猪IL-18在毕赤酵母中的分泌表达及生物学活性分析
|
摘要
白细胞介素-18是新近发现的一种细胞因子,它具有诱导T细胞和NK细胞产生IFN-γ,诱导GM-CSF的分泌,增强T细胞和NK细胞的细胞毒作用等免疫调节功能;能发挥显著的免疫佐剂作用;在抗肿瘤、病原微生物感染及抗超敏反应等方面有着潜在的应用前景。本论文取得的主要研究结果如下:
采用PCR技术从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/ L ,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验在国内首次在毕赤酵母中成功表达了具有生物学活性的pIL-18。
从发酵时间、不同诱导型、pH值、诱导剂量和接种量等方面对重组基因工程菌的发酵条件在摇床水平上进行了优化,进一步探索了酵母菌表达猪IL-18的发酵工艺。初步建立了一套较完善的在毕赤酵母中表达猪IL-18基因的生产工艺条件,为在发酵罐水平上大规模表达猪IL-18提供了依据。结果表明,摇瓶发酵时,诱导最佳时间为72h,甲醇最适浓度为20g/L,发酵pH范围为6.0～8.0,最适接种量1∶1;与单纯甲醇诱导相比,甘油/甲醇诱导后的表达量明显提高。
本研究为猪IL-18基因工程新制剂的生产和应用奠定了基础。
IL-18 was lately discovered, involved in induction of production of IFN-γand GM-CSF secreted by T and NK cell and in enforcement of cytotoxic effect of T and NK cell. It has promising prospects application in the fields of anti-tumor, infection of pathogenic microbes as well as hypersensitiveness. In this study, we have obtained the following results:
The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the fusion expression vector pPIC9K-IL-18 was constructed by inserting IL-18 fragment, and transformed to GS115 by electroporation. Multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, and SDS-PAGE was used to analyze the product. The bioactivity of IL-18 purified by Sephadex G-100, was measured by MTT assay. Experiment results showed the fusion protein of IL-18 secreted by GS115 and reached the expression peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in pichia pastoris.
Except the internal characters of heterogenous gene sequence itself, expression conditions also plays important roles in the expression yield. Different culturable time, inducible patterns, pH valves and the amount of inducible reagent were compared on effect of expression in this paper respectively. Experimental results revealed that the fermentation period induced with methanol was 72 h. The optimal concentration of methanol was 20g/L, pH range was 6.0–8.0 and the inoculation amount seed was 1: 1 in shake-flask fermentation. Compared with those induced only with methanol, pattern of inhibition/induction could improve the expression level significantly.
This study provides bases on development and application of new type reagent porcine IL-18.
采用PCR技术从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/ L ,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验在国内首次在毕赤酵母中成功表达了具有生物学活性的pIL-18。
从发酵时间、不同诱导型、pH值、诱导剂量和接种量等方面对重组基因工程菌的发酵条件在摇床水平上进行了优化,进一步探索了酵母菌表达猪IL-18的发酵工艺。初步建立了一套较完善的在毕赤酵母中表达猪IL-18基因的生产工艺条件,为在发酵罐水平上大规模表达猪IL-18提供了依据。结果表明,摇瓶发酵时,诱导最佳时间为72h,甲醇最适浓度为20g/L,发酵pH范围为6.0～8.0,最适接种量1∶1;与单纯甲醇诱导相比,甘油/甲醇诱导后的表达量明显提高。
本研究为猪IL-18基因工程新制剂的生产和应用奠定了基础。
IL-18 was lately discovered, involved in induction of production of IFN-γand GM-CSF secreted by T and NK cell and in enforcement of cytotoxic effect of T and NK cell. It has promising prospects application in the fields of anti-tumor, infection of pathogenic microbes as well as hypersensitiveness. In this study, we have obtained the following results:
The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the fusion expression vector pPIC9K-IL-18 was constructed by inserting IL-18 fragment, and transformed to GS115 by electroporation. Multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, and SDS-PAGE was used to analyze the product. The bioactivity of IL-18 purified by Sephadex G-100, was measured by MTT assay. Experiment results showed the fusion protein of IL-18 secreted by GS115 and reached the expression peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in pichia pastoris.
Except the internal characters of heterogenous gene sequence itself, expression conditions also plays important roles in the expression yield. Different culturable time, inducible patterns, pH valves and the amount of inducible reagent were compared on effect of expression in this paper respectively. Experimental results revealed that the fermentation period induced with methanol was 72 h. The optimal concentration of methanol was 20g/L, pH range was 6.0–8.0 and the inoculation amount seed was 1: 1 in shake-flask fermentation. Compared with those induced only with methanol, pattern of inhibition/induction could improve the expression level significantly.
This study provides bases on development and application of new type reagent porcine IL-18.
引文
[1] Zlotni A , Yoshie O. Chemokines : A new classification system and their role in immunity [J] . Immunity ,2000 ,12 (2) :121-127.
[2] 金伯泉. 细胞和分子免疫学[M] . 北京:科学出版社,2001. 131-239.
[3] 李元,陈松森,王渭池. 基因工程药物[M] . 北京:化学工业出版社,2003. 127-132.
[4] Lorberboum-Galski H , Fitz-Gerald D , Chaudhary V ,et al .Cytotoxic - activity of an interleukin-2-pseudomonas exotoxin chimeric protein produced in Escherichia col i [J] . Proc Natl Acad Sci USA ,1998 ,85 (6) :1922-1932.
