重组人CTLA4胞外区蛋白在酵母GS115中的表达、纯化及其体内外生物学活性研究
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摘要
背景：抗原特异性T细胞的活化在一些免疫相关疾病(如移植排斥反应、变应性疾病)的发生、发展中起着主导性的作用。而T细胞的活化除需要T细胞受体(TCR)与抗原呈递细胞(Antigen presenting cell,APC)表面的抗原肽-MHC复合物结合所形成的第一信号外，还需要T细胞和APC表面的其它膜分子结合所提供的共刺激信号(亦称第二信号或辅助刺激信号，Costimulatory signal)的参与。共刺激信号决定着T细胞与抗原反应的程度和结果。研究表明B7-CD28通路是最主要的刺激T细胞活化的共刺激信号通路，其不仅参与T细胞活化过程，还影响活化后T细胞的分化、细胞因子产生等。缺乏或阻断B7-CD28共刺激信号将导致T细胞活化增殖障碍、T细胞凋亡(Apoptosis)、克隆排除(Clone depletion)或进入一种无反应状态(无能，Anergy)，从而诱导针对该识别过程中特异性抗原的免疫耐受。CTLA4(Cytotoxic T lymphocyte associated antigen 4)作为一种主要的共刺激负调节剂，可竞争性结合B7分子，阻断B7-CD28途径、并通过产生抑制性信号使T细胞活化障碍。重组人CTLA4Ig融合蛋白已被运用于防治移植排斥反应、自身免疫性疾病和变应性疾病等的实验和临床研究，并取得了肯定的效果。CTLA4Ig融合蛋白是通过基因重组技术将编码CTLA4胞外区1-125个氨基酸序列的DNA片段与编码IgG Fc段的DNA片段融合后表达生成，其功能区是CTLA4胞外区。CTLA4胞外区序列决定了其空间结构和配体结合特异性。CTLA4胞外区DNA的表达蛋白由于不含有外源DNA表达产物，理论上与天然CTLA4蛋白的功能更为一致，也减少
了额外DNA表达产物的副作用。同时，由于CTLA4胞外区片段大小仅有
400hp，较CTLA4Ig融合蛋白＊．ZKb）在表达菌中的表达将更为稳定。GSlls
是毕赤酵母uichia pastoris）的一种菌株，己被用于多种外源蛋白的表达。
其可高密度生长，外源蛋白呈分泌性表达，具有外源蛋白产量高、纯化相
对容易的优点。为此，本实验选择 GSlls作为表达菌。
目的：获取具有生物学活性的重组人CTLA4胞外区蛋白（CTLA4e），
探讨其在变应性疾病和器官移植排斥反应防治中的作用和机理。
方法：1．重组人 CTLA4胞外区蛋白在酵母 GS 115中的表达和纯化：
首先通过基因操作技术，从pCTLA4八g质粒中扩增400hp的CTLA4胞外
区片段；将CTLA4胞外区片段插人pPICg质粒中获取单拷贝酵母表达质
粒pPICg－CTLA4125并进一步构建多拷贝酵母表达质粒pPICgK（。
利用限制性内切酶分析、＊＊A序列测定在＊＊A水平对质粒进行鉴定。将
pPICgK七 质粒电转化GSlls酵母；通过筛选获取稳定表达CTLA4
胞外区蛋白的表达菌。表达菌甲醇诱导发酵；发酵上清液经超滤、硫酸胺
粗分级分离以及阴离于交换层析，纯化出CTLA4 胞外区蛋白。利用
IJot．ELISA、SDA－PAGE、Western．blot等方法在蛋白水平对CTLA4胞外区
蛋白的表达进行了鉴定。
2．重组人 CTLA4胞外区蛋白离体生物学活性鉴定：我们通过h1dR
掺入法观察了CTLA4胞外区蛋白对人双向混合淋巴细胞反应（Mixed
lymphocyte reaction，MLR产 T细胞增殖的影响。
3．重组人 CTLA4胞外区蛋白体内生物学活性研究：l）观察 CTLA4
胞外区蛋白在小鼠变应性鼻炎中的作用。通过卵清蛋白（Ovalbumin，OVA）
致敏和激发诱导小鼠变应性鼻炎；60只小鼠随机分为变应性鼻炎组（OVA
组）、正常对照组（ Saline组）和 CTLA4e组（200 u g／只、l／日 X 7次，IP），
分别比较首次和一月后再次用抗原激发时各组动物鼻部症状、鼻粘膜形态
学改变，以及血清抗原特异性 IgE水平和鼻腔灌洗液（Nasal lavage fluid，
NLF）中EOtaxin（嗜酸粒细胞趋化素）水平。2）观察CTLA4e在移植排
斥反应防治中的作用。30只异基因气管移植大鼠随机分成CTLA4e组（500
·IX·
119／只、l／日 X 3次，IP）、Sgline组和环胞素 A对照组（CSA组，SSg／kg、
l／日 X 7次，IM），观察大鼠存活时间和术后 7d移植气管形态学改变。24
只异基因皮肤移植小鼠，随机分为 CTLAe组（200n g／只、l／日 X3次，IVL
Saline组、CsA组（sing／kg、l／日 X 7次，IM）和重组人 CTLA4Ig组（CTLA4Ig
组，200 p g／只、1／日X 3次，IV人 分别观察移植皮肤存活时间和术后7d、
14d组织形态学改变。
结果：1．成功构建CTLA4 胞外区蛋白的多拷贝酵母表达质粒
pPICgK－CTLA4125；获取整合pPICgK－CTLA4125质粒、并能表达CTLA4
胞外区蛋白的 GS表达菌；并从表达菌甲醇诱导发酵液中纯化出分子量
在28KDa、单体形式的重组人CTLA4胞外区蛋白。
2．重组人CTLA4胞外区蛋白还可显著抑制MLR中T细胞的增殖，抑
制率可高达95％。
3．重组人CTLA4胞外区蛋白可明显减轻变应性鼻炎小鼠的鼻部症状、
鼻粘膜形
