含硒单链抗体酶的人源化
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摘要
需氧生物在利用氧的过程中，会产生对机体有害的活性氧(ROS)，在正常情况下，机体ROS的产生和清除处于平衡状态，但在某些情况下，ROS的产生和清除不平衡时，过多ROS会损伤机体的各种生物大分子，如RNA、DNA、蛋白质、糖类和脂类，从而导致细胞功能紊乱。为了清除ROS对机体的损伤，机体建立了一系列防御系统，主要包括酶学和非酶学抗氧化体系。酶学体系由超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)和过氧化氢酶(CAT)等组成：非酶学体系包括一些抗氧化小分子，如α-生育酚、抗坏血酸(Vc)和还原型谷胱甘肽(GSH)等。GPX在机体抗氧化体系中起着十分重要的作用，它不仅能清除脂类氢过氧化物(ROOH)，并在过氧化氢酶(CAT)含量很少或过氧化氢(H_2O_2)产量很低的组织中，可代替CAT清除H_2O_2，所以GPX有防止畸变，预防衰老及参与前列腺素合成等重要生理作用。人体若缺乏GPX会导致由于H_2O_2损伤而引起的一系列疾病，如各种炎症、结肠癌、心脑血管疾病、生理性衰老等，因此，研究及开发GPX对酶催化机理的探索和疾病预防具有重大意义。由于天然GPX分子量大，不易纯化，半衰期短以及难于用基因工程方法获得等不利因素，严重限制了其应用。如何生产高GPX活力的模拟物是摆在生物学家面前的一个难题，各国科学家纷纷研制GPX的模拟物，其中比较著名的GPX模拟物为Ebselen(2-苯基-1，2-苯并异硒唑-3(2H)-酮)，在日本它已经进入Ⅲ期临床阶段，但由于GPX活力只有0.99U／μmol，并且水溶性差，导致其应用受到了有一定的限制。在近几年中，我们实验室制各并合成了一系列具有GPX活力鼠源抗体酶，并且在动物模型中表现出很好的抗氧化活性，但由于鼠源抗体应用于人体会存在严重的人—抗
中文摘要
..叫，....甲.......
鼠反应(HAMA)，所以抗体酶人源化势在必行。2001年，丁兰小组成功从
人源化半合成噬菌体抗体库中筛选出具有GPX结合活性的单链抗体，并在大
肠杆菌中表达、纯化，但遗憾的是当用化学诱变法引入催化基团一硒代半耽
氨酸(Sec)时，其GPX活力仅为80U/帅of，同普通的免疫球蛋白(I gG)分
子硒化后的GPX活力71 .4U/娜of基本相同，所以制备人源化的高GPX活力
抗体酶是巨大的挑战。
本文首次改造己有的经化学诱变后具有高GPX活力的鼠源单链抗体，并
展示于噬菌粒pFUW一80表面，设计半抗原，通过亲和筛选获得对半抗原特异
结合的单链抗体，在大肠杆菌Rosetta中成功表达。经纯化后的单链抗体通过
化学法引入催化基团一Sec，其GpX活力为88OU/卿ol，是第一个人源化GPX
抗体酶高11倍，这种高GPX活力的人源化抗体酶作为抗氧化剂具有广阔的
应用前景。
1、构建人源化噬菌体展示抗体库
对鼠源单链抗体的改形分重链和轻链两部分。在蛋白质数据库(PDB)
中，通过同源序列分析分别确定与重链和轻链同源性较高的人源抗体，对于
鼠源单链抗体的改造，除了完全保留CDR区外，还通过计算机模拟的抗原结
合位点的空间结构，确定与CDR结构在空间上有关的氨基酸残基，并与人同
源抗体该部位的氨基酸同时成对保留，形成人源化轻、重链抗体库。重链采
取分段全序列合成的方法连接而成，轻链则通过重迭 PCR方法扩增而成，用
连接肤(Gl外Ser)3把轻、重链连接起来，并克隆于噬菌粒载体pFUW一80中，
通过辅助噬菌体M13K07使人源化单链抗体展示于噬菌粒pFUw一80表面。
构建的抗体库的理论库容为连接产物转化后在有氨节抗性平板上的菌落数，
而实际库容为理论库容XPCR可扩增出单链抗体的比例。
2、具有GPX结合活性的单链抗体的筛选
用疏水腔修饰法，设计并合成半抗原GSH一S一DNP一Bu和GSH一S一DNP一Me，
进行抗体库的筛选。以噬菌体单链为模板进行PCR扩增，用BstNI酶切扩增
的单链抗体基因，通过DNA指纹法验证了单链抗体库的多样性，ELISA法验
证了筛选的阳性克隆。经4轮筛选，分别得到了对半抗原特异性结合的单链
抗体，从ELISA结果选出Bg和B14对GSH一S一DNP.Bu特异结合的抗体，通
中文摘要
过双酶切法和DNA序列分析法进行阳性鉴定。
3、定向进化构建次级库及特异结合性抗体的筛选
如何获得高亲和力抗体始终是噬菌体抗体库技术的一个发展方向。由于
构建的人源化抗体库库容较小，我们采用DNA改组技术在体外提高抗体的亲
和力，即通过有性和无性PCR扩增出特异性单链抗体基因后克隆于噬菌粒载
体pFUW一80中，形成次级抗体库，以GSH一s~DN甲一Bu为半抗原，进行正向有
意义突变的高亲和性抗体的筛选。经过3轮筛选后筛选出的单链抗体的ELISA
信号明显高于DNA改组前的两个阳性克隆，我们选ELISA信号最强的噬菌
体抗体克隆BS进行以后的表达。
4、单链抗体在大肠杆菌的表达及纯化
通过PCR扩增出单链抗体BS基因，然后克隆于载体pPe扭中，通过双
酶切法和DNA序列分析法鉴定连接产物，证实单链抗体基因已按正确方向连
入表达载体中。通过对表达菌株进行优化选择了最佳表达菌株，然后对诱导
剂加入时菌体浓度、诱导剂浓度和诱导表达时间进行优化，得到适合单链抗
体表达的最优条件，按此条件大量表达目的蛋白。通过Ni2+金属离子鳌合纯
化目的
