结核杆菌HSP70及其与HBsAg融合蛋白在Pichia pastoris 酵母中的表达和免疫学研究
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摘要
目的
从肿瘤组织中分离的热休克蛋白-肽复合物以及体外表达的热休克蛋白与抗原的融合蛋白均能诱导针对抗原肽的特异性免疫应答反应。本研究旨在开发应用于慢性乙型肝炎治疗的新型融合蛋白疫苗。通过构建结核杆菌HSP70及HSP70与HBsAg融合蛋白的酵母表达载体，将外源基因通过整合型载体整合到毕赤酵母（Pichia pastoris）GS115染色体上，获得遗传性稳定分泌表达株，以胞外分泌的方式表达融合蛋白，并对表达蛋白的生化性质进行鉴定，为研究其生物学功能奠定基础。
方法
1.结核杆菌HSP70及HSP70与HBsAg融合蛋白酵母表达载体的构建与鉴定：应用PCR和分子克隆技术，构建了一系列的酵母表达穿梭载体：pPIC9K/hsp70、pPIC9K/s、pPIC9K/hsp(1-370)-s、pPIC9K /hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370)，经酶切鉴定和DNA测序证实目的基因正确后，再转化受体菌GS115。
2.结核杆菌HSP70及HSP70与HBsAg融合蛋白在Pichia pastoris中的表达与鉴定：将上述构建的6种质粒经SacI酶切线性化后，用电转
的方法将其整合到酵母GS115的染色体上，PCR法筛选重组子，用0.5%甲醇诱导PCR阳性的重组子。表达上清用SDS-PAGE、ELISA和Western-blot鉴定。
结果
1.重组质粒的鉴定：经酶切和DNA测序证实上述6种质粒均构建正确。
2.重组子的诱导表达
2.1 GS115/hsp70成功地分泌表达hsp70，表达量为0.15mg/ml，占表达上清总蛋白量的30%以上，其分子量为70KD左右，与预计理论值相符；用亲和层析纯化目的蛋白经Western-blot鉴定，可被抗结核杆菌HSP70的单克隆抗体识别。
2.2其余的5种转化子经甲醇诱导后，用SDS-PAGE电泳观察，在预计分子量的地方没有相应的条带，说明在上清中没有分泌表达；但它们的菌体破碎液均能用ELISA的方法检测到HBsAg的阳性结果，因此它们的表达物可能堆积在胞内。
结论
1.成功构建pPIC9K/hsp70、pPIC9K/s、pPIC9K/hsp(1-370)-s、pPIC9K /hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370) 6种酵母穿梭表达载体。
2.首次筛选出高表达量的GS115/hsp70转化菌株，能分泌表达结核杆菌HSP70，为研究HSP70的应用和生物学功能奠定了基础。
Objective
It has been shown that hsp-peptide isolated from cancer and hsp fusion proteins can elicit protective antitumor or antiviral cellular immune response without any additional adjuvant. In this study, we explore the novel protein vaccines to cure HBV chronic infection. Hsp70 and hsp70 fused with HBsAg yeast expression vectors were constructed. The exogenous genes can be integrated into Pichia pastoris chromosome by integrative expression plasmid generating genetic state secretion expression strain. Expression proteins were characterized as a basis of further studying their biological functions.
Methods
Construction and identification of hsp70 and hsp70 fused with HBsAg yeast expression vectors: Applied PCR and molecular clone technology, the yeast shuttle plasmids including pPIC9K/hsp70、pPIC9K/s、
1. pPIC9K/hsp(1-370)-s、pPIC9K/hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370) were constructed. Restriction enzyme digestion and DNA sequencing analysis confirmed whether the recombinant plasmids were correct.
2. Expression of hsp70 and hsp70 fusion proteins in Pichia pastoris system: First,the plasmids were linearized with SacI; Second,Pichia pastoris strain GS115 was transformed by electroportion; Third,the transformants were selected by PCR; Last,the recombinant Pichia strains were induced by 0.5% methanol.The supernatants from cultures were identified by SDS-PAGE、ELISA and western-blot analysis.