Backgroud: The activation of antigen-specific T cells plays a vital role in the initiation and development of some immune-related diseases, such as transplant rejection and allergic diseases. Effective activation of antigen-specific T cells not only requires the first signal transduction through T-cell receptor(TCR) binding with peptide-MHC complex on the antigen presenting cell (APC), but also needs the second signal, termed costimulation. Costimulation critical to the degree and consequence of T cell activation is provided by interaction between soluble factors or cell-surface molecules on the T cell and on the APC. It has been demonstrated that B7-CD28 pathway is a prominent costimulation pathway which leads full T cell activation, induces differentiation and regulates the production of cytokines. Lacking and blocking of B7-CD28 pathway will result in blockage of the T cell activation and proliferation. T cell will be apopotosis, clone depletion and /or anergy, in turn inducing an antigen-specific tolerance. CTLA4 (Cytotoxic T lymphocyte associated antigen 4), a prominent negative costimulator, can competitively bind with B7, and block B7-CD28 pathway and T cell activation by producing inhibitory singals. Recombinated human CTLA4Ig fusion protein had been used experimentally and clinically in prevention and treatment of transplant rejection, autoimmune diseases and allergic diseases, and positive curiative results were obtained. CTLA4Ig fusion protein was expressed by genetically fusing the DNA fragment encoding 1-125 amino acid residues of CTLA4 extracellular domain with DNA fragment encoding an IgG Fc region. Its
in
functional region is the extracellular domain of CTLA4. The sequence of CTLA4 extracellular domain determines its space construction and binding specific with its ligand. Without heterologous DNA, the function of protein of CTLA4 extracellular domain will be more identical to that of natural CTLA4 theoretically. Meanwhile, because the fragment of CTLA4 extracellular domain is only 400bp, its expression will be more stable than that of CTLAIg fusion protein( 1.2kb). Yeast GS115, one kind of Pichia pastoris, has been used to express varied heterologous proteins. GS115 can grow in a high density and produce heterologous proteins by secretion, which are in a high-level and easy to be purified. Hence, yeast GS115 was used to express the target protein in this study.
