Objective To prepare a selenium-containing single-chain abzyme with glutathione peroxidase(GPX)activity.Methods The genes were amplified from the heavy(VH)and light(VL)chain variable regions of hybridoma cell strain 2F3 by RT-PCR and identified by DNA sequencing. The recombinant plasmid pTMF-scFv was constructed with the genes and transformed to E. coli ,and expressed product was purified by Co2+ -IMAC affinity chromatography. A selenium-containing single-chain abzyme with glutathione peroxidase activity was obtained by the renaturation and chemical mutagenesis of the purified expressed product. Results SDS-PAGE showed that the expressed scFv existed in the form of inclusion body with a molecular weight of 30000.The scFv expressed in E. coli .1M109(DE3) ,B121(DE3)and BL21(coden plus)contained about 5% - 10% ,15% - 20% and 25% - 30% of total protein respectively.The GPX activity of the selenium-containing single-chain abzyme( 3400U/μmol) was close to that of natural GPX enzyme. Conclusion The study laid a foundation of industrial production of selenium-containing single-chain abzyme with glutathione peroxidase activity.

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240–1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251–255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5–10%, 15–20% and 25–30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8M urea by Co2+-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46×105M−1 and 1.03×105M−1 respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394units/μmol, which approaches the activity of rabbit liver GPX.

利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker（Gly4 Ser1） 3 构建成单链抗体 （scFv）表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1（DE3） ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数

目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因.方法提取杂交瘤细胞的总RNA,分离mRNA,反转录合成cDNA.经PCR扩增VH基因和VL基因,将VH基因和VL基因与载体pGEM-T连接后,进行酶切鉴定和序列分析.结果构建了2个分别含有VH和VL基因的重组质粒.序列分析表明,VH和VL分别属于ousc heavy chain subgroupIII和mouse light chain subgroupV亚群,长度为372和324bp,编码124和108个氨基酸,在高变区分别有4和2个丝氨酸.结论克隆的VH和VL基因符合功能性重排的鼠抗体可变区基因特征,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料.

Glutathione was modified selectively by 2,4-dinitrochlorobenzene, giving S-substituted dinitrophenyl glutathione (GSH-S-DNP). GSH-S-DNP was further esterified by iso-butanol, hexanol, cyclohexanol and benzylalcohol. Four haptens used to generate abzyme with glutathione peroxidase (GPX) activity were synthesized. They are GSH-DNP-biesters: GSH-DNP-diiso-butyl ester (GSH-DNP-IBU), GSH-DNP-bihexyl ester(GSH-DNP-HE), GSH-DNP-bicyclohexyl ester(GSH-DNP-CH), GSH-DNP-bibenzylmethyl ester(GSH-DNP-BE). The structures of the haptens were characterized by means of elemental analysis, IR and H-1 NMR.

Annals of the New York Academy of SciencesVolume 864, Issue 1 p. 284-287 Antioxidant Effects of Superoxide Dismutase and Horseradish Peroxidase on Lipid Peroxidation SHU-JUAN GAO, SHU-JUAN GAO State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorGUANG-CHENG ZHAO, GUANG-CHENG ZHAO First Teaching Hospital; Norman Bethune University of Medicine Science; Changchun 130021, ChinaSearch for more papers by this authorGUI-MIN LUO, GUI-MIN LUO State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorTONG-SHU YANG, TONG-SHU YANG State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorJIA-CONG SHEN, JIA-CONG SHEN Key Lab of Supermolecular Structure and Spectrum; Jilin University; Changchun 130023, ChinaSearch for more papers by this author SHU-JUAN GAO, SHU-JUAN GAO State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorGUANG-CHENG ZHAO, GUANG-CHENG ZHAO First Teaching Hospital; Norman Bethune University of Medicine Science; Changchun 130021, ChinaSearch for more papers by this authorGUI-MIN LUO, GUI-MIN LUO State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorTONG-SHU YANG, TONG-SHU YANG State Lab of Enzyme Engineering; Jilin University; Changchun 130023, ChinaSearch for more papers by this authorJIA-CONG SHEN, JIA-CONG SHEN Key Lab of Supermolecular Structure and Spectrum; Jilin University; Changchun 130023, ChinaSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10323.xCitations: 8Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1 Wayner, D. D. M.. et al 1987. The relative contributions of vitamin E, urate, ascorbate, and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasms. Biochim. Biophys. Acta 924: 408–419. 2 Luacia, C. 1984. Comparison of cumene hydroperoxide and NADPH/Fe3+/ADP-induced lipid peroxidation in heart and liver submitochondrial particles. Biochem. Biophys. Acta 795: 466–472. 3 Boveris, A. et al. 1976. Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 156: 435–444. 4 Lowry, O. H. et al. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275. 5 Hiroshi, O. et al. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351–358. 6 Takashi, Y. et al. 1965. Studies on cytochrome oxidase: VI. Kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome oxidase. J. Biol. Chem. 240: 3392–3398. Citing Literature Volume864, Issue1ENZYME ENGINEERING XIVDecember 1998Pages 284-287 ReferencesRelatedInformation