[5] 毕美霞,杨渝珍,舒平,等. IL-2 重组毒素的克隆、表达及对IL-2R+ 淋巴细胞的特异性细胞毒效应[J] . 中国免疫学杂志,2000 ,16(3) :115-118.
[6] 孙卫民,王惠琴. 细胞因子研究方法学[M] . 北京:民卫生出版社,1999. 1-20.
[7] Ryu D D , Nam D H. Recent progress in biomolecular engineering [J] . Biotechnol Prog ,2000 ,16 (1) :2-16.
[8] Schijns V E , Claassen I J , Vermeulen A A , et al .Modulation of antiviral immune responses by exogenous cytokines : effect s of tumour necrosis factoralpha , interleukin-1 alpha , interleukin-2 and interferon-gamma on the immunogenicity of an inactivated rabies vaccine [J ] . J Gen Virol ,1994 ,75 (1) :55-63.
[9] Sasaki M G, Foccacia R , Messias2Reason I J . Efficacy of granulocyte- macrophage colony-stimulating factor( GM-CSF) as a vaccine adjuvant for hepatitis B virus in patient s with HIV infection [J ] . Vaccine , 2003 , 21(31) :4545-4549.
[10] Heaney M L , Golde D W. Soluble receptors in human disease [J] . J Leukoc Bio , 1998 , 64 (2) : 135-146.
[11] 王斐, 历永建, 孙卫民. 细胞因子与全身性炎症反应综合征发生机理的研究进展[J] . 国外医学免疫学分册,1999 ,22 (4) :177-180.
[12] 窦永喜, 景志忠, 才学鹏 . 细胞因子及其应用的研究进展[J] . 中国兽医科技,2005 ,35 (3) :233-238
[13] Yan H , Krishnan K, LimJ T E et al . Molecular Characterization of an α-IFNR1 subunit domain required for Tyk2 binding and signal transduction. Mol Cell Biol , 1996 , 16(5) : 2074～2082
[14] 贺平,汤钊猷. 细胞因子受体超家族与信号转导[J ] .生物化学与生物物理进展,1998 ; 25 (3): 201-204
[15] 朱锦芳,郑仲承,刘新桓. 细胞因子受体及其介导的信号转导 [J ] .生命的化学,1996;16(3): 14-17
[16] Krebs DL ,Hilton DJ. SOCS proteins : negative regulators of cytokine signaling[J]. Stem Cells ,2001 ,19 :378.
[17] O’Shea JJ ,Gadina M,Schreiber. Cytokine signaling in 2002 :new surprises in the Jak/ Stat pathway[J ]. Cell ,2002 ,109 :S121.
[18] Alexander WS. Suppressors of cytokine signaling (SOCS) in the immune system[J ]. Nat Rev Immunol ,2002 ,2 :410.
[19] 印 彤 . 细 胞 因 子 信 号 抑 制 蛋 白 与 细 胞 因 子 信 号 转 导 [J] . 现 代 免 疫 学 , 2004 ,24(2):164-168
[20] Auccoutorier J, Dupuis L, Ganne V. Adjuvants designed for veterinary and humman vaccines[J].Vaccine, 2001, 19 (17-19):2666-2672
[21] Chow Y H, Huang VVL, et al. Improvement of hepatitis B virus DNA vaccine by plasmids coexpressing hepatitis B surface antigen and interleukin-2 [J] .J Virol, 1997, 71(1):169-178
[22] Billaut-mulot 0, Idriorek T, Loyens M, et al. Modulation of cellular and humoral immune responses to a multipitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost[J}.Vaccine,2001, 19 (20-22):2803-2811
[23] Playfair H L and Desouza J B. Recombinant gamma interferon is a potent adjuvant for a malaria vaccine in mice. Clinical Experimental Immunolnev 1987, 67:5一10
[24] Heath AW, Playfair JH. Conjugation of interferon-gamma to antigen enhances its djuvanticity[J].Immunology. 1990, 71(3) :454-456.
[25] Sin JL, Kim JJ, Boyer J D, et al. In vivo modulation of vaccine-induced immune response toward a Thl phenotype increases potency and vaccine effedctiveness in a herpes simplex virus type 2 mouse model .J Virol. 1999, 73:501-507
[26] Heath A W, Playfair J H. Cytokines as immunological adjuvants. Vaccine 1992, 10 (7):427-434
[27] Micallef MJ, Ohtsuki K, Kohno F, et al. Interferon-gamma-inducing factor enhances T helper lcytokine production by stimulated human Tce1l:Synergism with interleukin-12 for interferon-gamma production [J].Eur J Immunol,1996, 2 (7):1647-1651
[28] Guidotti LG, Huang W K, et al. Improvemnet of hepatitis B virus DNA vaccines by plasmid co-expressing hepatitis B surface antigen and interleukin-18. Hepatology, 1998, 28:202-210
[29] Chowm Chiang BL, Lee YL,et a1. Development of Thl and Th2 population and the nature of immune responses to hepatitis B virus DNA vaccinescan be modulated by code livery of various cytokine genes,J Immunol, 1998,160:1320 一 1329
[30] Kimm JJ, Yang J S, Vcncort T C, et al. Modulation of antigen-specific humoral responses in Rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants[J].J, Virol, 2000, 74 (7):3427-3429
[31] 蔡雷.DNA 免疫的基础性研究进展及应用.国外医学免疫学分册,2001, 24: 121-125
[32] 文艳君.增强 DNA 免疫效应的研究进展.国外医学免疫学分册,1999,22:320-323
[33] Irvine K R, Rao JB, Rosenberg S A. et al. Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J Immunol, 1996, 156:238-245
[34] Meuer SCH, Dumann KH, Buschenfelda M, et al. Low dose interleukin-2 induces systemic immune responses against HBsAg in immunodeficient to hepatitis B vaccination[J].Lancet, 1989, 1:15-17
[35] Quiroga JA, Castillo I, Porres JC, et al. Recombinant Y 一 interferon as adjuvant to hepatitis B vaccine in hemodialysis patients.Hepatology 1990,12:662-663
[36] Good MF, Pombo D,Lunde MN, et a1. Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides:correlation of effect with biologic activity of IL-2. J Immunol,1988; 141:972-977
[37] Blecha F,D N Reddy,C G Chitko-Mckown et al. Influence of recombinant bovine interleukin-1 and interleukin-2 in pig vaccinated and challenged with streptococcus suis.Vet Immunopathal,1995, 44:329-346.