Objection: To obtain the biological activated recombianated protein of huaman CTLA4 extracellular domain(CTLA4e), and to explore its effects on allergic diseases and transplant rejection.
Methods: 1.Expression and purification of recombinant human CTLA4 extracellular domain: a 400bp DNA fragment of CTLA4 extracellular domain was obtained from the pCTLA4/Ig plasmid with genetical technique. Then, this DNA fragment was inserted into pPIC9 plasmid to construct the single copy plasmid of yeast expression system (pPIC9-CTLA4125). Furthermore, the multi-copy plasmid (pPIC9K-CTLA4125) was constructed. After the plasmid was assayed for DNA sequence, it was transformed into GS115 by electroporation. The stable expression yeast was obtained by screening and was induced to ferment by methanol. After ultrafiltration and rough separation with ammonium sulfate and anion-exchange chromatography, the recombination protein of CTLA4 extracelluar domain was rectified from the ferment supernatants. The expression of target protein was identified with Dot-ELISA, SDS-PAGE and Western blotting.
2. Inspection for the biological activities of recombination protein of
?
CTLA4 extracellular domain in vitro: We investigated the role of CTLA4e in T cell proliferation of MLR with 3H-TdR incorporation.
3. Investigation for the biological activities of recombination protein of CTLA4 extracellular domain in vivo: First, we observed the effects of CTLA4e on allergic rhinitis in mice. After sensitized and challenged by ovalbumin(OVA), 60 mice were equally divided into allergic rhinitis group(OVA group), Saline group and CTLAe group(200 U g/mice,
了额外DNA表达产物的副作用。同时，由于CTLA4胞外区片段大小仅有
400hp，较CTLA4Ig融合蛋白＊．ZKb）在表达菌中的表达将更为稳定。GSlls
是毕赤酵母uichia pastoris）的一种菌株，己被用于多种外源蛋白的表达。
其可高密度生长，外源蛋白呈分泌性表达，具有外源蛋白产量高、纯化相
对容易的优点。为此，本实验选择 GSlls作为表达菌。
目的：获取具有生物学活性的重组人CTLA4胞外区蛋白（CTLA4e），
探讨其在变应性疾病和器官移植排斥反应防治中的作用和机理。
方法：1．重组人 CTLA4胞外区蛋白在酵母 GS 115中的表达和纯化：
首先通过基因操作技术，从pCTLA4八g质粒中扩增400hp的CTLA4胞外
区片段；将CTLA4胞外区片段插人pPICg质粒中获取单拷贝酵母表达质
粒pPICg－CTLA4125并进一步构建多拷贝酵母表达质粒pPICgK（。
利用限制性内切酶分析、＊＊A序列测定在＊＊A水平对质粒进行鉴定。将
pPICgK七 质粒电转化GSlls酵母；通过筛选获取稳定表达CTLA4
胞外区蛋白的表达菌。表达菌甲醇诱导发酵；发酵上清液经超滤、硫酸胺
粗分级分离以及阴离于交换层析，纯化出CTLA4 胞外区蛋白。利用
IJot．ELISA、SDA－PAGE、Western．blot等方法在蛋白水平对CTLA4胞外区
蛋白的表达进行了鉴定。
2．重组人 CTLA4胞外区蛋白离体生物学活性鉴定：我们通过h1dR
掺入法观察了CTLA4胞外区蛋白对人双向混合淋巴细胞反应（Mixed
lymphocyte reaction，MLR产 T细胞增殖的影响。
3．重组人 CTLA4胞外区蛋白体内生物学活性研究：l）观察 CTLA4
胞外区蛋白在小鼠变应性鼻炎中的作用。通过卵清蛋白（Ovalbumin，OVA）
致敏和激发诱导小鼠变应性鼻炎；60只小鼠随机分为变应性鼻炎组（OVA
组）、正常对照组（ Saline组）和 CTLA4e组（200 u g／只、l／日 X 7次，IP），
分别比较首次和一月后再次用抗原激发时各组动物鼻部症状、鼻粘膜形态
学改变，以及血清抗原特异性 IgE水平和鼻腔灌洗液（Nasal lavage fluid，
NLF）中EOtaxin（嗜酸粒细胞趋化素）水平。2）观察CTLA4e在移植排
斥反应防治中的作用。30只异基因气管移植大鼠随机分成CTLA4e组（500
·IX·
119／只、l／日 X 3次，IP）、Sgline组和环胞素 A对照组（CSA组，SSg／kg、
l／日 X 7次，IM），观察大鼠存活时间和术后 7d移植气管形态学改变。24
只异基因皮肤移植小鼠，随机分为 CTLAe组（200n g／只、l／日 X3次，IVL
Saline组、CsA组（sing／kg、l／日 X 7次，IM）和重组人 CTLA4Ig组（CTLA4Ig
