重组Ig结合分子噬菌体展示文库的构建及选择研究
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摘要
定向分子进化是探索研究生物大分子结构与功能的重要手段，同时也是改造、优化生物大分子特性的有效方法。噬菌体展示及筛选和DNA重排是体外定向分子进化的重要核心技术。proteinA和protein L是两种不同的细菌表面蛋白，均可与Ig结合，但各自有不同的特点。本研究应用DNA重排技术将protein A的单个抗体结合结构域和protein L的单个抗体结合结构域随机组合，构建了重组Ig结合多肽PALn分子文库，并采用噬菌体展示技术结合体外定向进化IgG亲和筛选，探究Ig结合分子的结构和功能的关系，同时为定向改造Ig结合分子打下基础。本研究分四个部分：一、新型噬菌体展示载体pCANTAB5S的构建；二、噬菌体展示Ig结合多肽PLn分子文库的构建及筛选；三、噬菌体展示Ig结合多肽PALn分子文库的构建及筛选；四、(MDPL-MDPA)n代表分子的原核表达、纯化及功能鉴定。
一、新型噬菌体展示载体pCANTAB5S的构建
通过基因操作，将限制酶切位点SacI引入到噬菌体展示载体pCANTAB5L，并校正了pCANTAB5L载体Stu Ⅰ、Sal Ⅰ等位点的读框。具体过程是：以pCANTAB5X-IFN-α2b重组质粒为模板，在上游引物中引入XbaⅠ、Sac Ⅰ酶切位点，在下游引物中引入Sac Ⅰ、StuⅠ、Sal Ⅰ酶切位点，将用该引物扩增的IFN-α2b衔接片段PCR产物克隆到pMD18-T中，再将该片段用Xba Ⅰ和Sal Ⅰ酶切并将之插入pCANTAB5L的Xba Ⅰ和Sal Ⅰ位点中，经SacⅠ酶切，线性载体片段自连，构成新型噬菌体展示载体pCANTAB5S。序列分析证明pCANTAB5S在pCANTAB5L原有Xba Ⅰ和Stu Ⅰ之间引入Sac Ⅰ位点，并校正了原Stu Ⅰ的读框，有利于各种外源随机多肽和功能蛋白的有效展示。
二、噬菌体展示Ig结合多肽PLn分子文库的构建及筛选
用含Sac Ⅰ位点的特定引物PCR扩增protein L的B3抗体结合结构域(L)，Sac Ⅰ酶切后，经DNA重排连接形成各种不同长度的PLn随机分子库，并将该文库呈现在噬菌体表面，构建了噬菌体展示Ig结合多肽PLn文库。所建肽库容量为3×10~5个菌落形成单位，滴度为6.2×10~(10)TU／ml。通过三轮IgG亲和筛选，选择后的12个阳性克隆DNA测序分析显示8个为2L序列，1个为L序列，证实了protein L的单个B结构域具有Ig结合功能，2个重复protein L的B结构域序列形成的分子具有良好的IgG结合能力。结果表
安徽医科大学硕士学位论文
明应用分子进化手段研究Ig结合分子的结构和功能是可行的。
三、噬菌体展示Ig结合多肤PALn分子文库的构建及筛选
用含Sacl位点的特定引物PCR分别扩增ProteinA的A，B，C，D4个抗体结合结构域
和proteinL的B3抗体结合结构域，Sacl酶切后，经DNA重排连接形成各种不同长度
的PALn随机分子文库，并将该文库呈现在噬菌体表面，构建了噬菌体展示19结合多肤
队Ln文库。所建肤库容量为2.3、106个菌落形成单位，滴度为4、1、10”Tu/ml。通过四轮
IgG亲和筛选，选择后的36个阳性克隆序列分析显示:20个序列为特征性的由proteinL
的单结构域和ProteinA的单结构域间隔重组排列而成的(MDPL一MDPA)n结构。结果证实
分子进化技术是研究Ig结合分子的结构和功能关系的有效方法和手段，同时发现
(MDPL一MD队)n结构可能是具IgG结合能力的分子的一种重要的结构形式。
四、(MDPL一MDPA)n代表分子的原核表达、纯化及功能鉴定
PCR扩增(MDPL一MD队)n代表分子一LD3，LDS序列及PLn(PALn)库选择阳性序列
ZL，在上游引物中引入Neol位点，PCR扩增产物用Neol和sall酶切，ZL，LD3，LDS
序列被克隆于表达载体PET32a(+)的Ncol和Sall位点中，原核表达N端带His一Tag
的融合蛋白，Ni一NTA柱亲和层析纯化，W己stem Blot鉴定，ELISA检测证实
(MDPL一MDPA)n分子有很好的IgG结合活性，与队Ln噬菌体展示文库的筛选结果一致;
纯化蛋白IgG结合活性趋势比较:LDS>LD3>S队>ZL，结果显示结构域组成数越多，
IgG结合活性越高，为研究Ig结合分子结构和功能的关系提供了一些依据。
本研究首次构建ProteinA和ProteinL的单结构域重组分子文库，并与噬菌体筛选技
术相结合研究Ig结合分子的结构和功能，最终成功获得多种Ig结合多肤序列，发现了一
种新的Ig结合分子结构形式(MDPL一MDPA)n，原核表达、纯化了(MDPL一MD队)n代表
分子，证实其具备良好的IgG结合活性，并揭示了其功能和结构的一些联系。本研究为
应用分子进化手段研究蛋白质分子的结构和功能的关系提供了成功实例，为噬菌体展示
技术在蛋白质分子进化中的应用提供了新的可借鉴方法;同时为Ig结合分子的定向改造
和高亲和力Ig结合分子的获得奠定了基础。
In vitro molecular directed evolution is important means to research on the relationship between structure and function of biomacromolecule. It can chage and optimize the properties of biomacromolecule under laboratorial conditions. Phage display and DNA shuffling are important and efficient techniques for directed molecular evolution . Protein A and protein L are two different bacterial