Reactive oxygen species(ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage biomacromolecules such as RNA, DNA, protein, sugared and lipids, therefore this results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, aged-related disease, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. GPX plays an important role in antioxidant defense system of enzymatic action. Howe
ver, due to the shortcomings of native GPX (solution instability, poor availability, short half-lives and proteolytic digestion), mimics with high GPX efficiency were prepared by many biochemists. One of the well-known GPX mimics is Ebselen (2-phenyl-l,2- benziososelenazol-3 (2H)-one) . Although this intresting molecule is undergoing phase III clinical trial in Japan as antioxidant, it has some drawbacks such as its low GPX activity and its insolubility in water. We have successfully generated several mouse catalytic antibodies, and expressed mouse single-chain variable region antibodies (ScFv's) in Escherichia coli (E.coli)
by protein engineering and found high GPX efficiencies by chemical modified serine into selenocysteine (Sec). Although some antibodies from murine sources have good affinity and showed excellent results in animal models, the problem with human antimurine (HAMA) antibodies must be resolved. In 2001, the full human antibodies were selected from a semi-synthetic antibody library and the GPX efficiency of the catalytic antibody was found to be 80 U/umol, upon Sec was introduced. But this efficiency was almost as much as that of Se- y IgG
In this study, we described the construction of a library displayed human antibody fragments that can bind the specific substrate-glutathione (GSH). Our strategy was to construct a single-chain variable fragments (ScFv) library by reforming a mouse fragment variant region into a phagemid vector pFUW-80. The constructed library was a chimeric one that contained both conservative fragments of mouse ScFv and incorporated the human homologous fragments. Two designed haptens were synthesized GSH-S-DNP-Me and GSH-S-DNP-Bu and were coupled to BSA by glutaradehyde. The selected ScFv's were identified by ELISA and were cloned into the expression vector pPelB. The expressed protein was purified, identified and the catalytic group, Sec was incorporated into the protein by chemical mutation. The GPX activity of the single-chain antibody was 880 U/umol, which is 11-fold that of first reported human glutathione peroxidase mimic. The selenium-containing human single-chain abzyme with GPX activity shows great prospects in medicine.