Results
1. Restriction digestion and DNA sequence analysis confirmed the six vector correct.
Expression of recombinant Pichia strains:Hsp70 was secreted into the supernatant,western-blot analysis showed that the positive band with about 70KD molecular weight was recongnized by anti-Mt.hsp70 antibody.Yeild of expression was up to 0.15mg/ml and the expression amount was more than 30% of the supernatant total proteins.However,the other five recombinant Pichia strains didn't secret the expected fusion proteins into the supernatant.The cell lysates were positive by measure with HBsAg ELISA.According to these,they
2. maybe accumulated expression protein in cell.
Conclusion
1. We succeeded in constructing six yeast shuttle expression vectors.
2. We utilized the Pichia expression system to secret hsp70 at first time and got a high expression recombinant Pichia strain,which would provide a opportunity to further study the application and biologic function of hsp70.
从肿瘤组织中分离的热休克蛋白-肽复合物以及体外表达的热休克蛋白与抗原的融合蛋白均能诱导针对抗原肽的特异性免疫应答反应。本研究旨在开发应用于慢性乙型肝炎治疗的新型融合蛋白疫苗。通过构建结核杆菌HSP70及HSP70与HBsAg融合蛋白的酵母表达载体，将外源基因通过整合型载体整合到毕赤酵母（Pichia pastoris）GS115染色体上，获得遗传性稳定分泌表达株，以胞外分泌的方式表达融合蛋白，并对表达蛋白的生化性质进行鉴定，为研究其生物学功能奠定基础。
方法
1.结核杆菌HSP70及HSP70与HBsAg融合蛋白酵母表达载体的构建与鉴定：应用PCR和分子克隆技术，构建了一系列的酵母表达穿梭载体：pPIC9K/hsp70、pPIC9K/s、pPIC9K/hsp(1-370)-s、pPIC9K /hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370)，经酶切鉴定和DNA测序证实目的基因正确后，再转化受体菌GS115。
2.结核杆菌HSP70及HSP70与HBsAg融合蛋白在Pichia pastoris中的表达与鉴定：将上述构建的6种质粒经SacI酶切线性化后，用电转
的方法将其整合到酵母GS115的染色体上，PCR法筛选重组子，用0.5%甲醇诱导PCR阳性的重组子。表达上清用SDS-PAGE、ELISA和Western-blot鉴定。
结果
1.重组质粒的鉴定：经酶切和DNA测序证实上述6种质粒均构建正确。
2.重组子的诱导表达
2.1 GS115/hsp70成功地分泌表达hsp70，表达量为0.15mg/ml，占表达上清总蛋白量的30%以上，其分子量为70KD左右，与预计理论值相符；用亲和层析纯化目的蛋白经Western-blot鉴定，可被抗结核杆菌HSP70的单克隆抗体识别。
2.2其余的5种转化子经甲醇诱导后，用SDS-PAGE电泳观察，在预计分子量的地方没有相应的条带，说明在上清中没有分泌表达；但它们的菌体破碎液均能用ELISA的方法检测到HBsAg的阳性结果，因此它们的表达物可能堆积在胞内。
结论
1.成功构建pPIC9K/hsp70、pPIC9K/s、pPIC9K/hsp(1-370)-s、pPIC9K /hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370) 6种酵母穿梭表达载体。
2.首次筛选出高表达量的GS115/hsp70转化菌株，能分泌表达结核杆菌HSP70，为研究HSP70的应用和生物学功能奠定了基础。
Objective
It has been shown that hsp-peptide isolated from cancer and hsp fusion proteins can elicit protective antitumor or antiviral cellular immune response without any additional adjuvant. In this study, we explore the novel protein vaccines to cure HBV chronic infection. Hsp70 and hsp70 fused with HBsAg yeast expression vectors were constructed. The exogenous genes can be integrated into Pichia pastoris chromosome by integrative expression plasmid generating genetic state secretion expression strain. Expression proteins were characterized as a basis of further studying their biological functions.
Methods
Construction and identification of hsp70 and hsp70 fused with HBsAg yeast expression vectors: Applied PCR and molecular clone technology, the yeast shuttle plasmids including pPIC9K/hsp70、pPIC9K/s、
1. pPIC9K/hsp(1-370)-s、pPIC9K/hsp(161-370)-s、pPIC9K/s-hsp(1-370)、pPIC9K/s-hsp(161-370) were constructed. Restriction enzyme digestion and DNA sequencing analysis confirmed whether the recombinant plasmids were correct.