[38] 吴丹,郭滚军,王庆敏等。猪白细胞介素-4 的基因克隆及其在囊尾蝴 DNA 疫苗中的应用[J].第二军医大学学报,2002, 23 (11 ): 1192-1194
[39] Lim YS, Kang BY, Kim EJ etal. Potentiation of antigen-specific, Th1 immune response by multiple DNA vaccination with an ovalbumin/interferon-It hybrid construct [J].Immunol, 1998, 94:135-141
[40] Mbawuike IN, Enhancement of the protecting efficacy of inactivated influenza A vaccine in aged mice by IL-2-liposomes[J].Vaccine, 1990, 8:347-352
[41] Operschall E, Schuh T, Heinzerling L, et al. Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.J Clin Virol,1999,13:17-22
[42] 杜德伟,周永兴,冯志华等.IL-12 及 HBV 基因疫苗共同免疫小鼠的效果. 世界华人消化杂志 2000, 8 (2):128-130
[43] Lee SW, Cho J H, Sung YC et al.饰 timal induction of hepatitis C virusenvelope- specific immunity by bicistronic plasmid DNA inoculation with thegranulocyte-macrophage colony-stimulating factor gene.J Virol, 1998, 72:8430-8436
[44] 胡慧琼. 四川山地乌骨鸡 IL-2、15、18、IFN-γ 基因克隆测序及分子佐剂效应研究[D].四川农业大学,2006.
[45] Marton MJ ,Dekisi JL ,Bennett HA ,et al. 1998 ;4 (11) :1293～1301
[46] 张瑞军. IL-18 对 NK 细胞的调节剂抗肿瘤效应[J]. 国外医学肿瘤学分册,2000,27(3):148～150
[47] Dao T ,Ohashi K,Kayano T ,et al1Interferon-γ-inducing factor ,a novel cytokine ,enhances Fas ligand-mediated cytotoxicity ofmurine T helper 1 cells1Cell Immunol11996 ;173 :230
[48] Yoshimono T ,Okamura H ,Tagawa YI ,et al.Interleukin 18 to2gether with interleukin 12 inhibits IgE production by inductionof interferon-γ production from activated B cells1Proc NatlAcad Sci USA ,1997 ;94 :3948
[49] 陆意. IL-18 的研究进展[J].浙江海洋学院学报,2001,20(3):244～247
[50] HashimotoW, Osaki T, Okamura H, et al. Differential antitumoreffects of administration of recombinant IL-18 or recombinant IL-12are mediated primarily by Fas-Fas ligand and perforin induced tumorapop tosis, respectively[16] HashimotoW, Osaki T, Okamura H, et al. Differential antitumor
[51] 王萍等. IL-18对肝细胞损伤的作用研究进展[J].河南科技大学学报,2004,22(4):306～308
[52] Tone M et al. J Immunol,1997,159(12):6156
[53] Robinson D et al. Immunity,1997,7(4):571
[54] Okamura H ,Nagata K,Komatsu H ,et al.A novel costimulatoryfactor for gamma interferon induction found in the livers of micecauses endotoxic shock. Infect Immun ,1995 ;63 :3966
[55] 将定文等.IL-18 信号转导途径研究进展[J].国外医学分子生物学分册,2000,22(5):301～304
[56] Cregg JM,Ceregino JL , Shi J , et al . Recombinant protein expression in Pichia Pastoris[J]. Molecular Biotechnology 2000 , 16 : 23-52.