suface proteins which have affinity for Ig molecules, but they have special characteristics respectively. In this study, we randomly combined each mono-domain of protein A and mono-domain of protein L to construct the molecular directed evolutional library of the Ig-binding peptides PALn, using phage display technique combined with in vitro molecular directed evolutional selection in order to explore the relationship between structure and function of IgG-binding molecule, and to lay a foundation for directed improvement of IgG-binding molecule as well. So we carried out the following four-part work. 1. Construction of a nove
l phagemid pCANTABSS
A new cloning site of restriction enzyme Sac I was added to phagemid pCANTAB5S and the open reading frame of the clone sites Stu I, Sal I, Kpn I etc. in phagemid pCANTAB5L was corrected by gene operation. Firstly, The recombinant plasmid pCANTAB5X-IFN- -2b was provided as the amplification template, new cloning site Sac I were added to upstream primer , and Sac I, Stu I and Sal I was added to downstream primer for linker IFN- -2b PCR amplification. The PCR product was cloned into pMD18-T. This DNA fragment was cut out by disgestion of restriction enzymes Xba I and Sal I, and inserted into the clone sites in phagemid pCANTAB5L, by digestion of restriction enzyme Sac I, the linear vector fragment was isolated and ligated to construct the the new phage display vector pCANTAB5S. Sequence analysis showed pCANTAB5S had a new cloning site of Sac I which was added between Xba I and Stu I sites in phagemid pCANTAB5L and the open reading frame of the clone site Stu I in phagemid pCANTAB5L was corrected for
more effectively displaying the foreign random peptide and function protein .
2. Display of Ig-binding peptides PLn molecular library and selection for positive
peptides from this phage displayed library
A pair of primers containing Sac I sequence were synthesized to amplify B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PLn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4 107 phage clones and 6.2x1010TU/ml, respectively. After three rounds IgG affinity selection , Ig-binding peptides 2L was obtained, sequence analysis showed 1 of 12 inserts were L sequences , which suggests the a single B-domain of protein L has affinity for Ig; 8 of 12 inserts were 2L sequences , which suggests the twin B-domain of protein L repeats has good Ig-binding activity. It indicates the application of molecular directed evolution technonogy is a feasible method to explore the structure and function of Ig-binding molecules.