1. Construction of ScFv antibody library
The heavy chain (VH) and light chain (VL) of the reshaping antibody of variable fragment were designed respectively. By homology sequence analysis from protein data bank (PDB), homology replacements of the VH and VL fragments regions were chosen. There is a need to select a correct pair of variable regions to achieve the right specificity. Key residues from the mouse single-chain antibody (heavy chain CDR3 sequences and sometimes also CDR2) are incorporated into human repertoires in order to preserve the desired binding function. By computer
stimulation, the conservative residues, which are relevant to the structure of the non-human antibodies, as well as CDRs, were retained. The VH genes were ligated by the synthesized VH fragments, while the VL genes were amplified by over-lap PCR with the primers o
中文摘要
..叫，....甲.......
鼠反应(HAMA)，所以抗体酶人源化势在必行。2001年，丁兰小组成功从
人源化半合成噬菌体抗体库中筛选出具有GPX结合活性的单链抗体，并在大
肠杆菌中表达、纯化，但遗憾的是当用化学诱变法引入催化基团一硒代半耽
氨酸(Sec)时，其GPX活力仅为80U/帅of，同普通的免疫球蛋白(I gG)分
子硒化后的GPX活力71 .4U/娜of基本相同，所以制备人源化的高GPX活力
抗体酶是巨大的挑战。
本文首次改造己有的经化学诱变后具有高GPX活力的鼠源单链抗体，并
展示于噬菌粒pFUW一80表面，设计半抗原，通过亲和筛选获得对半抗原特异
结合的单链抗体，在大肠杆菌Rosetta中成功表达。经纯化后的单链抗体通过
化学法引入催化基团一Sec，其GpX活力为88OU/卿ol，是第一个人源化GPX
抗体酶高11倍，这种高GPX活力的人源化抗体酶作为抗氧化剂具有广阔的
应用前景。
1、构建人源化噬菌体展示抗体库
对鼠源单链抗体的改形分重链和轻链两部分。在蛋白质数据库(PDB)
中，通过同源序列分析分别确定与重链和轻链同源性较高的人源抗体，对于
鼠源单链抗体的改造，除了完全保留CDR区外，还通过计算机模拟的抗原结
合位点的空间结构，确定与CDR结构在空间上有关的氨基酸残基，并与人同
源抗体该部位的氨基酸同时成对保留，形成人源化轻、重链抗体库。重链采
取分段全序列合成的方法连接而成，轻链则通过重迭 PCR方法扩增而成，用
连接肤(Gl外Ser)3把轻、重链连接起来，并克隆于噬菌粒载体pFUW一80中，
通过辅助噬菌体M13K07使人源化单链抗体展示于噬菌粒pFUw一80表面。
构建的抗体库的理论库容为连接产物转化后在有氨节抗性平板上的菌落数，
而实际库容为理论库容XPCR可扩增出单链抗体的比例。
2、具有GPX结合活性的单链抗体的筛选
用疏水腔修饰法，设计并合成半抗原GSH一S一DNP一Bu和GSH一S一DNP一Me，
进行抗体库的筛选。以噬菌体单链为模板进行PCR扩增，用BstNI酶切扩增
的单链抗体基因，通过DNA指纹法验证了单链抗体库的多样性，ELISA法验
证了筛选的阳性克隆。经4轮筛选，分别得到了对半抗原特异性结合的单链
抗体，从ELISA结果选出Bg和B14对GSH一S一DNP.Bu特异结合的抗体，通
中文摘要
过双酶切法和DNA序列分析法进行阳性鉴定。
3、定向进化构建次级库及特异结合性抗体的筛选
如何获得高亲和力抗体始终是噬菌体抗体库技术的一个发展方向。由于
构建的人源化抗体库库容较小，我们采用DNA改组技术在体外提高抗体的亲
和力，即通过有性和无性PCR扩增出特异性单链抗体基因后克隆于噬菌粒载
体pFUW一80中，形成次级抗体库，以GSH一s~DN甲一Bu为半抗原，进行正向有
意义突变的高亲和性抗体的筛选。经过3轮筛选后筛选出的单链抗体的ELISA
信号明显高于DNA改组前的两个阳性克隆，我们选ELISA信号最强的噬菌
体抗体克隆BS进行以后的表达。
4、单链抗体在大肠杆菌的表达及纯化
通过PCR扩增出单链抗体BS基因，然后克隆于载体pPe扭中，通过双
酶切法和DNA序列分析法鉴定连接产物，证实单链抗体基因已按正确方向连
入表达载体中。通过对表达菌株进行优化选择了最佳表达菌株，然后对诱导
剂加入时菌体浓度、诱导剂浓度和诱导表达时间进行优化，得到适合单链抗
体表达的最优条件，按此条件大量表达目的蛋白。通过Ni2+金属离子鳌合纯
化目的
Reactive oxygen species(ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage biomacromolecules such as RNA, DNA, protein, sugared and lipids, therefore this results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, aged-related disease, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. GPX plays an important role in antioxidant defense system of enzymatic action. Howe
ver, due to the shortcomings of native GPX (solution instability, poor availability, short half-lives and proteolytic digestion), mimics with high GPX efficiency were prepared by many biochemists. One of the well-known GPX mimics is Ebselen (2-phenyl-l,2- benziososelenazol-3 (2H)-one) . Although this intresting molecule is undergoing phase III clinical trial in Japan as antioxidant, it has some drawbacks such as its low GPX activity and its insolubility in water. We have successfully generated several mouse catalytic antibodies, and expressed mouse single-chain variable region antibodies (ScFv's) in Escherichia coli (E.coli)