[57] 王勇. 毕赤酵母表达外源基因研究进展[J].微生物学免疫学进展,2004, 32(1):62～64
[58] 周则迅,袁汉英,何炜.乙肝病毒表面抗原SA-28融合基因在酵母中的组成型表达.复旦学报(自然科学版),2000,39(3):264～268
[59] David R, Higgins, James M et al. Pichia Protocols. New jersey, Humana Press Inc, 1998, 193～200
[60] MarcW T. High-yield secretion of recombinant gelation by pichia pastoris. yeast, 1999,15:1087~1096
[61] Eckhart L, Raffelsberger W, Ferko B, Klima et al. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.J Gen Virol,1996,77:2001～2008
[62] Wegner G.Emerging applications of methytrophic yeasts FEMS Microbiol Rev,1990,87:279～284
[63] Siegel R S, Buckholz R G, Thill G P et al. Production of epidermal growth factor in methylogrophic yeast cells.European Patent Application W090/10697,1990
[64] Scorer C A, Clare JJ, Mccombie W R et al. Rapid selection using G418 of high copy number tramsformants of pichia pastoris for high-level foreign gene expression. Bio/Technology, 1994, 12: 181～174
[65] Tschopp J F, Brust p f, cregg j m et al. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris.Nucleic Acids Res,1987,15:3859～3876
[66] Fahnestock S R, Bedzyk L A. Production of synthetic spider dragline slik protein in Pichia pastoris .Appl Microbiol Biotechnol,1997,47:33～39
[67] Cregg J M, Madden K R, Barringer K J et al. Functional characterization of the two alcoholoxidase genes from the yeast pichia pastoris. Mol Cell Biol, 1989, 9: 1316～1323
[68] Koutz P, Davis G R, Stillman C et al. Structural comparison of the Pichia Pastoris alcohol oxidase genes. Yeast, 1989, 5: 167～177
[69] Waterham H R, Digan M E, Koutz P J et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate deydyogenase gene and regulation and use of its promoter. Gene,1997,186:37～44
[70] 范乔,刘宏迪.甲醇营养型酵母表达外源蛋白的特点及策略[J].微生物学通报 ,1999,26(4):304～306
[71] Tschopp J F, Sverlow G, Kosson R et al. High-level secretion of glycosylated invertase in the methylotrophic yeast,Pichia pastoris.Bio/Technology , 1987,5:1305～1308
[72] Julius D, Brake A, Blair L et al. Isolation of putative structural gene for the lysine- arginine-cleaving endopeptidese required for processing of yeast prepro-a-factor. Cell, 1985, 37: 1075
[73] Sreekrishna K, Tschopp J F, FucR M. Invertase gene (SUC2) of saccharomyces cere uisiae as a dominant marker for transformation of pichia pastoris. Gene, 1987, 59(1): 115～125
[74] White C E, Hunter M J, Meininger D P et al. Large-scale expression,purification and charaterization of small fragments of thrombomodulin:the roles of the sixth domain and of methionine 388. protein Eng,1995,8:1177～1187
[75] Cregg J M, Madden K R, Barringer K J et al. Functional characterization of the two alcohol oxidase genes from the yeast pichia pastoris. Mol Cell Biol, 1989, 9: 1316～1323
[76] 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 the B7-2 counter-receptor CTLA-4 FEBS Lett, 1997,407:63～68
[77] 黄志清.猪干扰素-γ基因在毕赤酵母中的分泌表达及其抗猪瘟病毒活性研究[D].中国农业大学硕士学位论文,2005
[78] 彭毅,杨希才,康良仪.影响甲醇酵母中外源蛋白表达的因素[J].生物技术通报,2000,(4):33～36
[79] Scorer C A, Buckholz R G, Clare J J,et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene, 1993, 136:111～119
[80] Clare J J, Rayment F B, Ballantine S P et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology, 1991 ,9:455～460
[81] Paul M S. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Research, 1986, 14(3): 5125～5143
[82] Andre H. Codon relacemont in the PAK1 gene: Experimental approach to study the role of based codan usage in gene expression. Molecular and cellular biology, 1987, 2914～2924
[83] 赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3):308～311
[84] 姚斌,张春义等.高效表达具有生物学活性的植酸酶的毕赤酵母[J].中国科学(C 辑),1998,28(3):237～243
[85] Wulfing C,PlucKthun A.Correctly folded T-cell receptor fragment in the periplasm of Escherichia Coli. J MolBiol,1994,243(3):655～669
[86] Steidler L, Fiers W, Remaut E. Efficient specific release of Poriplasmic Proteins from Escherichia coli using temperature induction of cloned Kil gen of pMB9. Biotech Bioeng, 1994, 44(9): 1074～1082
[87] Leif.Lactase from the white-rot fungs trametes versicolor:cDNA cloning of lccl and expression in Pichia pastoris.Curr Gene,1997,32:425～430
[88] Henri P F. Overexpression and characterization of Aspergillus awamori wildtype and mutant glucoamylase secreted by the methylotrophic yeast pichia pastoris: Comparison with wild-type recombinant glucoamylast. Protein Expression and purification, 1997, 9:199～170
[89] 圣栋主编.现代分子生物学实验技术.北京:中国协和医科大学出版社,1999
[90] LaVallie E R, DiBlasion EA, Kovacic S et al. A thioredoxin gene fusion expression system that circumvents clusion body formation in the E.Coli cytoplasm. Bio/Technology, 1993, 11(2): 187～193
[91] 辛利,张励,徐韧等.人血管抑素在毕氏酵母中的表达及其活性测定[J].生物化学与生物物理学报,2001,33(3):237～343
[92] Nakamura K,Okamura H ,Nagata K,et al. Purification of a factor which provides a costim- ulatory signal for gamma interferon production[J]. Infect Immun,1993 ;61-64.
[93] Oamura H , Nagata K, Komatsu T ,et al. A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock [J]. Infect Immun ,1995 ;63 (10):3966-3972.
[94] Dinarello CA. Interleukin-18, aproinflammatory cytokine [J]. Eur Cytokine N etw, 2000; 11 (3) : 483 - 486.
[95] Ushio S,Namba M,Okura T,et al.Cloning of the cDNA for human IFN-γinducing factor,expression in Eschetichia coli,and studies on the biologic activities of the protein[J].J Immonol,1996,156:4274-4279.
