绿脓杆菌外毒素A与人组蛋白H3融合蛋白的表达与纯化
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摘要
绿脓杆菌外毒素A(Pseudomonas Exotoxin A,PEA)是绿脓杆菌的主要毒力因子之一，其毒性作用必须在毒素分子进入细胞后才发挥出来。PEA为一条单链毒蛋白，具有三个功能区，分别为功能区Ⅰa，功能区Ⅰb，功能区Ⅱ和功能区Ⅲ。功能区Ⅰa负责识别和结合细胞，功能区Ⅱ负责跨膜移位，功能区Ⅲ具有ADP-核糖基化活性，可通过对延伸因子EF-2的ADP-核糖基化作用而抑制真核细胞的蛋白合成。人组蛋白H3是碱性核蛋白，富含精氨酸，在生理条件下，H3的精氨酸带正电荷，而DNA的磷酸基团带负电荷，所以组蛋白和DNA分子主要依靠静电引力相结合。
本实验在已成功获得的PEA功能区Ⅰa，功能区Ⅱ基因片段与人组蛋白H3基因片段的克隆菌株的基础上，构建原核表达载体，将克隆质粒pMD-18T-PEA经Nco Ⅰ，Mlu Ⅰ双酶切，回收酶切产物；将克隆质粒pMD-18T-H3经Mlu Ⅰ，Xho Ⅰ双酶切，回收酶切产物。同时将原核表达载体pET-28c用Nco Ⅰ，Xho Ⅰ双酶切，回收酶切产物，将回收的酶切产物PEA，H3，载体进行连接，并转入DH5α感受态细胞内，培养12-18小时后，挑取阳性菌落，经Nco Ⅰ，Xho Ⅰ双酶切分析及PCR检测，筛选到阳性克隆，其质粒测序结果表明成功地构建了毒性基因缺失的PEA与人组蛋白H3融合基因的原核表达载体。
提取重组质粒并转化大肠杆菌BL21(DE3)，经IPTG诱导阳性菌，对菌体总蛋白进行SDS-PAGE电泳分析，结果表明BL21(DE3)能有效表达目的蛋白，薄层扫描计算分析，显示表达蛋白带密度积分占总蛋白量的16.28％。大量诱导培养表达菌，制备包涵体，经侧带法纯化后加入弗氏佐剂制成免疫原，对獭兔进行3次(每次间隔14天)注射免疫，通过ELISA法测定，该重组蛋白能够诱导獭兔产生抗体，免疫后7-14天达到峰值，并初步制备了终点滴度为1：1600的兔抗PEA-H3抗血清。大量制备包涵体，并对包涵体制备方法优化。利用融合蛋白带有6个组氨酸标签的特点采用金属螯和亲和层析对变性溶解的融合蛋白进行纯化，纯化结果含有少量杂带。用鲑鱼精DNA和质粒与表达蛋白作用，进行表达蛋白与DNA的凝胶迁移率变动分析实验，证明了融合蛋白与DNA的结合效果非常好。采用溴化氰活化的
Sepharose 4 Fast Flow进行亲和层析。用聚乙二醇（PEG）10000浓缩，G－250法测
含量，计算蛋白产率为30％。表明亲和层析纯化效果非常理想。从而为进一步的重
组蛋白功能分析和基因转染，基因治疗实验奠定了基础。
Pseudomonas exotoxin A (PEA) is a single chain toxin,PEA is made up of three domains. The pHysical boundaries indicated that domain Ia is composed of residues l-252;domain II, residues 253-364; domainIb, residues 365-404; and domain III, residues 405-613. It has been shown that domain Ia is responsible for cell recognition, domain II is to be involved in translocation of the toxin across membranes,and domain HI catalyzes the ADP-ribosylation of elongation factor2, Which arrests protein synthesis and results in cell death. Arginine rich human histone H3 is a kind of basic nucleosome protein.In the medium of pHysiological condition,by electrostatic interaction,histone H3 which arginine impart to it a positive charge binds to DNA which pHospHate groups impart to it a negative charge.In this paper,In order to establish a new technology of transfection, the functional domains la and II gene segments of PEA were lined with histone H3 gene segment, then a recombinant fusion protein was constructed which serves as c
arrier for transfer of DNA via receptor-mediated endocytosis.