by protein engineering and found high GPX efficiencies by chemical modified serine into selenocysteine (Sec). Although some antibodies from murine sources have good affinity and showed excellent results in animal models, the problem with human antimurine (HAMA) antibodies must be resolved. In 2001, the full human antibodies were selected from a semi-synthetic antibody library and the GPX efficiency of the catalytic antibody was found to be 80 U/umol, upon Sec was introduced. But this efficiency was almost as much as that of Se- y IgG
In this study, we described the construction of a library displayed human antibody fragments that can bind the specific substrate-glutathione (GSH). Our strategy was to construct a single-chain variable fragments (ScFv) library by reforming a mouse fragment variant region into a phagemid vector pFUW-80. The constructed library was a chimeric one that contained both conservative fragments of mouse ScFv and incorporated the human homologous fragments. Two designed haptens were synthesized GSH-S-DNP-Me and GSH-S-DNP-Bu and were coupled to BSA by glutaradehyde. The selected ScFv's were identified by ELISA and were cloned into the expression vector pPelB. The expressed protein was purified, identified and the catalytic group, Sec was incorporated into the protein by chemical mutation. The GPX activity of the single-chain antibody was 880 U/umol, which is 11-fold that of first reported human glutathione peroxidase mimic. The selenium-containing human single-chain abzyme with GPX activity shows great prospects in medicine.
1. Construction of ScFv antibody library
The heavy chain (VH) and light chain (VL) of the reshaping antibody of variable fragment were designed respectively. By homology sequence analysis from protein data bank (PDB), homology replacements of the VH and VL fragments regions were chosen. There is a need to select a correct pair of variable regions to achieve the right specificity. Key residues from the mouse single-chain antibody (heavy chain CDR3 sequences and sometimes also CDR2) are incorporated into human repertoires in order to preserve the desired binding function. By computer
stimulation, the conservative residues, which are relevant to the structure of the non-human antibodies, as well as CDRs, were retained. The VH genes were ligated by the synthesized VH fragments, while the VL genes were amplified by over-lap PCR with the primers o
引文
1. Gavilondo J. and Larrick, J.W. Antibody engineering at the millennium, Biotech. 2000, 29:128-136.
2．蔓尼阿蒂斯，萨姆布鲁克，弗里奇．分子克隆实验指南(第二版)，1989
3. Griffiths A.D., Malmqvist M., Marks J.D., Bye J.M., Embleton M.J., McCafferty J., Baier M., Holliger K.P., Gorick B.D. and Hughes-Jones N.C. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 1993, 12(2): 725-34.
4. Fan Z.C., Shah L., Guddat L.W., He X.M., Gray W.R., Raison R.L. and Edmundaon A.B. Three-dimensional structure of an Fv from a human IgM immtmoglobulin, J Mol Biol, 1992, 228: 188-207.