[96] 景志忠 , 窦永喜 , 侯俊琳等 . 猪 IL-18 基因的克隆和序列分析 [J]. 中国兽医科技2004,34(7):3～7.
[97] Yoshihiro Muneta, Yasuyuki Mori, Yoshihiro Shimoji. Yuichi Yokomizo. Porcine Inte- rleukin 18: Cloning, Characterization of the cDNA and Expression with the Baculovirus System[J]. Cytokine, 2000;12(6): 566–572.
[98] Yoshihiro Muneta*, Yoshihiro Shimoji, Yuichi Yokomizo, Yasuyuki Mori. Production ofmonoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purific- ation of recombinant porcine interleukin-18[J]. Journal of Immunological Methods , 236 (2000) 99–104.
[99] 陈芳,孙红立,李震等.细胞因子的研究进展[J].上海畜牧兽医通讯,2003,(5):6～8.
[100] apamichail M, Perez SA, Gritzapis AD, et al. Natural killer lymphocytes: Biology, development and function [J]. Cancer Immuno Imunother, 2004; 53 (3): 176 – 186.
[101] Schmidt OI, Morganti-Kossmann MC, Heyde CE, et al. Tumor necrosis factor mediated inhibition of interleukin-18 in the brain: A clinical and experimental study in head-injured patients and in a murine model of closed head injury[J]. J N euroinflamm, 2004; 1 (1) : 13.
[102] Cregg JM, Ceregino JL , Shi J , et al . Recombinant protein expression in Pichia Pastori[J]. Molecular Biotechnology, 2000, 16: 23-52.
[103] 裴仉福,陈瑞爱,温纳相等.猪 IL-18 基因原核表达载体的构建和表达[J]. 动物医学进展,2006,27(10):73-77.
[104] 韩雪清 ,刘湘涛 ,任芳丽等.猪瘟病毒E2 蛋白在酵母中分泌表达条件的优化[J]. 畜牧兽医学报,2003 ,34 (6) ,567-572
[105] Wenhui W , Mark A B ,Bradley A P , et al. Modeling pichia pastoris growth on methanol and optimizing the production of a recombinant protein. the Heavy chainfragment C of Botulinum neurotoxin、serotype A [J] .Biothchnology and Bieng - neering ,2000 ,20(1) :1～8.
[106] 傅安静,岳华,刘树玲等. 免疫佐剂的研究进展[J] .西南民族大学学报,2006,73-77
[2] 金伯泉. 细胞和分子免疫学[M] . 北京:科学出版社,2001. 131-239.
[3] 李元,陈松森,王渭池. 基因工程药物[M] . 北京:化学工业出版社,2003. 127-132.
[4] Lorberboum-Galski H , Fitz-Gerald D , Chaudhary V ,et al .Cytotoxic - activity of an interleukin-2-pseudomonas exotoxin chimeric protein produced in Escherichia col i [J] . Proc Natl Acad Sci USA ,1998 ,85 (6) :1922-1932.
[5] 毕美霞,杨渝珍,舒平,等. IL-2 重组毒素的克隆、表达及对IL-2R+ 淋巴细胞的特异性细胞毒效应[J] . 中国免疫学杂志,2000 ,16(3) :115-118.
[6] 孙卫民,王惠琴. 细胞因子研究方法学[M] . 北京:民卫生出版社,1999. 1-20.
[7] Ryu D D , Nam D H. Recent progress in biomolecular engineering [J] . Biotechnol Prog ,2000 ,16 (1) :2-16.
[8] Schijns V E , Claassen I J , Vermeulen A A , et al .Modulation of antiviral immune responses by exogenous cytokines : effect s of tumour necrosis factoralpha , interleukin-1 alpha , interleukin-2 and interferon-gamma on the immunogenicity of an inactivated rabies vaccine [J ] . J Gen Virol ,1994 ,75 (1) :55-63.
[9] Sasaki M G, Foccacia R , Messias2Reason I J . Efficacy of granulocyte- macrophage colony-stimulating factor( GM-CSF) as a vaccine adjuvant for hepatitis B virus in patient s with HIV infection [J ] . Vaccine , 2003 , 21(31) :4545-4549.
[10] Heaney M L , Golde D W. Soluble receptors in human disease [J] . J Leukoc Bio , 1998 , 64 (2) : 135-146.
[11] 王斐, 历永建, 孙卫民. 细胞因子与全身性炎症反应综合征发生机理的研究进展[J] . 国外医学免疫学分册,1999 ,22 (4) :177-180.
[12] 窦永喜, 景志忠, 才学鹏 . 细胞因子及其应用的研究进展[J] . 中国兽医科技,2005 ,35 (3) :233-238
[13] Yan H , Krishnan K, LimJ T E et al . Molecular Characterization of an α-IFNR1 subunit domain required for Tyk2 binding and signal transduction. Mol Cell Biol , 1996 , 16(5) : 2074～2082
[14] 贺平,汤钊猷. 细胞因子受体超家族与信号转导[J ] .生物化学与生物物理进展,1998 ; 25 (3): 201-204
[15] 朱锦芳,郑仲承,刘新桓. 细胞因子受体及其介导的信号转导 [J ] .生命的化学,1996;16(3): 14-17
[16] Krebs DL ,Hilton DJ. SOCS proteins : negative regulators of cytokine signaling[J]. Stem Cells ,2001 ,19 :378.