2. Expression of hsp70 and hsp70 fusion proteins in Pichia pastoris system: First,the plasmids were linearized with SacI; Second,Pichia pastoris strain GS115 was transformed by electroportion; Third,the transformants were selected by PCR; Last,the recombinant Pichia strains were induced by 0.5% methanol.The supernatants from cultures were identified by SDS-PAGE、ELISA and western-blot analysis.
Results
1. Restriction digestion and DNA sequence analysis confirmed the six vector correct.
Expression of recombinant Pichia strains:Hsp70 was secreted into the supernatant,western-blot analysis showed that the positive band with about 70KD molecular weight was recongnized by anti-Mt.hsp70 antibody.Yeild of expression was up to 0.15mg/ml and the expression amount was more than 30% of the supernatant total proteins.However,the other five recombinant Pichia strains didn't secret the expected fusion proteins into the supernatant.The cell lysates were positive by measure with HBsAg ELISA.According to these,they
2. maybe accumulated expression protein in cell.
Conclusion
1. We succeeded in constructing six yeast shuttle expression vectors.
2. We utilized the Pichia expression system to secret hsp70 at first time and got a high expression recombinant Pichia strain,which would provide a opportunity to further study the application and biologic function of hsp70.
引文
1． Marie L M, Delphine L.DNA vaccines for prophylactic or therapeutic immunization against Hepatitis B.Intervirology,2001,44:78-87.
2． Hoofnagle J H,di Bisceglie A M.The treatment of chronic viral hepatitis. N Engl J Med, 1997,336:347-356.
3． Lai C L,Chien R N,Leung N W,et al.A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med, 1998,339:61-68.
4． 张申英。慢性乙型肝炎治疗性疫苗的应用研究。国外医学流行病学传染病学分册。2001,Vol28(2):53-56.
5． Heather L D,Cynthia L,Brazolot M.DNA-based immunization against hepatitis B virus. Springer Semin Immunopathol,1997,19:195-209.
6． Chow Y H,et al. Improvement of HBV DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.J of Virology,1997, 71:169.
7． Srivastava P K, A Deleo, L J Old. Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc Natl Acad Sci USA ,1986,83: 3407-3411.
8． Ullrich S J, E A Robinson, L W Law, et al. A mouse tumor-specific transplanttion antigen is a heat shock-related protein. Proc Natl Acad Sci USA,1986,83:3121-3125.
9． Udono H, Srivastava P K.Hsp70-associated peptides elicit specific cancer immunity.J Exp Med,1993,178:1391-1396.
10.Suzue K, Young R A. Adjuvant-free hsp70 fusion protein system elicits humoral and cellular immune responses to HIV-1 p24.J Immunol,1996, 156:873-879.
11.Qian H, J F L Richmond, Suzue K, et al. In vivo cytotoxic T lymphocyte elicitation by mycobacterial heat shock protein 70 fusion proteins maps to a discrete domain and is CD4+T cell independent.J Exp Med,2000, vol191(2):403-408.
12.Udono H,Yamano T,Kawabata Y,et al.Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70.Inter Immunol.2001,Vol13(10):1233-1242.
13.J L Cereghino,J M Cregg. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 2000(24):45-66.
14.张学峰，葛忠良，孙丽亚等。rhGM-CSF/LIF融合蛋白基因克隆与表达。生物化学与生物物理学报，1997，Vol29(5):425-431.
15.何诚，朱运松。甲醇营养型酵母表达系统的研究进展。生物工程进展，1998，Vol18(3):7-11.
16.Brake A J, Merryweather J P,Coit D J,et al.α-Factor directed systhesis and secretion of mature foreign proteins in Saccharomyces cerevisiae, Proc Natl Acad Sci USA,1944,81:4642-4646.
17.Rosenfeld S T,Nadeau D,Tirado J,et al.Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris ,Protein Expression and Purification,1996,8:476-482.
