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国家自然科学基金(30200050)

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具有GPX活性单链抗体的制备及其抗氧化效应被引量:4
2004年
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.
李维佳魏景艳孙晔牟颖吕绍武闫岗林罗贵民
关键词:谷胱甘肽过氧化物酶单链抗体可溶性表达抗氧化
Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)被引量:2
2007年
Glutathione peroxidase(GPX) is a selenoenzyme that protects the biomembrane and other cellular components {against} oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3(Se-scFv2F3) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. {Se-scFv2F3} is generated by the chemical modification of the single chain Fv fragment of monoclonal antibody 2F3(scFv2F3), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates. The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.
DU Xiu-boSUN YeLIN FengZHENG Ke-yanWANG Ke-weiLIN Ting-tingLIU Jun-qiuSHEN jia-congLUO Gui-min
关键词:谷胱甘肽过氧化物酶
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