[17] O’Shea JJ ,Gadina M,Schreiber. Cytokine signaling in 2002 :new surprises in the Jak/ Stat pathway[J ]. Cell ,2002 ,109 :S121.
[18] Alexander WS. Suppressors of cytokine signaling (SOCS) in the immune system[J ]. Nat Rev Immunol ,2002 ,2 :410.
[19] 印 彤 . 细 胞 因 子 信 号 抑 制 蛋 白 与 细 胞 因 子 信 号 转 导 [J] . 现 代 免 疫 学 , 2004 ,24(2):164-168
[20] Auccoutorier J, Dupuis L, Ganne V. Adjuvants designed for veterinary and humman vaccines[J].Vaccine, 2001, 19 (17-19):2666-2672
[21] Chow Y H, Huang VVL, et al. Improvement of hepatitis B virus DNA vaccine by plasmids coexpressing hepatitis B surface antigen and interleukin-2 [J] .J Virol, 1997, 71(1):169-178
[22] Billaut-mulot 0, Idriorek T, Loyens M, et al. Modulation of cellular and humoral immune responses to a multipitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost[J}.Vaccine,2001, 19 (20-22):2803-2811
[23] Playfair H L and Desouza J B. Recombinant gamma interferon is a potent adjuvant for a malaria vaccine in mice. Clinical Experimental Immunolnev 1987, 67:5一10
[24] Heath AW, Playfair JH. Conjugation of interferon-gamma to antigen enhances its djuvanticity[J].Immunology. 1990, 71(3) :454-456.
[25] Sin JL, Kim JJ, Boyer J D, et al. In vivo modulation of vaccine-induced immune response toward a Thl phenotype increases potency and vaccine effedctiveness in a herpes simplex virus type 2 mouse model .J Virol. 1999, 73:501-507
[26] Heath A W, Playfair J H. Cytokines as immunological adjuvants. Vaccine 1992, 10 (7):427-434
[27] Micallef MJ, Ohtsuki K, Kohno F, et al. Interferon-gamma-inducing factor enhances T helper lcytokine production by stimulated human Tce1l:Synergism with interleukin-12 for interferon-gamma production [J].Eur J Immunol,1996, 2 (7):1647-1651
[28] Guidotti LG, Huang W K, et al. Improvemnet of hepatitis B virus DNA vaccines by plasmid co-expressing hepatitis B surface antigen and interleukin-18. Hepatology, 1998, 28:202-210
[29] Chowm Chiang BL, Lee YL,et a1. Development of Thl and Th2 population and the nature of immune responses to hepatitis B virus DNA vaccinescan be modulated by code livery of various cytokine genes,J Immunol, 1998,160:1320 一 1329
[30] Kimm JJ, Yang J S, Vcncort T C, et al. Modulation of antigen-specific humoral responses in Rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants[J].J, Virol, 2000, 74 (7):3427-3429
[31] 蔡雷.DNA 免疫的基础性研究进展及应用.国外医学免疫学分册,2001, 24: 121-125
[32] 文艳君.增强 DNA 免疫效应的研究进展.国外医学免疫学分册,1999,22:320-323
[33] Irvine K R, Rao JB, Rosenberg S A. et al. Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J Immunol, 1996, 156:238-245
[34] Meuer SCH, Dumann KH, Buschenfelda M, et al. Low dose interleukin-2 induces systemic immune responses against HBsAg in immunodeficient to hepatitis B vaccination[J].Lancet, 1989, 1:15-17
[35] Quiroga JA, Castillo I, Porres JC, et al. Recombinant Y 一 interferon as adjuvant to hepatitis B vaccine in hemodialysis patients.Hepatology 1990,12:662-663
[36] Good MF, Pombo D,Lunde MN, et a1. Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides:correlation of effect with biologic activity of IL-2. J Immunol,1988; 141:972-977
[37] Blecha F,D N Reddy,C G Chitko-Mckown et al. Influence of recombinant bovine interleukin-1 and interleukin-2 in pig vaccinated and challenged with streptococcus suis.Vet Immunopathal,1995, 44:329-346.
[38] 吴丹,郭滚军,王庆敏等。猪白细胞介素-4 的基因克隆及其在囊尾蝴 DNA 疫苗中的应用[J].第二军医大学学报,2002, 23 (11 ): 1192-1194
[39] Lim YS, Kang BY, Kim EJ etal. Potentiation of antigen-specific, Th1 immune response by multiple DNA vaccination with an ovalbumin/interferon-It hybrid construct [J].Immunol, 1998, 94:135-141
[40] Mbawuike IN, Enhancement of the protecting efficacy of inactivated influenza A vaccine in aged mice by IL-2-liposomes[J].Vaccine, 1990, 8:347-352
[41] Operschall E, Schuh T, Heinzerling L, et al. Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.J Clin Virol,1999,13:17-22
[42] 杜德伟,周永兴,冯志华等.IL-12 及 HBV 基因疫苗共同免疫小鼠的效果. 世界华人消化杂志 2000, 8 (2):128-130
[43] Lee SW, Cho J H, Sung YC et al.饰 timal induction of hepatitis C virusenvelope- specific immunity by bicistronic plasmid DNA inoculation with thegranulocyte-macrophage colony-stimulating factor gene.J Virol, 1998, 72:8430-8436
[44] 胡慧琼. 四川山地乌骨鸡 IL-2、15、18、IFN-γ 基因克隆测序及分子佐剂效应研究[D].四川农业大学,2006.