18.Curtis B M, Willian D E, Broxmeyer H E, et al. Enhanced hematopoietio activity of a human granulocytemacrophage colony-stimulating factor-interleukin 3 fusion protein.PNAS,1991,88:5809.
19.Cregg J M, Tschopp J F, Stillman C,et al. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris.Bio/technology,1987,Vol5:479-485.
20.杨剑莹，金菁，孔玉英等。毕赤酵母表达的重组乙肝表面抗原SS1纯化及性质鉴定。生物化学与生物物理学报，2000，32（5）：503-508.
21.Eble B E, Lingappa V R, Ganem D. The N-terminal(pre-S2)domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences.J Virology,1990,Vol64(3):1414-1419.
22.范乔，刘宏迪。甲醇营养型酵母表达外源蛋白的特点及策略。微生物学通报，1999，26（4）：304-306.
23.Hendrick J P, Hartl F U. Molecular chaperone function of heat shock proteins. Annu Rev Biochem,1993,62:349-384.
24.Georgopoulos C, Welch W J. Role of the major heat shock proteins as molecular chaperons. Annu Rev Cell Biol,1993,9:601-634.
25.Boston R S, Viitanen P V, Vierling E. Molecular chaperones and protein folding in plant. Plant Mol Biol,1996,32(1-2):191-222.
26.Li Z. Priming of T cells by heat shock protein-peptide complexes as the basis of tumor vaccines. Sem Immunol ,1997,9:315-322.
27.Srivastava P K, Udono H, Blachere N E, et al.Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics ,1994,39: 93-98
28.Blachere N E, Srivastava P K. Heat shock protein-based cancer vaccines and related thoughts on immunogenicity of human tumors. Seminars in Immunology 1995,6 : 349-355.
29.Masoud H M,Xiang Y W,Juneui P,et al.Immunotherapy of cancer using heat shock proteins.Frontiers in Bioscience 7,2002,Jan.1:d43-52.
30.Perraut R,Lussow A R,Gavoille S, et al.Successful primate immunization with peptides conjugated to purified protein derivative or Mycobacterial heat shock proteins in the absence of adjuvants. Clin Exp Immunol,1993,93(3):382-6.
31.Maria C K, Robert G, Svetiana G, et al. The role of heat shock protein
(hsp70) in dendritic cell maturation:Hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors. Eur J Immunol,2001,31:1602-1609.
32.Janetzki S,Blachere N E,Srivastava P K. Generation of tumor-specific cytotoxic T lymphocytes and memory T cells by immunization with tumor- derived heat shock protein gp96. J Immunother 1998,21:269-276.
33.Penna A,Chisari F V,Bertoletti A, et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis nucleocapsid antigen.J Exp Med,1991,174(6):1565-70.
34.Nayersina R, Fowler P, Guilhot S,et al.HLA-A2-restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.J Immunol,1993,150(10):4659-71.
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2． Hoofnagle J H,di Bisceglie A M.The treatment of chronic viral hepatitis. N Engl J Med, 1997,336:347-356.
3． Lai C L,Chien R N,Leung N W,et al.A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med, 1998,339:61-68.
4． 张申英。慢性乙型肝炎治疗性疫苗的应用研究。国外医学流行病学传染病学分册。2001,Vol28(2):53-56.
5． Heather L D,Cynthia L,Brazolot M.DNA-based immunization against hepatitis B virus. Springer Semin Immunopathol,1997,19:195-209.
6． Chow Y H,et al. Improvement of HBV DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.J of Virology,1997, 71:169.
7． Srivastava P K, A Deleo, L J Old. Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc Natl Acad Sci USA ,1986,83: 3407-3411.
8． Ullrich S J, E A Robinson, L W Law, et al. A mouse tumor-specific transplanttion antigen is a heat shock-related protein. Proc Natl Acad Sci USA,1986,83:3121-3125.
9． Udono H, Srivastava P K.Hsp70-associated peptides elicit specific cancer immunity.J Exp Med,1993,178:1391-1396.
10.Suzue K, Young R A. Adjuvant-free hsp70 fusion protein system elicits humoral and cellular immune responses to HIV-1 p24.J Immunol,1996, 156:873-879.