3. Display of Ig-binding peptides PALn molecular library and selection for positive
peptides from this phage displayed library
A pair of primers containing Sac I sequence were synthesized to amplify A,B,C,D Ig-binding domain of protein A and B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PALn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4xl07phage clones and 6.2x1010TU/ml, respectively. After four rounds IgG affinity selection , a variety of Ig-binding peptides was obtained, sequence analysis showed 20 of 36 inserts were the characteristic (MDPL-MDPA)n structure which consisls of mono-domain of protein A
一、新型噬菌体展示载体pCANTAB5S的构建
通过基因操作，将限制酶切位点SacI引入到噬菌体展示载体pCANTAB5L，并校正了pCANTAB5L载体Stu Ⅰ、Sal Ⅰ等位点的读框。具体过程是：以pCANTAB5X-IFN-α2b重组质粒为模板，在上游引物中引入XbaⅠ、Sac Ⅰ酶切位点，在下游引物中引入Sac Ⅰ、StuⅠ、Sal Ⅰ酶切位点，将用该引物扩增的IFN-α2b衔接片段PCR产物克隆到pMD18-T中，再将该片段用Xba Ⅰ和Sal Ⅰ酶切并将之插入pCANTAB5L的Xba Ⅰ和Sal Ⅰ位点中，经SacⅠ酶切，线性载体片段自连，构成新型噬菌体展示载体pCANTAB5S。序列分析证明pCANTAB5S在pCANTAB5L原有Xba Ⅰ和Stu Ⅰ之间引入Sac Ⅰ位点，并校正了原Stu Ⅰ的读框，有利于各种外源随机多肽和功能蛋白的有效展示。
二、噬菌体展示Ig结合多肽PLn分子文库的构建及筛选
用含Sac Ⅰ位点的特定引物PCR扩增protein L的B3抗体结合结构域(L)，Sac Ⅰ酶切后，经DNA重排连接形成各种不同长度的PLn随机分子库，并将该文库呈现在噬菌体表面，构建了噬菌体展示Ig结合多肽PLn文库。所建肽库容量为3×10~5个菌落形成单位，滴度为6.2×10~(10)TU／ml。通过三轮IgG亲和筛选，选择后的12个阳性克隆DNA测序分析显示8个为2L序列，1个为L序列，证实了protein L的单个B结构域具有Ig结合功能，2个重复protein L的B结构域序列形成的分子具有良好的IgG结合能力。结果表
安徽医科大学硕士学位论文
明应用分子进化手段研究Ig结合分子的结构和功能是可行的。
三、噬菌体展示Ig结合多肤PALn分子文库的构建及筛选
用含Sacl位点的特定引物PCR分别扩增ProteinA的A，B，C，D4个抗体结合结构域
和proteinL的B3抗体结合结构域，Sacl酶切后，经DNA重排连接形成各种不同长度
的PALn随机分子文库，并将该文库呈现在噬菌体表面，构建了噬菌体展示19结合多肤
队Ln文库。所建肤库容量为2.3、106个菌落形成单位，滴度为4、1、10”Tu/ml。通过四轮
IgG亲和筛选，选择后的36个阳性克隆序列分析显示:20个序列为特征性的由proteinL
的单结构域和ProteinA的单结构域间隔重组排列而成的(MDPL一MDPA)n结构。结果证实
分子进化技术是研究Ig结合分子的结构和功能关系的有效方法和手段，同时发现
(MDPL一MD队)n结构可能是具IgG结合能力的分子的一种重要的结构形式。
四、(MDPL一MDPA)n代表分子的原核表达、纯化及功能鉴定
PCR扩增(MDPL一MD队)n代表分子一LD3，LDS序列及PLn(PALn)库选择阳性序列
ZL，在上游引物中引入Neol位点，PCR扩增产物用Neol和sall酶切，ZL，LD3，LDS
序列被克隆于表达载体PET32a(+)的Ncol和Sall位点中，原核表达N端带His一Tag
的融合蛋白，Ni一NTA柱亲和层析纯化，W己stem Blot鉴定，ELISA检测证实
(MDPL一MDPA)n分子有很好的IgG结合活性，与队Ln噬菌体展示文库的筛选结果一致;
纯化蛋白IgG结合活性趋势比较:LDS>LD3>S队>ZL，结果显示结构域组成数越多，
IgG结合活性越高，为研究Ig结合分子结构和功能的关系提供了一些依据。
本研究首次构建ProteinA和ProteinL的单结构域重组分子文库，并与噬菌体筛选技
术相结合研究Ig结合分子的结构和功能，最终成功获得多种Ig结合多肤序列，发现了一
种新的Ig结合分子结构形式(MDPL一MDPA)n，原核表达、纯化了(MDPL一MD队)n代表
分子，证实其具备良好的IgG结合活性，并揭示了其功能和结构的一些联系。本研究为
应用分子进化手段研究蛋白质分子的结构和功能的关系提供了成功实例，为噬菌体展示
技术在蛋白质分子进化中的应用提供了新的可借鉴方法;同时为Ig结合分子的定向改造
和高亲和力Ig结合分子的获得奠定了基础。