[45] Marton MJ ,Dekisi JL ,Bennett HA ,et al. 1998 ;4 (11) :1293～1301
[46] 张瑞军. IL-18 对 NK 细胞的调节剂抗肿瘤效应[J]. 国外医学肿瘤学分册,2000,27(3):148～150
[47] Dao T ,Ohashi K,Kayano T ,et al1Interferon-γ-inducing factor ,a novel cytokine ,enhances Fas ligand-mediated cytotoxicity ofmurine T helper 1 cells1Cell Immunol11996 ;173 :230
[48] Yoshimono T ,Okamura H ,Tagawa YI ,et al.Interleukin 18 to2gether with interleukin 12 inhibits IgE production by inductionof interferon-γ production from activated B cells1Proc NatlAcad Sci USA ,1997 ;94 :3948
[49] 陆意. IL-18 的研究进展[J].浙江海洋学院学报,2001,20(3):244～247
[50] HashimotoW, Osaki T, Okamura H, et al. Differential antitumoreffects of administration of recombinant IL-18 or recombinant IL-12are mediated primarily by Fas-Fas ligand and perforin induced tumorapop tosis, respectively[16] HashimotoW, Osaki T, Okamura H, et al. Differential antitumor
[51] 王萍等. IL-18对肝细胞损伤的作用研究进展[J].河南科技大学学报,2004,22(4):306～308
[52] Tone M et al. J Immunol,1997,159(12):6156
[53] Robinson D et al. Immunity,1997,7(4):571
[54] Okamura H ,Nagata K,Komatsu H ,et al.A novel costimulatoryfactor for gamma interferon induction found in the livers of micecauses endotoxic shock. Infect Immun ,1995 ;63 :3966
[55] 将定文等.IL-18 信号转导途径研究进展[J].国外医学分子生物学分册,2000,22(5):301～304
[56] Cregg JM,Ceregino JL , Shi J , et al . Recombinant protein expression in Pichia Pastoris[J]. Molecular Biotechnology 2000 , 16 : 23-52.
[57] 王勇. 毕赤酵母表达外源基因研究进展[J].微生物学免疫学进展,2004, 32(1):62～64
[58] 周则迅,袁汉英,何炜.乙肝病毒表面抗原SA-28融合基因在酵母中的组成型表达.复旦学报(自然科学版),2000,39(3):264～268
[59] David R, Higgins, James M et al. Pichia Protocols. New jersey, Humana Press Inc, 1998, 193～200
[60] MarcW T. High-yield secretion of recombinant gelation by pichia pastoris. yeast, 1999,15:1087~1096
[61] Eckhart L, Raffelsberger W, Ferko B, Klima et al. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.J Gen Virol,1996,77:2001～2008
[62] Wegner G.Emerging applications of methytrophic yeasts FEMS Microbiol Rev,1990,87:279～284
[63] Siegel R S, Buckholz R G, Thill G P et al. Production of epidermal growth factor in methylogrophic yeast cells.European Patent Application W090/10697,1990
[64] Scorer C A, Clare JJ, Mccombie W R et al. Rapid selection using G418 of high copy number tramsformants of pichia pastoris for high-level foreign gene expression. Bio/Technology, 1994, 12: 181～174
[65] Tschopp J F, Brust p f, cregg j m et al. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris.Nucleic Acids Res,1987,15:3859～3876
[66] Fahnestock S R, Bedzyk L A. Production of synthetic spider dragline slik protein in Pichia pastoris .Appl Microbiol Biotechnol,1997,47:33～39
[67] Cregg J M, Madden K R, Barringer K J et al. Functional characterization of the two alcoholoxidase genes from the yeast pichia pastoris. Mol Cell Biol, 1989, 9: 1316～1323
[68] Koutz P, Davis G R, Stillman C et al. Structural comparison of the Pichia Pastoris alcohol oxidase genes. Yeast, 1989, 5: 167～177
[69] Waterham H R, Digan M E, Koutz P J et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate deydyogenase gene and regulation and use of its promoter. Gene,1997,186:37～44
[70] 范乔,刘宏迪.甲醇营养型酵母表达外源蛋白的特点及策略[J].微生物学通报 ,1999,26(4):304～306
[71] Tschopp J F, Sverlow G, Kosson R et al. High-level secretion of glycosylated invertase in the methylotrophic yeast,Pichia pastoris.Bio/Technology , 1987,5:1305～1308
[72] Julius D, Brake A, Blair L et al. Isolation of putative structural gene for the lysine- arginine-cleaving endopeptidese required for processing of yeast prepro-a-factor. Cell, 1985, 37: 1075
[73] Sreekrishna K, Tschopp J F, FucR M. Invertase gene (SUC2) of saccharomyces cere uisiae as a dominant marker for transformation of pichia pastoris. Gene, 1987, 59(1): 115～125
[74] White C E, Hunter M J, Meininger D P et al. Large-scale expression,purification and charaterization of small fragments of thrombomodulin:the roles of the sixth domain and of methionine 388. protein Eng,1995,8:1177～1187
[75] Cregg J M, Madden K R, Barringer K J et al. Functional characterization of the two alcohol oxidase genes from the yeast pichia pastoris. Mol Cell Biol, 1989, 9: 1316～1323