On the basis of cyclodextrin, 6-selenium bridged β-cyclodextrin (6-β-CD-Se-Se-β-CD, known as 6-SeCD) was synthesized by the selective tosylation of β-cyclodextrin and nucleophilic displacement by sodium hydroselenide to imitate glutathione peroxidase (GPX). The GPX activity of diselenide 6-SeCD is 4.3 times that of PZ51. The structure of the mimic 6-SeCD was characterized by means of laser mass spectroscopy, elemental analysis, IR and1H NMR. The selenium content and its valence in 6-SeCD were determined by means of X-ray photoelectron spectra. Kinetics of the mimic showed that its enzymatic behavior was similar to that of native GPX.

Annals of the New York Academy of SciencesVolume 864, Issue 1 p. 280-283 Kinetic Studies of Abzyme with Glutathione Peroxidase Activity SHU-JUAN GAO, SHU-JUAN GAO State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorMO CHEN, MO CHEN State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorWEI LIN, WEI LIN State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorGUI-MIN LUO, GUI-MIN LUO State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorJIA-CONG SHEN, JIA-CONG SHEN Key Lab of Supermolecular Structure and Spectrum Jilin University Changchun 130023, ChinaSearch for more papers by this author SHU-JUAN GAO, SHU-JUAN GAO State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorMO CHEN, MO CHEN State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorWEI LIN, WEI LIN State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorGUI-MIN LUO, GUI-MIN LUO State Lab of Enzyme Engineering, Jilin University Changchun 130023, ChinaSearch for more papers by this authorJIA-CONG SHEN, JIA-CONG SHEN Key Lab of Supermolecular Structure and Spectrum Jilin University Changchun 130023, ChinaSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10322.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1 Peter, G. G. et al. 1993. Selenoperoxidase-mediated cytoprotection against the damaging effects of tert-butyl hydroperoxide on leukemia cells. Free Radical Biol. Med. 14: 251– 266. 2 Vasanthy, N. & S. Helmut. 1990. Oxidative damage to mitochondria and protection by ebselen and other antioxidants. Biochem. Pharmacol. 40: 1623– 1629. 3 Zhen-Ping, W. & H. Donald. 1990. Selenosubtilisin as a glutathione peroxidase mimic. J. Am. Chem. Soc. 112: 5647– 5648. 4 Luo, G. M. et al. 1994. Generation of selenium-containing abzyme by using chemical mutation. Biochem. Biophys. Res. Commun. 198: 1240– 1247. 5 Ian, M. et al. 1993. Kinetic studies on the peroxidase activity of selenosubtilisin. Biochemistry 32: 3754– 3762. 6 Lowry, O. H. et al. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265– 275. 7 Zhen-Ping, W. & H. Donald. 1989. Conversion of a protease into an acyl transferase: selenosubtilisin. J. Am. Chem. Soc. 111: 4513– 4514. Citing Literature Volume864, Issue1ENZYME ENGINEERING XIVDecember 1998Pages 280-283 ReferencesRelatedInformation