In this study,the recombinant plasmid pMD-18T-PEA-H3 was cleavaged with NcoI ,XhoI and inserted into the expression vector pET-28c and subsequently subjected to restriction endonuclease analysis and sequencing, the result indicated that the prokaryotic expression vector pET-28c-PEA-H3 was constructed successfully.After the expression plasmid was extracted and transformed into expression hosts BL21(DE3) of E.coli,the transformed hosts were induced by IPTG,bySDS-PAGE and ELISA analysis of host protein.the expression of the
objective gene was detected ,and it could account for 16.28% of the total host protein .inclusion body was prepared from the incubating expression hosts induced by IPTG. After purification and absolv in 8mol/L urea .And then injected rabbits 3 times with 14 days interval.By analysis of indirect ELISA, the anti-PEA-H3 antibody appear in the rabbit serum induced by the recombinant fusion protein,the highest antibody level was observed during 7-14days after the final inoculation .
Incubate the genetic engineering bacteria in LB medium(Kana) and induce the inclusion body with IPTG ,then sonicated the bacterial cells and then subjected to denaturation in 8mol/L urea.Then purified inclusion body with His trap?metal affinity chromatograpHy.In order to obtain higher purity the expressed protein.Protein was further purified by CNBr-activated SepHarose 4 Fast Flow with nonspecial affinity chromatograpHy .At last,PEA-H3protein eventually collect 30%,it was prepared for next experiments functional analysis of the fusion protein
and genetic transfaction.
本实验在已成功获得的PEA功能区Ⅰa，功能区Ⅱ基因片段与人组蛋白H3基因片段的克隆菌株的基础上，构建原核表达载体，将克隆质粒pMD-18T-PEA经Nco Ⅰ，Mlu Ⅰ双酶切，回收酶切产物；将克隆质粒pMD-18T-H3经Mlu Ⅰ，Xho Ⅰ双酶切，回收酶切产物。同时将原核表达载体pET-28c用Nco Ⅰ，Xho Ⅰ双酶切，回收酶切产物，将回收的酶切产物PEA，H3，载体进行连接，并转入DH5α感受态细胞内，培养12-18小时后，挑取阳性菌落，经Nco Ⅰ，Xho Ⅰ双酶切分析及PCR检测，筛选到阳性克隆，其质粒测序结果表明成功地构建了毒性基因缺失的PEA与人组蛋白H3融合基因的原核表达载体。
提取重组质粒并转化大肠杆菌BL21(DE3)，经IPTG诱导阳性菌，对菌体总蛋白进行SDS-PAGE电泳分析，结果表明BL21(DE3)能有效表达目的蛋白，薄层扫描计算分析，显示表达蛋白带密度积分占总蛋白量的16.28％。大量诱导培养表达菌，制备包涵体，经侧带法纯化后加入弗氏佐剂制成免疫原，对獭兔进行3次(每次间隔14天)注射免疫，通过ELISA法测定，该重组蛋白能够诱导獭兔产生抗体，免疫后7-14天达到峰值，并初步制备了终点滴度为1：1600的兔抗PEA-H3抗血清。大量制备包涵体，并对包涵体制备方法优化。利用融合蛋白带有6个组氨酸标签的特点采用金属螯和亲和层析对变性溶解的融合蛋白进行纯化，纯化结果含有少量杂带。用鲑鱼精DNA和质粒与表达蛋白作用，进行表达蛋白与DNA的凝胶迁移率变动分析实验，证明了融合蛋白与DNA的结合效果非常好。采用溴化氰活化的
Sepharose 4 Fast Flow进行亲和层析。用聚乙二醇（PEG）10000浓缩，G－250法测
含量，计算蛋白产率为30％。表明亲和层析纯化效果非常理想。从而为进一步的重
组蛋白功能分析和基因转染，基因治疗实验奠定了基础。
Pseudomonas exotoxin A (PEA) is a single chain toxin,PEA is made up of three domains. The pHysical boundaries indicated that domain Ia is composed of residues l-252;domain II, residues 253-364; domainIb, residues 365-404; and domain III, residues 405-613. It has been shown that domain Ia is responsible for cell recognition, domain II is to be involved in translocation of the toxin across membranes,and domain HI catalyzes the ADP-ribosylation of elongation factor2, Which arrests protein synthesis and results in cell death. Arginine rich human histone H3 is a kind of basic nucleosome protein.In the medium of pHysiological condition,by electrostatic interaction,histone H3 which arginine impart to it a positive charge binds to DNA which pHospHate groups impart to it a negative charge.In this paper,In order to establish a new technology of transfection, the functional domains la and II gene segments of PEA were lined with histone H3 gene segment, then a recombinant fusion protein was constructed which serves as c
arrier for transfer of DNA via receptor-mediated endocytosis.
