S-腺苷高半胱氨酸水解酶（SAHH）的克隆表达纯化、酶促反应动力学分析及其抑制剂筛选

发布时间：2018-05-15 05:30

本文选题：S-腺苷高半胱氨酸水解酶(SAHH) + 高半胱氨酸(Hcy)　；参考：《济南大学》2017年硕士论文

【摘要】：当体内高半胱氨酸(Hcy)含量异常升高时会严重损害DNA的修复机制,诱导细胞的凋亡,进一步就会引起阿尔茨海默综合症(AD)、高半胱氨酸血症、冠心病等。体内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。细胞中Hcy的生成只有水解SAH这唯一的一条代谢途径,所以体内Hcy含量的高低与SAHH蛋白酶有着密切的关系。本实验旨在通过体外合成具有生物活性的SAHH,并且以该SAHH为研究基础筛选得到可以抑制SAHH活性的抑制剂。此抑制剂的发现为有效地治疗以及防止AD、高半胱氨酸血症、冠心病等老年性疾病提供了新的、更有效的临床治疗方法。本论文通过基因克隆技术将sahh克隆到p Pic9k质粒中构建p PIC9K-sahh重组表达质粒。该重组表达质粒在限制性内切酶——Bgl II的作用下酶切,酶切后目的基因通过电转化的方法整合到毕赤酵母菌(P.pastoris)GS115的基因组。为了得到高抗性的转化子,我们利用不同梯度浓度的G418遗传霉素进行高抗性筛选,最终筛选得到高拷贝、高抗性的转化子。将筛选到的高拷贝转化子通过菌体的PCR进行扩增,鉴定其为阳性的转化子。得到的阳性转化子在1%的甲醇诱导下表达目的蛋白SAHH。随后收集目的蛋白进行镍柱纯化,纯化后的SAHH用DTNB定量的检测其酶活,其比活为105 U/mg。为了进一步更好的了解此酶,本实验还进行了酶促反应动力学研究,探究得到该水解酶的K_m=21.8μM,V_(max)=22.9μM/min,最适的T=41℃,最适p H=6.5。我们利用Chem Mapper、Sci Finder Scholar等信息平台来进行抑制剂的初步筛选。通过DTNB试剂检测其酶活的变化情况来进一步的筛选,从而确定具有抑制效果的SAHH的抑制剂。最终成功筛选得到一种抑制效果相对较高的小分子化合物——4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol(松柏醇),其半抑制浓度(half maximal inhibitory concentration,IC_(50))为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.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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