组，200 p g／只、1／日X 3次，IV人 分别观察移植皮肤存活时间和术后7d、
14d组织形态学改变。
结果：1．成功构建CTLA4 胞外区蛋白的多拷贝酵母表达质粒
pPICgK－CTLA4125；获取整合pPICgK－CTLA4125质粒、并能表达CTLA4
胞外区蛋白的 GS表达菌；并从表达菌甲醇诱导发酵液中纯化出分子量
在28KDa、单体形式的重组人CTLA4胞外区蛋白。
2．重组人CTLA4胞外区蛋白还可显著抑制MLR中T细胞的增殖，抑
制率可高达95％。
3．重组人CTLA4胞外区蛋白可明显减轻变应性鼻炎小鼠的鼻部症状、
鼻粘膜形
Backgroud: The activation of antigen-specific T cells plays a vital role in the initiation and development of some immune-related diseases, such as transplant rejection and allergic diseases. Effective activation of antigen-specific T cells not only requires the first signal transduction through T-cell receptor(TCR) binding with peptide-MHC complex on the antigen presenting cell (APC), but also needs the second signal, termed costimulation. Costimulation critical to the degree and consequence of T cell activation is provided by interaction between soluble factors or cell-surface molecules on the T cell and on the APC. It has been demonstrated that B7-CD28 pathway is a prominent costimulation pathway which leads full T cell activation, induces differentiation and regulates the production of cytokines. Lacking and blocking of B7-CD28 pathway will result in blockage of the T cell activation and proliferation. T cell will be apopotosis, clone depletion and /or anergy, in turn inducing an antigen-specific tolerance. CTLA4 (Cytotoxic T lymphocyte associated antigen 4), a prominent negative costimulator, can competitively bind with B7, and block B7-CD28 pathway and T cell activation by producing inhibitory singals. Recombinated human CTLA4Ig fusion protein had been used experimentally and clinically in prevention and treatment of transplant rejection, autoimmune diseases and allergic diseases, and positive curiative results were obtained. CTLA4Ig fusion protein was expressed by genetically fusing the DNA fragment encoding 1-125 amino acid residues of CTLA4 extracellular domain with DNA fragment encoding an IgG Fc region. Its
in
functional region is the extracellular domain of CTLA4. The sequence of CTLA4 extracellular domain determines its space construction and binding specific with its ligand. Without heterologous DNA, the function of protein of CTLA4 extracellular domain will be more identical to that of natural CTLA4 theoretically. Meanwhile, because the fragment of CTLA4 extracellular domain is only 400bp, its expression will be more stable than that of CTLAIg fusion protein( 1.2kb). Yeast GS115, one kind of Pichia pastoris, has been used to express varied heterologous proteins. GS115 can grow in a high density and produce heterologous proteins by secretion, which are in a high-level and easy to be purified. Hence, yeast GS115 was used to express the target protein in this study.
Objection: To obtain the biological activated recombianated protein of huaman CTLA4 extracellular domain(CTLA4e), and to explore its effects on allergic diseases and transplant rejection.