In this study,the recombinant plasmid pMD-18T-PEA-H3 was cleavaged with NcoI ,XhoI and inserted into the expression vector pET-28c and subsequently subjected to restriction endonuclease analysis and sequencing, the result indicated that the prokaryotic expression vector pET-28c-PEA-H3 was constructed successfully.After the expression plasmid was extracted and transformed into expression hosts BL21(DE3) of E.coli,the transformed hosts were induced by IPTG,bySDS-PAGE and ELISA analysis of host protein.the expression of the
objective gene was detected ,and it could account for 16.28% of the total host protein .inclusion body was prepared from the incubating expression hosts induced by IPTG. After purification and absolv in 8mol/L urea .And then injected rabbits 3 times with 14 days interval.By analysis of indirect ELISA, the anti-PEA-H3 antibody appear in the rabbit serum induced by the recombinant fusion protein,the highest antibody level was observed during 7-14days after the final inoculation .
Incubate the genetic engineering bacteria in LB medium(Kana) and induce the inclusion body with IPTG ,then sonicated the bacterial cells and then subjected to denaturation in 8mol/L urea.Then purified inclusion body with His trap?metal affinity chromatograpHy.In order to obtain higher purity the expressed protein.Protein was further purified by CNBr-activated SepHarose 4 Fast Flow with nonspecial affinity chromatograpHy .At last,PEA-H3protein eventually collect 30%,it was prepared for next experiments functional analysis of the fusion protein
and genetic transfaction.
引文
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[3] Avramoglu, R.K.,Nimpf, et al. 1998, J.Biol. Chem.273:6057-6065
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[9] Marie-Pierre Taupiac,Magali Bebien,Meriem A.et al.A deletion within the translocation domain of Pseudomonas exotoxin A enhances transloeation efficiency and cytotoxicity concomitantly.1999,Molecular microbiology.31:1385-1393
[10] Madshus, I.H.,and Collier, R.J.Effeets of eliminating a disulfide bridge within domain Ⅱ of PEA. 1989,Infect Immun. 57:1873-1878
[11] Kasturi,S.,Kihara, A.,FitzGerald,et al.Alanine scanning mutagenesis identifies surface amino acids on domain Ⅱ of Pseudomonas Exotoxin required for eytotoxicity, proper folding and secretion into periplasm.1992, J.Biol. Chem.267:23427-23433
[12] Jinno,Y.,Ogata, M.,Chaudhary, V.K., et al. Domain Ⅱ mutants of Pseudomonas Exotoxin deficient in translocation. 1989, J.Biol. Chem.264:15953-15959
[13] Chraudhrary VK, Jinno Y, FitzGerald D. Pseudomonas exotoxin contains a specific sequence at thre carboxyl terminus that is required for eytotoxieity. Proc Natl Acad Sci USA. 1990 Jan;87(1):308-12.
[14] Carroll SF, et al:J Bioi then 1987 262:8707～8711.
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[18] 郭学军，刘晓明，朱平，等．绿脓杆菌外毒素A受体结合亚单位基因的克隆与表达 中国兽医学报 1997，17(3)：226-229
[19] Jesus Fominaya,Winfried Wels. Target Cell-specific DNA Transfer Mediated by a Chimeric Multidomain Protein. The American Society for Biochemistry and Molecular Biology. 271:10560-10568
[20] Epstein SE, Siegall CB, Biro S. Cytotoxic effects of a recombinant chrimeric toxin on rapidly proliferating vascular smoothr muscle cells. Circulation. 1991 Aug;84(2):778-87.
[21] Weltman JK, Pedroso P, Johrnson SA. Rapid screening withr indirect immunotoxin for monoclonal antibodies against hruman small cell lung cancer.Cancer Res. 1987 Nov 1;47(21):5552-6.