In vitro molecular directed evolution is important means to research on the relationship between structure and function of biomacromolecule. It can chage and optimize the properties of biomacromolecule under laboratorial conditions. Phage display and DNA shuffling are important and efficient techniques for directed molecular evolution . Protein A and protein L are two different bacterial suface proteins which have affinity for Ig molecules, but they have special characteristics respectively. In this study, we randomly combined each mono-domain of protein A and mono-domain of protein L to construct the molecular directed evolutional library of the Ig-binding peptides PALn, using phage display technique combined with in vitro molecular directed evolutional selection in order to explore the relationship between structure and function of IgG-binding molecule, and to lay a foundation for directed improvement of IgG-binding molecule as well. So we carried out the following four-part work. 1. Construction of a nove
l phagemid pCANTABSS
A new cloning site of restriction enzyme Sac I was added to phagemid pCANTAB5S and the open reading frame of the clone sites Stu I, Sal I, Kpn I etc. in phagemid pCANTAB5L was corrected by gene operation. Firstly, The recombinant plasmid pCANTAB5X-IFN- -2b was provided as the amplification template, new cloning site Sac I were added to upstream primer , and Sac I, Stu I and Sal I was added to downstream primer for linker IFN- -2b PCR amplification. The PCR product was cloned into pMD18-T. This DNA fragment was cut out by disgestion of restriction enzymes Xba I and Sal I, and inserted into the clone sites in phagemid pCANTAB5L, by digestion of restriction enzyme Sac I, the linear vector fragment was isolated and ligated to construct the the new phage display vector pCANTAB5S. Sequence analysis showed pCANTAB5S had a new cloning site of Sac I which was added between Xba I and Stu I sites in phagemid pCANTAB5L and the open reading frame of the clone site Stu I in phagemid pCANTAB5L was corrected for
more effectively displaying the foreign random peptide and function protein .
2. Display of Ig-binding peptides PLn molecular library and selection for positive
peptides from this phage displayed library
A pair of primers containing Sac I sequence were synthesized to amplify B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PLn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4 107 phage clones and 6.2x1010TU/ml, respectively. After three rounds IgG affinity selection , Ig-binding peptides 2L was obtained, sequence analysis showed 1 of 12 inserts were L sequences , which suggests the a single B-domain of protein L has affinity for Ig; 8 of 12 inserts were 2L sequences , which suggests the twin B-domain of protein L repeats has good Ig-binding activity. It indicates the application of molecular directed evolution technonogy is a feasible method to explore the structure and function of Ig-binding molecules.
