具有GPX活性单链抗体的制备及其抗氧化效应

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（lys S）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、 Western Blot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2 530 U/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.

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

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 t he pRose at the N end of 2F3. The 2F3 antibody was expressed as a soluble prote in in the periplasm cavity of E. coli BL21 (lys S). The purified product was con firmed by Western Blotting. Se-2F3-ScFv is generated by the chemical mutatio n 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 investigat ed. It was found that Se-2F3-ScFv possessed strong ability to prevent epidermal cells from UVB damage and was an effective membrane peroxidation inhibitor.