[22] 杜世或．绿脓杆菌外毒素A的结构，功能及其重组毒素，生物化学与生物物理进展，1995，22(2)112～117。
[23] Vorobev VI.,Birshtein TM.Relationship between the primary and secondary structure of histone.I.Analysis of the primary structure of historic f2al. 1971,Mol Biol.5:261-265
[24] Behe,M.J. The biology of nonspeeific DNA-protein interactions.CRC Press,1990,pp.229-249
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[29] Richmond, T. J., Finch, J. T., Rushton, B., et al. Structure of the nucleosome core particle at 7 A resolution. (1984) Nature 311,532-537
[30] ChristopHer, R.Adams and Rohinton, T.Kamakaka. Chromatin assembly:biochemical identities and genetic redundancy. 1999,Current Opinion in Genetics & Development. 9:185-190
[31] Annunziato,AT. Histone acetylation during ehromatin replication and nucleosome assembly. 1995,Nucleus, 1:31-56
[32] Gruss C,Sogo JM. Chromatin replication. 1992,Bioessays,14:1-8
[33] Arents, G., Burlingame, R. W., Wang, B. C., et al. The nucleosomal core histone octamer at 3.1 A° resolution. 1991, Proc. Nat.l Acad. Sc. U. S. A. 88:10148-10152
[34] Kathryn A.Bailey, John N.Reeve. DNA repeats and archaeal nuxleosome positioning.
1999,Res.Microbiol.150:701-709
[35] Wagner, E., Zenke, M., Cotten, M., et al. Transferrin-polycation conjugates as carriers for DNA uptake into cells.(1990) Proc. Natl. Acad. Sci. U.S.A. 87:3410-3414
[36] Yin, W., and Cheng, P.-W. Lectin conjugate-directed gene transfer to airway epithelial cells.(1994) Biochem. BiopHys. Res. Commun. 205:826-833
[37] Chen, S.-Y., Zani, C., Khouri, Y.,et al. Design of a genetic immunotoxin to eliminate toxin immunogenicity. 1995, Gene Ther. 2:116-123
[38] Sambrook J,Fritsch EF,Manitatis T.分子克隆实验．科学出版社．1992．
[39] F．奥斯伯，R．布伦特，R．E．金斯顿等．精编分子生物学实验指南．中国科学出版社．1998．
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[2] Kounnas,M.Z.,Morris,et al.The α 2 maeroglobulin receptor/low density lipoprotein receptor binds and internalizes Pseudomonas exotoxin A. 1992, J.Biol. Chem.267:12420-12423
[3] Avramoglu, R.K.,Nimpf, et al. 1998, J.Biol. Chem.273:6057-6065
[4] FitzGerald,D.J.,Fryling,et al. 1995,J.Cell Biol. 129:1533-1541
[5] Kondo,T.,FitzGerald D.J.et al. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain 1988, J Biol Chem.263:9470-9475
[6] Middlebrook JL, et al:Microbiol Rev 1984 48:199-221
[7] Gray GL,et.al;proe Nat Acad Sci USA 1984 81:2645～2649
[8] Allured VS, Collier R.J, Carroll SF. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci USA. 1986 Mar;83(5): 1320-4
[9] Marie-Pierre Taupiac,Magali Bebien,Meriem A.et al.A deletion within the translocation domain of Pseudomonas exotoxin A enhances transloeation efficiency and cytotoxicity concomitantly.1999,Molecular microbiology.31:1385-1393
[10] Madshus, I.H.,and Collier, R.J.Effeets of eliminating a disulfide bridge within domain Ⅱ of PEA. 1989,Infect Immun. 57:1873-1878
[11] Kasturi,S.,Kihara, A.,FitzGerald,et al.Alanine scanning mutagenesis identifies surface amino acids on domain Ⅱ of Pseudomonas Exotoxin required for eytotoxicity, proper folding and secretion into periplasm.1992, J.Biol. Chem.267:23427-23433
[12] Jinno,Y.,Ogata, M.,Chaudhary, V.K., et al. Domain Ⅱ mutants of Pseudomonas Exotoxin deficient in translocation. 1989, J.Biol. Chem.264:15953-15959
[13] Chraudhrary VK, Jinno Y, FitzGerald D. Pseudomonas exotoxin contains a specific sequence at thre carboxyl terminus that is required for eytotoxieity. Proc Natl Acad Sci USA. 1990 Jan;87(1):308-12.