[76] 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 the B7-2 counter-receptor CTLA-4 FEBS Lett, 1997,407:63～68
[77] 黄志清.猪干扰素-γ基因在毕赤酵母中的分泌表达及其抗猪瘟病毒活性研究[D].中国农业大学硕士学位论文,2005
[78] 彭毅,杨希才,康良仪.影响甲醇酵母中外源蛋白表达的因素[J].生物技术通报,2000,(4):33～36
[79] Scorer C A, Buckholz R G, Clare J J,et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene, 1993, 136:111～119
[80] Clare J J, Rayment F B, Ballantine S P et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology, 1991 ,9:455～460
[81] Paul M S. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Research, 1986, 14(3): 5125～5143
[82] Andre H. Codon relacemont in the PAK1 gene: Experimental approach to study the role of based codan usage in gene expression. Molecular and cellular biology, 1987, 2914～2924
[83] 赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3):308～311
[84] 姚斌,张春义等.高效表达具有生物学活性的植酸酶的毕赤酵母[J].中国科学(C 辑),1998,28(3):237～243
[85] Wulfing C,PlucKthun A.Correctly folded T-cell receptor fragment in the periplasm of Escherichia Coli. J MolBiol,1994,243(3):655～669
[86] Steidler L, Fiers W, Remaut E. Efficient specific release of Poriplasmic Proteins from Escherichia coli using temperature induction of cloned Kil gen of pMB9. Biotech Bioeng, 1994, 44(9): 1074～1082
[87] Leif.Lactase from the white-rot fungs trametes versicolor:cDNA cloning of lccl and expression in Pichia pastoris.Curr Gene,1997,32:425～430
[88] Henri P F. Overexpression and characterization of Aspergillus awamori wildtype and mutant glucoamylase secreted by the methylotrophic yeast pichia pastoris: Comparison with wild-type recombinant glucoamylast. Protein Expression and purification, 1997, 9:199～170
[89] 圣栋主编.现代分子生物学实验技术.北京:中国协和医科大学出版社,1999
[90] LaVallie E R, DiBlasion EA, Kovacic S et al. A thioredoxin gene fusion expression system that circumvents clusion body formation in the E.Coli cytoplasm. Bio/Technology, 1993, 11(2): 187～193
[91] 辛利,张励,徐韧等.人血管抑素在毕氏酵母中的表达及其活性测定[J].生物化学与生物物理学报,2001,33(3):237～343
[92] Nakamura K,Okamura H ,Nagata K,et al. Purification of a factor which provides a costim- ulatory signal for gamma interferon production[J]. Infect Immun,1993 ;61-64.
[93] Oamura H , Nagata K, Komatsu T ,et al. A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock [J]. Infect Immun ,1995 ;63 (10):3966-3972.
[94] Dinarello CA. Interleukin-18, aproinflammatory cytokine [J]. Eur Cytokine N etw, 2000; 11 (3) : 483 - 486.
[95] Ushio S,Namba M,Okura T,et al.Cloning of the cDNA for human IFN-γinducing factor,expression in Eschetichia coli,and studies on the biologic activities of the protein[J].J Immonol,1996,156:4274-4279.
[96] 景志忠 , 窦永喜 , 侯俊琳等 . 猪 IL-18 基因的克隆和序列分析 [J]. 中国兽医科技2004,34(7):3～7.
[97] Yoshihiro Muneta, Yasuyuki Mori, Yoshihiro Shimoji. Yuichi Yokomizo. Porcine Inte- rleukin 18: Cloning, Characterization of the cDNA and Expression with the Baculovirus System[J]. Cytokine, 2000;12(6): 566–572.
[98] Yoshihiro Muneta*, Yoshihiro Shimoji, Yuichi Yokomizo, Yasuyuki Mori. Production ofmonoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purific- ation of recombinant porcine interleukin-18[J]. Journal of Immunological Methods , 236 (2000) 99–104.
[99] 陈芳,孙红立,李震等.细胞因子的研究进展[J].上海畜牧兽医通讯,2003,(5):6～8.
[100] apamichail M, Perez SA, Gritzapis AD, et al. Natural killer lymphocytes: Biology, development and function [J]. Cancer Immuno Imunother, 2004; 53 (3): 176 – 186.
[101] Schmidt OI, Morganti-Kossmann MC, Heyde CE, et al. Tumor necrosis factor mediated inhibition of interleukin-18 in the brain: A clinical and experimental study in head-injured patients and in a murine model of closed head injury[J]. J N euroinflamm, 2004; 1 (1) : 13.
[102] Cregg JM, Ceregino JL , Shi J , et al . Recombinant protein expression in Pichia Pastori[J]. Molecular Biotechnology, 2000, 16: 23-52.
[103] 裴仉福,陈瑞爱,温纳相等.猪 IL-18 基因原核表达载体的构建和表达[J]. 动物医学进展,2006,27(10):73-77.
[104] 韩雪清 ,刘湘涛 ,任芳丽等.猪瘟病毒E2 蛋白在酵母中分泌表达条件的优化[J]. 畜牧兽医学报,2003 ,34 (6) ,567-572
[105] Wenhui W , Mark A B ,Bradley A P , et al. Modeling pichia pastoris growth on methanol and optimizing the production of a recombinant protein. the Heavy chainfragment C of Botulinum neurotoxin、serotype A [J] .Biothchnology and Bieng - neering ,2000 ,20(1) :1～8.
[106] 傅安静,岳华,刘树玲等. 免疫佐剂的研究进展[J] .西南民族大学学报,2006,73-77