11.Qian H, J F L Richmond, Suzue K, et al. In vivo cytotoxic T lymphocyte elicitation by mycobacterial heat shock protein 70 fusion proteins maps to a discrete domain and is CD4+T cell independent.J Exp Med,2000, vol191(2):403-408.
12.Udono H,Yamano T,Kawabata Y,et al.Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70.Inter Immunol.2001,Vol13(10):1233-1242.
13.J L Cereghino,J M Cregg. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 2000(24):45-66.
14.张学峰，葛忠良，孙丽亚等。rhGM-CSF/LIF融合蛋白基因克隆与表达。生物化学与生物物理学报，1997，Vol29(5):425-431.
15.何诚，朱运松。甲醇营养型酵母表达系统的研究进展。生物工程进展，1998，Vol18(3):7-11.
16.Brake A J, Merryweather J P,Coit D J,et al.α-Factor directed systhesis and secretion of mature foreign proteins in Saccharomyces cerevisiae, Proc Natl Acad Sci USA,1944,81:4642-4646.
17.Rosenfeld S T,Nadeau D,Tirado J,et al.Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris ,Protein Expression and Purification,1996,8:476-482.
18.Curtis B M, Willian D E, Broxmeyer H E, et al. Enhanced hematopoietio activity of a human granulocytemacrophage colony-stimulating factor-interleukin 3 fusion protein.PNAS,1991,88:5809.
19.Cregg J M, Tschopp J F, Stillman C,et al. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris.Bio/technology,1987,Vol5:479-485.
20.杨剑莹，金菁，孔玉英等。毕赤酵母表达的重组乙肝表面抗原SS1纯化及性质鉴定。生物化学与生物物理学报，2000，32（5）：503-508.
21.Eble B E, Lingappa V R, Ganem D. The N-terminal(pre-S2)domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences.J Virology,1990,Vol64(3):1414-1419.
22.范乔，刘宏迪。甲醇营养型酵母表达外源蛋白的特点及策略。微生物学通报，1999，26（4）：304-306.
23.Hendrick J P, Hartl F U. Molecular chaperone function of heat shock proteins. Annu Rev Biochem,1993,62:349-384.
24.Georgopoulos C, Welch W J. Role of the major heat shock proteins as molecular chaperons. Annu Rev Cell Biol,1993,9:601-634.
25.Boston R S, Viitanen P V, Vierling E. Molecular chaperones and protein folding in plant. Plant Mol Biol,1996,32(1-2):191-222.
26.Li Z. Priming of T cells by heat shock protein-peptide complexes as the basis of tumor vaccines. Sem Immunol ,1997,9:315-322.
27.Srivastava P K, Udono H, Blachere N E, et al.Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics ,1994,39: 93-98
28.Blachere N E, Srivastava P K. Heat shock protein-based cancer vaccines and related thoughts on immunogenicity of human tumors. Seminars in Immunology 1995,6 : 349-355.
29.Masoud H M,Xiang Y W,Juneui P,et al.Immunotherapy of cancer using heat shock proteins.Frontiers in Bioscience 7,2002,Jan.1:d43-52.
30.Perraut R,Lussow A R,Gavoille S, et al.Successful primate immunization with peptides conjugated to purified protein derivative or Mycobacterial heat shock proteins in the absence of adjuvants. Clin Exp Immunol,1993,93(3):382-6.
31.Maria C K, Robert G, Svetiana G, et al. The role of heat shock protein
(hsp70) in dendritic cell maturation:Hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors. Eur J Immunol,2001,31:1602-1609.
32.Janetzki S,Blachere N E,Srivastava P K. Generation of tumor-specific cytotoxic T lymphocytes and memory T cells by immunization with tumor- derived heat shock protein gp96. J Immunother 1998,21:269-276.
33.Penna A,Chisari F V,Bertoletti A, et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis nucleocapsid antigen.J Exp Med,1991,174(6):1565-70.
34.Nayersina R, Fowler P, Guilhot S,et al.HLA-A2-restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.J Immunol,1993,150(10):4659-71.
35.Guidotti L G,Chisari F V.Cytokine-induced viral purging-role in viral pathogenesis.Curr Opin Microbiol,1999,2:388-391.
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