本文選題：S-腺苷高半胱氨酸水解酶(SAHH) + 高半胱氨酸(Hcy)��； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：當(dāng)體內(nèi)高半胱氨酸(Hcy)含量異常升高時(shí)會(huì)嚴(yán)重?fù)p害DNA的修復(fù)機(jī)制,誘導(dǎo)細(xì)胞的凋亡,進(jìn)一步就會(huì)引起阿爾茨海默綜合癥(AD)、高半胱氨酸血癥、冠心病等。體內(nèi)Hcy的生成是由S-腺苷高半胱氨酸水解酶(S-adenosyl-homocysteine hydrolase,SAHH,E.C.3.3.1.1)催化S-腺苷高半胱氨酸(S-adenosylhomocystein,SAH)可逆水解生成腺苷(Ado)和Hcy。細(xì)胞中Hcy的生成只有水解SAH這唯一的一條代謝途徑,所以體內(nèi)Hcy含量的高低與SAHH蛋白酶有著密切的關(guān)系。本實(shí)驗(yàn)旨在通過(guò)體外合成具有生物活性的SAHH,并且以該SAHH為研究基礎(chǔ)篩選得到可以抑制SAHH活性的抑制劑。此抑制劑的發(fā)現(xiàn)為有效地治療以及防止AD、高半胱氨酸血癥、冠心病等老年性疾病提供了新的、更有效的臨床治療方法。本論文通過(guò)基因克隆技術(shù)將sahh克隆到p Pic9k質(zhì)粒中構(gòu)建p PIC9K-sahh重組表達(dá)質(zhì)粒。該重組表達(dá)質(zhì)粒在限制性內(nèi)切酶——Bgl II的作用下酶切,酶切后目的基因通過(guò)電轉(zhuǎn)化的方法整合到畢赤酵母菌(P.pastoris)GS115的基因組。為了得到高抗性的轉(zhuǎn)化子,我們利用不同梯度濃度的G418遺傳霉素進(jìn)行高抗性篩選,最終篩選得到高拷貝、高抗性的轉(zhuǎn)化子。將篩選到的高拷貝轉(zhuǎn)化子通過(guò)菌體的PCR進(jìn)行擴(kuò)增,鑒定其為陽(yáng)性的轉(zhuǎn)化子。得到的陽(yáng)性轉(zhuǎn)化子在1%的甲醇誘導(dǎo)下表達(dá)目的蛋白SAHH。隨后收集目的蛋白進(jìn)行鎳柱純化,純化后的SAHH用DTNB定量的檢測(cè)其酶活,其比活為105 U/mg。為了進(jìn)一步更好的了解此酶,本實(shí)驗(yàn)還進(jìn)行了酶促反應(yīng)動(dòng)力學(xué)研究,探究得到該水解酶的K_m=21.8μM,V_(max)=22.9μM/min,最適的T=41℃,最適p H=6.5。我們利用Chem Mapper、Sci Finder Scholar等信息平臺(tái)來(lái)進(jìn)行抑制劑的初步篩選。通過(guò)DTNB試劑檢測(cè)其酶活的變化情況來(lái)進(jìn)一步的篩選,從而確定具有抑制效果的SAHH的抑制劑。最終成功篩選得到一種抑制效果相對(duì)較高的小分子化合物——4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol(松柏醇),其半抑制濃度(half maximal inhibitory concentration,IC_(50))為34.04nM。
[Abstract]:When the content of homocysteine (Hcy) in the body is abnormal, it can seriously damage the mechanism of DNA repair, induce cell apoptosis, and further cause Alzheimer's syndrome (AD), hypercysteinemia, coronary heart disease and so on. The formation of Hcy in vivo is from S- adenosine homocysteine hydrolase (S-adenosyl-homocysteine hydrolase, SAHH, E.C.3.3.1.1). S- adenosine (S-adenosylhomocystein, SAH) catalyzes the reversible hydrolysis of adenosine (Ado) and the formation of Hcy in Hcy. cells only a metabolic pathway that hydrolyzes SAH, so the level of Hcy in the body is closely related to the SAHH protease. This experiment aims to synthesize SAHH with biological activity in vitro AHH provides a new and more effective clinical therapy for the effective treatment and prevention of AD, hypercysteinemia, coronary heart disease and other senile diseases. This paper constructs a p PIC9K-sahh by cloning SAHH into the P Pic9k plasmid by gene cloning technology. Recombinant expression plasmid, the recombinant expression plasmid was cut under the action of restriction endonuclease, Bgl II, and the target gene was integrated into the genome of P.pastoris GS115 by electrical transformation. In order to obtain highly resistant transformants, we used the G418 geniin of different levels of G418 for high resistance screening. The highly copied and highly resistant transformants were screened. The screened high copy transformants were amplified by the PCR of the mycelium to identify the positive transformants. The positive transformants expressed the target protein SAHH. under 1% methanol and then collected the target protein for the purification of the nickel column. The purified SAHH was quantified by DTNB to detect the enzyme. In order to get a better understanding of the enzyme, the activity is 105 U/mg. to further understand the enzyme. This experiment has also carried out a kinetic study of enzymatic reaction, exploring the K_m=21.8 M of the hydrolase, V_ (max) =22.9 mu M/min, the optimum T=41 C, and the optimum P H=6.5.. TNB reagent detects the change of the enzyme activity to further screening, thus determining the inhibition effect of SAHH inhibitors. Finally, a relatively high inhibitory effect of small molecular compound, 4- (3-Hydroxyprop-1-en-1-yl) -2-methoxyphenol (pine cedar alcohol), its semi inhibitory concentration (half maximal inhibitory concentra) has been successfully screened. Tion, IC_ (50)) is 34.04nM.

【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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