Methods: 1.Expression and purification of recombinant human CTLA4 extracellular domain: a 400bp DNA fragment of CTLA4 extracellular domain was obtained from the pCTLA4/Ig plasmid with genetical technique. Then, this DNA fragment was inserted into pPIC9 plasmid to construct the single copy plasmid of yeast expression system (pPIC9-CTLA4125). Furthermore, the multi-copy plasmid (pPIC9K-CTLA4125) was constructed. After the plasmid was assayed for DNA sequence, it was transformed into GS115 by electroporation. The stable expression yeast was obtained by screening and was induced to ferment by methanol. After ultrafiltration and rough separation with ammonium sulfate and anion-exchange chromatography, the recombination protein of CTLA4 extracelluar domain was rectified from the ferment supernatants. The expression of target protein was identified with Dot-ELISA, SDS-PAGE and Western blotting.
2. Inspection for the biological activities of recombination protein of
?
CTLA4 extracellular domain in vitro: We investigated the role of CTLA4e in T cell proliferation of MLR with 3H-TdR incorporation.
3. Investigation for the biological activities of recombination protein of CTLA4 extracellular domain in vivo: First, we observed the effects of CTLA4e on allergic rhinitis in mice. After sensitized and challenged by ovalbumin(OVA), 60 mice were equally divided into allergic rhinitis group(OVA group), Saline group and CTLAe group(200 U g/mice,
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44. Murasugi A, Asami Y, Mera-Kikuchi Y. Production of recombinant human
bile salt-stimulated lipase in Pichia pastoris. Protein Expr Purif. 2001 Nov;23(2):282-8.
45. Mwangi SM, Stabel J, Lee E, et al. Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type Ⅰ . Vet Immunol Immunopathol. 2000 Dec 29;77(3-4):233-41.
46．易绍萱，吴军，王锡华，等．CTLA4-Ig融合蛋白在COS-7中的表达、纯化及鉴定．第三军医大学学报，2000，22(6)：523-526．
47．易绍萱，吴军，姜庆，等．人CTLA4胞外区cDNA的克隆及Ig融合蛋白表达载体的构建[J]．第三军医大学学报，2000，22(9)：834-837．
48. Dariavach P, Mattei MG, Golstein P, et al. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains. Eur J Immunol. 1988 Dec; 18(12): 1901-5.
49．金冬雁，黎孟枫等编译。《分子克隆实验指南》第二版，科学出版社，北京，1993。
50．李永明，赵玉琪等主编。《实验分子生物学方法手册》，科学出版社，北京，1993。
51. Peach RJ, Bajorath J, Brady W, et al. Complementarity determining region 1(CDR1-) and CDR3- analogous regions in CTLA4 and CD28 determine the binding to B7-1. J Exp Med. 1994, 180(6): 2049-2058
52. Kariv I, Truneh A, Sweet RW. Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function. J Immunol. 1996, 157(1): 29-38
53. Linsley PS, Nadler SG, Bajorath J, et al. Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4(CTLA4). A disulfide-linked homodimer binds two CD86 molecules. J Biol Chem. 1995 Jun 23;270(25): 15417-24.
54. Gerstmayer B, Pessara U, Wels W. Construction and expression in the yeast
Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA4. FEBS. 1997, 407:63-68
55．朱厚础等编译。《蛋白质纯化与鉴定实验指南》。科学出版社，北京。2000。
56. Deutscher MP. Guide to protein purification methods in emqymology vl 82. Academic Press Inc. 1990.
57．颜子颖，王海林编译。《精编分子生物学实验指南》。科学出版社，北京。1999。
58. Wells, AD, Li XC, Li Y, et al. Requirement for T cell apoptosis in the inducetion of peripheral transplantation tolerance. Nat Med. 1999, 5(11):1303-1307
