quinone produced by dopamine oxidation can bind and modify the protein, which makes the protein aggregate to form a polymer, which reduces the protease activity. With the increase of treatment time, the concentration of dopamine increases, and the effect becomes more serious.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5

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相關(guān)期刊論文 前1條

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本文編號(hào)：1641658