[14] Carroll SF, et al:J Bioi then 1987 262:8707～8711.
[15] Douglas CM,et al:J Bacteriol 1987 169:4962～4966.
[16] Lukac M,et al:J Biol ehen 1988 263:6146～6149.
[17] 刘晓明，郭学军，马从林，等．含绿脓杆菌外毒素A受体结合区重组蛋白的纯化．中国兽医学报，1998，18(1)：38～41
[18] 郭学军，刘晓明，朱平，等．绿脓杆菌外毒素A受体结合亚单位基因的克隆与表达 中国兽医学报 1997，17(3)：226-229
[19] Jesus Fominaya,Winfried Wels. Target Cell-specific DNA Transfer Mediated by a Chimeric Multidomain Protein. The American Society for Biochemistry and Molecular Biology. 271:10560-10568
[20] Epstein SE, Siegall CB, Biro S. Cytotoxic effects of a recombinant chrimeric toxin on rapidly proliferating vascular smoothr muscle cells. Circulation. 1991 Aug;84(2):778-87.
[21] Weltman JK, Pedroso P, Johrnson SA. Rapid screening withr indirect immunotoxin for monoclonal antibodies against hruman small cell lung cancer.Cancer Res. 1987 Nov 1;47(21):5552-6.
[22] 杜世或．绿脓杆菌外毒素A的结构，功能及其重组毒素，生物化学与生物物理进展，1995，22(2)112～117。
[23] Vorobev VI.,Birshtein TM.Relationship between the primary and secondary structure of histone.I.Analysis of the primary structure of historic f2al. 1971,Mol Biol.5:261-265
[24] Behe,M.J. The biology of nonspeeific DNA-protein interactions.CRC Press,1990,pp.229-249
[25] VD Paponov, PS Gromov, NA Sokolov, et al. On mechanisms determining the interrelationships between DNA and histone components of chromatin. 1980,European Journal of Biochemistry, 107:113-122
[26] Bina-Stein, M., and Simpson, R. T. Specific folding and contraction of DNA by histories H3 and H4. (1977) Cell 11,609-618
[27] Camerini-Otero, R.D., Sollner-Webb, B., and Felsenfeld, G. The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel.(1976) Cell 8, 333-347
[28] Sollner-Webb, B., Camerini-Otero, R. D., and Felsenfeld, G. Chromatin structure as probed by nucleases and proteases: evidence for the central role of histones H3 and H4.(1976) Cell 9,179-193
[29] Richmond, T. J., Finch, J. T., Rushton, B., et al. Structure of the nucleosome core particle at 7 A resolution. (1984) Nature 311,532-537
[30] ChristopHer, R.Adams and Rohinton, T.Kamakaka. Chromatin assembly:biochemical identities and genetic redundancy. 1999,Current Opinion in Genetics & Development. 9:185-190
[31] Annunziato,AT. Histone acetylation during ehromatin replication and nucleosome assembly. 1995,Nucleus, 1:31-56
[32] Gruss C,Sogo JM. Chromatin replication. 1992,Bioessays,14:1-8
[33] Arents, G., Burlingame, R. W., Wang, B. C., et al. The nucleosomal core histone octamer at 3.1 A° resolution. 1991, Proc. Nat.l Acad. Sc. U. S. A. 88:10148-10152
[34] Kathryn A.Bailey, John N.Reeve. DNA repeats and archaeal nuxleosome positioning.
1999,Res.Microbiol.150:701-709
[35] Wagner, E., Zenke, M., Cotten, M., et al. Transferrin-polycation conjugates as carriers for DNA uptake into cells.(1990) Proc. Natl. Acad. Sci. U.S.A. 87:3410-3414
[36] Yin, W., and Cheng, P.-W. Lectin conjugate-directed gene transfer to airway epithelial cells.(1994) Biochem. BiopHys. Res. Commun. 205:826-833
[37] Chen, S.-Y., Zani, C., Khouri, Y.,et al. Design of a genetic immunotoxin to eliminate toxin immunogenicity. 1995, Gene Ther. 2:116-123
[38] Sambrook J,Fritsch EF,Manitatis T.分子克隆实验．科学出版社．1992．
[39] F．奥斯伯，R．布伦特，R．E．金斯顿等．精编分子生物学实验指南．中国科学出版社．1998．
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