59. Sorensen SF. The mixed lymphocyte culture interaction: techniques and immunogenetics. Denmark, 1972.
60. Steurer W, Nickerson PW, Steele AW, et al. Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J Immunol. 1995, 155(3):1165-1174
61. Zuberi RI, Apgar JR, Chen SS, et al. Role for IgE in airway secretions: IgE immume complexes are more potent inducers than antigen alone of airway inflammation in a murine model. J Immunol. 2000, 154:2667-2673
62．薛金梅，赵海亮，安云芳，等。大鼠变应性鼻炎模型鼻粘膜P物质受体mRNA的表达。中华耳鼻咽喉科杂志。2000，35(4)：247-250
63. Van Oosterhout A, Hofstra CL, Shields R, et al. Murine CTLA4Ig treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma. Am J Respir Cell Mol Biol. 1997, 17:386-39264
64. Fernvik E, Gronneberg R, Lundahl J, et al. Characterization of eosinophils and detection of eotaxin in skin chamber fluid after challenge with relevant allergen in patient with mild asthma. Clin Exp Allergy, 1999, 29(11):
1516-1525.
65.Ikonen TS, Brazehon TR, Berry CJ, et al. Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats. Transplantation. 2000, 70(6):857-63.
66.Maksoud-Filho JG, Rodrigues CJ, Tannuri U, et al. The effects of early and delayed immunosuppression in experimental tracheal transplantation with omentopexy. J Pediatr Surg. 1999 Aug;34(8): 1223-8.
67.Bolling SF, Lin H, Turka LA. The time course of CTLA4Ig effect on cardiac allograft rejection. J Surg Res. 1996, 63(1): 320-323
68.Tran HM, Nickerson PW, Restifo AC, et al. Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment. J Immunol. 1997, 159(5):2232-2239
69.Lin H, Bolling SF, Linsley PS, et al. Long-term acceptance of major histocompatitability complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J Exp Med. 1993, 178(5): 1801-1806
70.Akita S, Ishihara H, Mohammad AR, et al. Leukemia inhibitory factor gene improves skin allograft survival in the mouse model. Transplantation. 2000,70(7): 1026-1031
71.Khan LN, Kon OM, Macfarlane AJ, et al. Attenuation of the allergen-induced late asthmatic reaction by cyclosporin A is associated with inhibition of bronchial eosinophilis, interleukin- 5, granulocyte macrophage colony stimulating factor, and eotaxin. Am J Respir Crit Care Med. 2000, 162(4): 1377-1382
72.Thompson AG, Starzl TC. New immunosuppressive drugs: mechanistic insights and potential therapeutic advances. Immunol. 1993,136:71-98
73.Hogan SP. A novel T cell-regulated mechanism modulating
allergen-induced airways hyperreactivity in BALB/c mice independently of IL-4 and IL-5. J Immunol. 1998, 161; 1501-1509
74. Coyle A, Wagner C, Bertrand S, et al. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody. J Exp Med, 1996, 183(4): 1303-1310.
75. Gutierrez JC, Lloyd RC, Gonzalo JA. Eotaxin: from an eosinophillic chemokine to a major regulator of allergic reactions. Immunology Today. 1999, 20(11):500-504
76. Minshall EM, Cameron L, Lavigne F, et al. Eotaxin mRNA and protein expression in chronic sinusitis and allergen-induced nasal responses in seasonal allergic rhinitis. Am J Respir Cell Mol Biol, 1997,17: 683-690.
77. VandeRijn M, Mehlhop PD, Judkins A, et al. A murine model of allergic rhinitis: studies on the role of IgE in pathogenesis and analysis of the eosinophil influx elicited by allergen and eotaxin. J Allergy Clin Immunol, 1998, 102(1):65-74.
78. Nicolaidou E, Okada Y, Zuo XJ, et al. Prolongation of skin allograft survival is associated with reduced Th1 cytokine response in the WKY—F344 rat model. Transplantation. 1999, 68(9): 1393-1401
79. Eagar TN, Karandikar NJ, Bluestone JA, et al. The role of CTLA-4 in induction and maintenance of peripheral T cell tolerance. Eur J Immunol, 2002, 32(4):972.. 1997,13(4):250.
80．杨异，赵珩．同种异体气管移植研究进展．中华心胸外科杂志，1997，13(4)：250．
81. Yokomise Y, Inui K, Wada H, et al. Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability. J Thorac Cardiovasc Surg, 1996, 111:930.
