具有GPX活性单链抗体的制备及其抗氧化效应  

Generation of single-chain antibody with GPX activity and its antioxidant effect

作　　者：李维佳[1] 魏景艳[1] 孙晔[1] 牟颖[1] 吕绍武[1] 闫岗林[1] 罗贵民[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130023

出　　处：《吉林大学学报（理学版）》2004年第3期458-462,共5页Journal of Jilin University:Science Edition

基　　金：国家自然科学基金(批准号:30200050);吉林大学科研创新基金;青年教师基金(批准号:2000A18).

摘　　要：为实现单链抗体的可溶性表达,制备具有谷胱甘肽过氧化物酶(GPX)活力的单链抗体,在单链抗体表达载体pTMF 2F3上去除原2F3基因N端非必需的18个氨基酸,采用新的表达载体pRose质粒,在2F3的N端引入13个氨基酸的前导肽,转入BL21(lysS)中,使其分泌到大肠杆菌的周质腔中得以表达,获得可溶性表达产物.产物经过分离纯化、WesternBlot印迹和硒化测活确定具有GPX活性的鼠单链抗体,其GPX活力为2530U/μmol.以脂质过氧化、细胞存活率和细胞膜完整性为指标的抗氧化实验研究表明,具有GPX活性的单链抗体对大鼠乳鼠表皮细胞有抗紫外线损伤的作用,是膜脂质过氧化的有效抑制剂.To get soluble form of single-chain Fv of antibody (scFv) and generate selenium-containing scFv with glutathione peroxidase (GPX) activity, scFv expression vector pTMF-2F3 was replaced by a new vector pRose-2F3. Non-essential amino acids of 18 were removed from pTMF-2F3 and a signal peptide was introduced in the pRose at the N end of 2F3. The 2F3 antibody was expressed as a soluble protein in the periplasm cavity of E. coli BL21 (lys S). The purified product was confirmed by Western Blotting. Se-2F3-ScFv is (generated) by the chemical mutation of 2F3-ScFv. The GPX activity of Se-2F3-ScFv is (2　530 U/μmol). For (studying) its protection of epidermal cell from UVB damage, the effects of UVB and Se-2F3-ScFv on lipid (peroxidation,) the cell viability and cell membrane integrity were investigated. It was found that Se-2F3-ScFv possessed strong ability to prevent epidermal cells from UVB damage and was an effective membrane peroxidation inhibitor.

关 键 词：谷胱甘肽过氧化物酶 单链抗体 可溶性表达 抗氧化 

分 类 号：Q511[生物学—生物化学]