Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized c...Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.展开更多
目的运用计算机辅助蛋白质分子设计的方法设计针对蓖麻毒素A链（RTA）的拮抗肽，实现在大肠杆菌BL21中的可溶性表达，并对其生物学活性进行评价。方法根据RTA的晶体结构、RTA-rRNA相互作用复合物模型，在CVFF（consistent-valence force...目的运用计算机辅助蛋白质分子设计的方法设计针对蓖麻毒素A链（RTA）的拮抗肽，实现在大肠杆菌BL21中的可溶性表达，并对其生物学活性进行评价。方法根据RTA的晶体结构、RTA-rRNA相互作用复合物模型，在CVFF（consistent-valence force field）、Amber力场下，对RTA的空间构象进行理论模拟，初步确定其生物活性功能域；然后针对该功能域设计小分子拮抗肽，并借助人抗体重链可变区骨架，在CDR3区对拈抗肽进行展示，用重叠延伸PCR全基因合成人源化的单域抗体并克隆至载体pET-32a（＋）；双酶切和DNA测序技术对构建的载体进行鉴定；IPTG诱导人源化的单域抗体表达，用镍离子亲和层析纯化，竞争ELISA和MTr法分别进行结合和中和活性检测。结果从头搭建并设计合成了人源化的单域抗体，实现了其原核表达，并进行了生物活性检测；建立了基于人源化的单域抗体的RTA和蓖麻毒素检测方法。结论研究结果为新型蓖麻毒素小分子拈抗剂的研制奠定了理论和实验基础。展开更多
基金Supported by the National Natural Science Foundation of China(Nos 20072010 and 20572035) and the Science Foundation ofJilin University(Nos419070100087 and 01208006)
文摘Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.
文摘目的运用计算机辅助蛋白质分子设计的方法设计针对蓖麻毒素A链（RTA）的拮抗肽，实现在大肠杆菌BL21中的可溶性表达，并对其生物学活性进行评价。方法根据RTA的晶体结构、RTA-rRNA相互作用复合物模型，在CVFF（consistent-valence force field）、Amber力场下，对RTA的空间构象进行理论模拟，初步确定其生物活性功能域；然后针对该功能域设计小分子拮抗肽，并借助人抗体重链可变区骨架，在CDR3区对拈抗肽进行展示，用重叠延伸PCR全基因合成人源化的单域抗体并克隆至载体pET-32a（＋）；双酶切和DNA测序技术对构建的载体进行鉴定；IPTG诱导人源化的单域抗体表达，用镍离子亲和层析纯化，竞争ELISA和MTr法分别进行结合和中和活性检测。结果从头搭建并设计合成了人源化的单域抗体，实现了其原核表达，并进行了生物活性检测；建立了基于人源化的单域抗体的RTA和蓖麻毒素检测方法。结论研究结果为新型蓖麻毒素小分子拈抗剂的研制奠定了理论和实验基础。