82. Murakawa T, Nakajima J, Motomura N, et al. Successful
allotransplantation of cryopreserved tracheal grafts with preservation of the pars membranacea in nonhuman primates. J Thorac Cardiovasc Surg, 2002, 123(1):153.
83.Masaoka T, Oizumi H, Fujishima T, et al. Removal of cartilage rings prevents graft stenosis in extended tracheal allotransplantation with omentopexy and immunosuppression: an experimental study. J Heart Lung Transplant, 2002, 21(4): 485.
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42. Akita S, Ishihara H, Mohammad AR, et al. Leukemia inhibitory factor gene improves skin allograft survival in the mouse model. Transplantation. 2000,70(7): 1026-1031
43．张惠展编著。基因工程概论。华东理工大学出版社，上海。2000。
44. Murasugi A, Asami Y, Mera-Kikuchi Y. Production of recombinant human
bile salt-stimulated lipase in Pichia pastoris. Protein Expr Purif. 2001 Nov;23(2):282-8.
45. Mwangi SM, Stabel J, Lee E, et al. Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type Ⅰ . Vet Immunol Immunopathol. 2000 Dec 29;77(3-4):233-41.
46．易绍萱，吴军，王锡华，等．CTLA4-Ig融合蛋白在COS-7中的表达、纯化及鉴定．第三军医大学学报，2000，22(6)：523-526．
47．易绍萱，吴军，姜庆，等．人CTLA4胞外区cDNA的克隆及Ig融合蛋白表达载体的构建[J]．第三军医大学学报，2000，22(9)：834-837．
48. Dariavach P, Mattei MG, Golstein P, et al. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains. Eur J Immunol. 1988 Dec; 18(12): 1901-5.
49．金冬雁，黎孟枫等编译。《分子克隆实验指南》第二版，科学出版社，北京，1993。
50．李永明，赵玉琪等主编。《实验分子生物学方法手册》，科学出版社，北京，1993。
51. Peach RJ, Bajorath J, Brady W, et al. Complementarity determining region 1(CDR1-) and CDR3- analogous regions in CTLA4 and CD28 determine the binding to B7-1. J Exp Med. 1994, 180(6): 2049-2058
52. Kariv I, Truneh A, Sweet RW. Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function. J Immunol. 1996, 157(1): 29-38
53. Linsley PS, Nadler SG, Bajorath J, et al. Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4(CTLA4). A disulfide-linked homodimer binds two CD86 molecules. J Biol Chem. 1995 Jun 23;270(25): 15417-24.
54. Gerstmayer B, Pessara U, Wels W. Construction and expression in the yeast
Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA4. FEBS. 1997, 407:63-68
55．朱厚础等编译。《蛋白质纯化与鉴定实验指南》。科学出版社，北京。2000。
56. Deutscher MP. Guide to protein purification methods in emqymology vl 82. Academic Press Inc. 1990.
57．颜子颖，王海林编译。《精编分子生物学实验指南》。科学出版社，北京。1999。
58. Wells, AD, Li XC, Li Y, et al. Requirement for T cell apoptosis in the inducetion of peripheral transplantation tolerance. Nat Med. 1999, 5(11):1303-1307
59. Sorensen SF. The mixed lymphocyte culture interaction: techniques and immunogenetics. Denmark, 1972.
60. Steurer W, Nickerson PW, Steele AW, et al. Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J Immunol. 1995, 155(3):1165-1174
61. Zuberi RI, Apgar JR, Chen SS, et al. Role for IgE in airway secretions: IgE immume complexes are more potent inducers than antigen alone of airway inflammation in a murine model. J Immunol. 2000, 154:2667-2673
62．薛金梅，赵海亮，安云芳，等。大鼠变应性鼻炎模型鼻粘膜P物质受体mRNA的表达。中华耳鼻咽喉科杂志。2000，35(4)：247-250
63. Van Oosterhout A, Hofstra CL, Shields R, et al. Murine CTLA4Ig treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma. Am J Respir Cell Mol Biol. 1997, 17:386-39264
64. Fernvik E, Gronneberg R, Lundahl J, et al. Characterization of eosinophils and detection of eotaxin in skin chamber fluid after challenge with relevant allergen in patient with mild asthma. Clin Exp Allergy, 1999, 29(11):
1516-1525.
