【作者】 于扬；

【导师】 魏景艳；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 谷胱甘肽过氧化物酶(Glutathione Peroxidase, GPx)是生物体内一类重要的抗氧化酶,它能够清除机体内的脂质氢过氧化物(ROOH)以及促进过氧化氢(H2O2)分解,保护组织免受过氧化损伤,对防治由ROS引起的多种疾病有潜在的药用价值。但由于天然GPx来源有限、稳定性差以及分子量较大等缺点,使其无法通过基因工程方法大量表达。因此人们转向研究GPx的人工模拟,但由于没有考虑到底物的结合作用,早期GPx模拟物如Ebselen等活力普遍偏低。单链抗体酶技术是产生具有高活力GPx模拟物的有效方法,本课题组通过噬菌体展示技术筛选得到抗GSH人源单链抗体scFv-2D8,利用化学突变引入硒代半胱氨酸(Sec),获得具有GPx活力的人源单链抗体酶。为找出该抗体酶催化位点,我们以scFv-2D8为基础,与吉林大学理论化学计算国家重点实验室郑清川老师课题组合作,通过同源模建法及分子对接模拟推测出scFv-2D8的底物结合位点和活性基团,通过定点突变技术将预测出的关键氨基酸突变为无关氨基酸。将野生型单链抗体酶及其突变体进行化学诱变后,比较GPx活力变化,进而确定Se-scFv-2D8的催化位点。更多还原

【Abstract】 Reactive oxygen species (ROS) are reactive molecules that contain the oxygen atom. ROS coming from normal metabolism are important to the life activities of the tissues and cells in vivo. When the metabolic balance between the generation and the elimination of ROS is broken, which leads to excessive accumulation of it, great damage will be caused at molecular and cell levels. For example, cell necrosis and apoptosis will happen owing to the damage of DNA, proteins and lipid caused by ROS. Many diseases are related to the damage, such as atherosclerosis, Parkinsonism, Alzheimer’s disease and so on. Glutathione peroxidase (GPx, EC. 1. 11. 1. 9) is an important member of antioxidant enzymes in organism, which clear up lipid hydroperoxides and reduce free hydrogen peroxide to water to protect the organism from oxidative damage. Therefore much attention has been placed on GPx because of its potential medicinal value. Due to the disadvantages of limited availability, poor stability and high molecular weight of native GPx, the therapeutic use of GPx is limited. In recent years, a large number of studies have focused attention on artificial imitation of GPx. One effective method for producing GPx mimics with high GPx activity is the preparation of selenium-containing abzyme. A single chain antibody variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. Compared to the normal abzyme, scFv is suitable as drugs for its lower molecular weight. In previous studies new human single chain antibody scFv-2D8 rose against GSH-S-DNPBu, which is synthesized as GSH derivative, has been acquired by phage display technology. We convert active-site serine residue into seleno-cysteine residue by chemical modification, then human selenium-containing abzyme Se-scFv-2D8 with GPx activity is obtained. In order to investigate the catalytic group,We cooperate with group of Zheng from Theoretical Chemistry Research Institute of Jinlin University on structural analysis of scFv-2D8. The three-dimensional structure of Se-scFv-2D8 is predicted using the knowledge-based homology modeling approach. And two possible GSH-binding sites are found by molecular docking simulation, which are known as Site 1 and Site 2. Through the conformation of the complex that GSH binds to Site 1 and Site 2 created by Insight II/Affinity, we find that scFv-2D8 forms two hydrogen bonds with GSH in Site 1 while four hydrogen bonds in Site 2. Because of the Sec introduced by substituting for serine, it is significant to locate the right active-site serine for the study. The interaction energies of GSH with each of the residues in the active site of scFv-2D8 are calculated and two serines have the lowest energy, which are Ser 207 (-3.50kcal/mol) in Site 1 and Ser65 (-4.05kcal/mol) in Site 2. Based on the view of the data of the interaction energy and the quantitative of hydrogen bonds, it’s likely that Sec65 contributes more to the GPx activity. Then two mutants S207A (serine to alanie) and S65A (serine to alanie) are gained by QuikChange site-directed mutagenesis. Soluble proteins are expressed in E.coli Rosetta and purified by immobilized metal affinity chromatography (IMAC). After chemically converting the active Sers into Secs, the GPx activity of wild-type human selenium-containing scFv-2D8 is detected to be 1684 U/μmol. In comparison with Se-scFv-2D8-S207A, the activity of mutant Se-scFv-2D8-S65A decreases from 1684 U/μmol to 659 U/μmol, the GPx activity of the latter declines greater than the anterior one, this indicates that Sec65 probably plays more important role in the catalytic reaction.更多还原