5. Kabat E.A., Sequence of proteins of Immunological Interest 5th, U S Department of Health and Human Services, 1991
6．刘喜富，博士论文，中国科学院遗传研究所，1995，6
7. Weng N.E, Yu-lee L.Y., Sanz I., Patten B.M. and Marcus D.M. Structure and specificities of anti-ganglioside autoantibodies associated with motor neuropathies. J Immunol. 1992, 149(7): 2518-2529.
8. Portolano S., Mclachlan S.M. and Rapoport B. High affinity, thyroid-specific human autoantibodies displayed on the surface of filamentous phage use V genes similar to other autoantibodies. J Immunol. 1993, 151(5): 2839-2851.
9. Jones P.T., Dear P.H., Foote J., Meuberger M.S. and Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986, 321: 522-525.
10. Tempest P.R., Barbanti E., Bremner P,, Carr F.J., Ghislieri M., Rifaldi B. and Marcucci F. A humanized anti-tumor necrosis factor-alpha monoclonal antibody that acts as a partial, competitive antagonist of the template antibody. Hybridoma. 1994, 13(3): 183-190.
11. Ohtomo T., Tsuchiya M., Sato K., Shimizu K., Moriuchi S., Miyao Y., Akimoto T. and Akamatsu K. Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions. Mol Immunol. 1995, 32(6): 407-416.
12. Couto J.R., Padlan E.A., Blank E.W., Peterson LA. and Ceriani R.L. Humanization of KC4G3, an anti-human carcinoma antibody, Hybridoma. 1994, 13(3): 215-219.
13. Sato K., Tsuchiya M., Saldanha J., Koishihara Y., Ohsugi Y., Kishimoto T. and Bendig M.M. Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions. Mol Immunol. 1994, 31(5): 371-381.
14. Couto J.R., Blank E.W., Peterson J.A. and Ceriani R.L. Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization. Cancer Res. 1995, 55(8): 1717-1722.
15. Chothia C., Lesk A.M., Tramontano A., Levitt M., Smith-Gill S.L, Air G, Sheriff S., Padlan E.A., Davies D., Tulip W.R., Colman P.M., Spinelli S., Alzari P.M. and Poljak R.J. Conformations of immunoglobulin hypervariable regions. Nature. 1989, 342: 877-883.
16. Singer Ⅱ, Kawka D.W., DeMartino LA., Daugherty B.L., Elliston K.O., Alves K., Bush B.L., Cameron P.M., Cuca GC. and Davies P. Optimal humanization of 1B4, an anti-CD18 routine monoclonal antibody, is achieved by correct choice of human V-region framework sequences. J Imrnunol. 1993, 150(7): 2844～2857.
2．蔓尼阿蒂斯，萨姆布鲁克，弗里奇．分子克隆实验指南(第二版)，1989
3. Griffiths A.D., Malmqvist M., Marks J.D., Bye J.M., Embleton M.J., McCafferty J., Baier M., Holliger K.P., Gorick B.D. and Hughes-Jones N.C. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 1993, 12(2): 725-34.
4. Fan Z.C., Shah L., Guddat L.W., He X.M., Gray W.R., Raison R.L. and Edmundaon A.B. Three-dimensional structure of an Fv from a human IgM immtmoglobulin, J Mol Biol, 1992, 228: 188-207.
5. Kabat E.A., Sequence of proteins of Immunological Interest 5th, U S Department of Health and Human Services, 1991
6．刘喜富，博士论文，中国科学院遗传研究所，1995，6
7. Weng N.E, Yu-lee L.Y., Sanz I., Patten B.M. and Marcus D.M. Structure and specificities of anti-ganglioside autoantibodies associated with motor neuropathies. J Immunol. 1992, 149(7): 2518-2529.
8. Portolano S., Mclachlan S.M. and Rapoport B. High affinity, thyroid-specific human autoantibodies displayed on the surface of filamentous phage use V genes similar to other autoantibodies. J Immunol. 1993, 151(5): 2839-2851.
9. Jones P.T., Dear P.H., Foote J., Meuberger M.S. and Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986, 321: 522-525.
10. Tempest P.R., Barbanti E., Bremner P,, Carr F.J., Ghislieri M., Rifaldi B. and Marcucci F. A humanized anti-tumor necrosis factor-alpha monoclonal antibody that acts as a partial, competitive antagonist of the template antibody. Hybridoma. 1994, 13(3): 183-190.
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