3. Display of Ig-binding peptides PALn molecular library and selection for positive
peptides from this phage displayed library
A pair of primers containing Sac I sequence were synthesized to amplify A,B,C,D Ig-binding domain of protein A and B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PALn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4xl07phage clones and 6.2x1010TU/ml, respectively. After four rounds IgG affinity selection , a variety of Ig-binding peptides was obtained, sequence analysis showed 20 of 36 inserts were the characteristic (MDPL-MDPA)n structure which consisls of mono-domain of protein A
引文
1. Boyle,M.D.R(Ed.) Bacterial Immunoglobulin-Binding Proteins,Vols. Ⅰ and Ⅱ.Academic Press,San Diego,USA. 1990
2. Tashiro M, Montelione GT. Structures of bacterial immunoglobulin-binding domains and their complexes with immunoglobulins. Curr Opin Struct Biol. 1995 Aug;5(4):471-81
3. Bjorck, L. Protein L, a novel bacterial cell wall protein with affinity for Ig L chains. J.Immunol, 1988;140:1194-1197
4. Akerstrom, B., Bjorck, L. Protein L: A light chain-binding bacterial protein.J.Biol.Chem, 1989;264:19740-19746
5. Kastern,W.,Sjobring, U., et al. Structure of peptostreptococcal protein L and identification of a repeated immurnoglobulin light chain-binding domain.J.Biol.Chem. 1992;267:12820-12825
6. Nilson, B.H.K.,Solomon,A., et al. Protein L from Peptostreptococcus magnus binds to the κ light chain variable domain.J.Biol.Chem. 1992;267:2234-2239
7. Housden NG, Harrison S, et al. Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus. Biochem Soc Trans. 2003 Jun;31(3):716-8.
8. M. Tashiro, R. Tejero, et al.High Resolution Solution NMR Structure of the Z Domain of Stapphylococcal Protein A.J. Mol. Biol. 1997272:573-579
9. Rarnirez-Alvarado M, Serrano L.Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold. Protein Science, 1997, 6:162-174
10. Svensson HG, Hoogenboom HR, Sjobring U. Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties.Eur J Biochem. 1998 Dec 1;258(2):890-896.
11. Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994;370:389-391
12. Crameri A, Raillard SA, et al. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature, 1998;391(6664):288-292
13．田波，李传昭．分子进化工程，北京：科学出版社．1999，第一版．
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25. Wikstrom,M., Sjobring, U. et al. Mapping of the immunoglobulin light chain-binding site of protein L. J.Biol.Chem. 1995;250:128-133
26. Sjodahl, J. Repetitive sequences in protein A from Staphylococcus aureus.Arrangement of five regions within the protein,four being highly homologous and Fc-binding. Eur.J.Biochem. 1977, 73:343-351
27. Ramirez-Alvarado M, Serrano L.Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold. Protein Science, 1997, 6:162-174
28．Sambrook T, Russell.D.W.,著，黄培堂等译．分子克隆实验指南，北京：科学出版社．1999，第三版．
2. Tashiro M, Montelione GT. Structures of bacterial immunoglobulin-binding domains and their complexes with immunoglobulins. Curr Opin Struct Biol. 1995 Aug;5(4):471-81
3. Bjorck, L. Protein L, a novel bacterial cell wall protein with affinity for Ig L chains. J.Immunol, 1988;140:1194-1197
4. Akerstrom, B., Bjorck, L. Protein L: A light chain-binding bacterial protein.J.Biol.Chem, 1989;264:19740-19746
5. Kastern,W.,Sjobring, U., et al. Structure of peptostreptococcal protein L and identification of a repeated immurnoglobulin light chain-binding domain.J.Biol.Chem. 1992;267:12820-12825
6. Nilson, B.H.K.,Solomon,A., et al. Protein L from Peptostreptococcus magnus binds to the κ light chain variable domain.J.Biol.Chem. 1992;267:2234-2239
7. Housden NG, Harrison S, et al. Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus. Biochem Soc Trans. 2003 Jun;31(3):716-8.
8. M. Tashiro, R. Tejero, et al.High Resolution Solution NMR Structure of the Z Domain of Stapphylococcal Protein A.J. Mol. Biol. 1997272:573-579
9. Rarnirez-Alvarado M, Serrano L.Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold. Protein Science, 1997, 6:162-174
10. Svensson HG, Hoogenboom HR, Sjobring U. Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties.Eur J Biochem. 1998 Dec 1;258(2):890-896.
11. Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994;370:389-391
12. Crameri A, Raillard SA, et al. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature, 1998;391(6664):288-292
13．田波，李传昭．分子进化工程，北京：科学出版社．1999，第一版．
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