65.Ikonen TS, Brazehon TR, Berry CJ, et al. Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats. Transplantation. 2000, 70(6):857-63.
66.Maksoud-Filho JG, Rodrigues CJ, Tannuri U, et al. The effects of early and delayed immunosuppression in experimental tracheal transplantation with omentopexy. J Pediatr Surg. 1999 Aug;34(8): 1223-8.
67.Bolling SF, Lin H, Turka LA. The time course of CTLA4Ig effect on cardiac allograft rejection. J Surg Res. 1996, 63(1): 320-323
68.Tran HM, Nickerson PW, Restifo AC, et al. Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment. J Immunol. 1997, 159(5):2232-2239
69.Lin H, Bolling SF, Linsley PS, et al. Long-term acceptance of major histocompatitability complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J Exp Med. 1993, 178(5): 1801-1806
70.Akita S, Ishihara H, Mohammad AR, et al. Leukemia inhibitory factor gene improves skin allograft survival in the mouse model. Transplantation. 2000,70(7): 1026-1031
71.Khan LN, Kon OM, Macfarlane AJ, et al. Attenuation of the allergen-induced late asthmatic reaction by cyclosporin A is associated with inhibition of bronchial eosinophilis, interleukin- 5, granulocyte macrophage colony stimulating factor, and eotaxin. Am J Respir Crit Care Med. 2000, 162(4): 1377-1382
72.Thompson AG, Starzl TC. New immunosuppressive drugs: mechanistic insights and potential therapeutic advances. Immunol. 1993,136:71-98
73.Hogan SP. A novel T cell-regulated mechanism modulating
allergen-induced airways hyperreactivity in BALB/c mice independently of IL-4 and IL-5. J Immunol. 1998, 161; 1501-1509
74. Coyle A, Wagner C, Bertrand S, et al. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody. J Exp Med, 1996, 183(4): 1303-1310.
75. Gutierrez JC, Lloyd RC, Gonzalo JA. Eotaxin: from an eosinophillic chemokine to a major regulator of allergic reactions. Immunology Today. 1999, 20(11):500-504
76. Minshall EM, Cameron L, Lavigne F, et al. Eotaxin mRNA and protein expression in chronic sinusitis and allergen-induced nasal responses in seasonal allergic rhinitis. Am J Respir Cell Mol Biol, 1997,17: 683-690.
77. VandeRijn M, Mehlhop PD, Judkins A, et al. A murine model of allergic rhinitis: studies on the role of IgE in pathogenesis and analysis of the eosinophil influx elicited by allergen and eotaxin. J Allergy Clin Immunol, 1998, 102(1):65-74.
78. Nicolaidou E, Okada Y, Zuo XJ, et al. Prolongation of skin allograft survival is associated with reduced Th1 cytokine response in the WKY—F344 rat model. Transplantation. 1999, 68(9): 1393-1401
79. Eagar TN, Karandikar NJ, Bluestone JA, et al. The role of CTLA-4 in induction and maintenance of peripheral T cell tolerance. Eur J Immunol, 2002, 32(4):972.. 1997,13(4):250.
80．杨异，赵珩．同种异体气管移植研究进展．中华心胸外科杂志，1997，13(4)：250．
81. Yokomise Y, Inui K, Wada H, et al. Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability. J Thorac Cardiovasc Surg, 1996, 111:930.
82. Murakawa T, Nakajima J, Motomura N, et al. Successful
allotransplantation of cryopreserved tracheal grafts with preservation of the pars membranacea in nonhuman primates. J Thorac Cardiovasc Surg, 2002, 123(1):153.
83.Masaoka T, Oizumi H, Fujishima T, et al. Removal of cartilage rings prevents graft stenosis in extended tracheal allotransplantation with omentopexy and immunosuppression: an experimental study. J Heart Lung Transplant, 2002, 21(4): 485.