禽传染性支气管炎病毒M基因在毕赤酵母中表达与蛋白活性检测
|
摘要
禽传染性支气管炎(Avian Infectious Bronchitis，IB)是由禽传染性支气管炎病毒(IBV)引起的鸡的一种急性、高度接触性的传染病。IBV的变异大，血清型多，是IB免疫预防的一大难题。IBVM蛋白比较保守，在病毒粒子中所占的比例最大，同时具有膜蛋白的典型特点，是重要的免疫原基因。M蛋白的这些特性为M蛋白作为IB的诊断抗原提供了重要的分子生物学及免疫学依据。
本研究根据本课题组在GenBank中注册的禽传染性支气管炎病毒中国分离株SAIBK株的M基因序列(注册号AY302742)及毕赤酵母分泌型表达载体pPIC9K的序列设计引物，通过鸡胚培养收获禽传染性支气管炎病毒，提取病毒RNA，用RT-PCR方法扩增出IBV M基因，构建了克隆载体pMD18-M，通过菌落PCR和酶切对克隆载体进行了鉴定。将克隆载体pMD18-M和酵母表达载体pPIC9K分别用限制性内切酶EcoR I和Not I进行双酶切，回收目的片段，用T4 DNA连接酶将两者连接后转化大肠杆菌，筛选得到酵母表达载体，经酶切鉴定M基因已经正确地连接到pPIC9K上。用限制性内切酶Sac I将表达质粒pPIC9K-M线性化，然后用电转化的方法导入毕赤酵母GS115中。将在MD平板上生长的转化子经过PCR鉴定、表型筛选和不同G418抗性浓度筛选后，获得整合型阳性重组菌株，命名为GS115／pPIC9K-MHis~+Mut~+。将重组菌株在1％的甲醇中进行诱导分泌表达，通过收集不同时间段的表达产物进行SDS-PAGE分析表明，在诱导72小时后目的蛋白表达量最大，表达蛋白占上清中总蛋白量的13.3％，表达蛋白的分子量约为33KD。
通过Western-blot检测显示，表达蛋白能与IBV阳性血清特异性结合。对表达蛋白进行初步纯化后，以M蛋白为包被抗原的ELISA检测结果显示表达蛋白与特异性抗体反应良好，表明表达的M蛋白生物学活性良好。目前国内外还没有利用毕赤酵母分泌型表达载体pPIC9K来表达IBV M蛋白的报道。本研究表达的IBV M蛋白可作为制备IB诊断抗原的基础材料，对IB的防治有重要理论和实用价值。
IBV is a fast and highly—touched infectious disease which has been variating frequently.It has much serological type and was difficult to prevent. IBV M protein was ratherconservative, concerned with replication of viruses it was an important immunogen gene.In addition, M gene holds a largest proportion in IBV particle and has typical feature ofmembrane protein. The characteristics of M protein provided molecular biology andimmunology base for M protein becaming IB diagnostic antigen.
A pair of M gene expression fragment PCR primers were designed according toisolated IBV's M gene sequence which was accepted by Genbank and the sequence ofPichia pastoris expression vector pPIC9K's Multiple Cloning Site(MCS). Virus wereobtained by chicken embryo cultivating and the RNA of IBV virus was extracted. M genewas amplified by RT-PCR and cloned into the vector pMD18.Then the cloned vectorpMD18-M was identified by colony-PCR and enzyme digestion. The M fragment,producing from pMD18-M by EcoR I and Not I, was ligated with the yeast expressingvector pPIC9K which was also digested by EcoR I and Not I,then the ligation wastransformed to E.coli. The results of PCR and enzyme digestion of plasmid proved thatrecombination vector was obtained (named pPIC9K-M). The expressing plasmidpPIC9K-M was linearized with Sac I, then it was transformed into Pichia pastoris GS115by electroporation. After the transformants were selected by PCR、Methanol-utilization-plus and different G418 concentrations YPD medium, the positiverecombination yeast was obtained(named GS115/ pPIC9K-M His~+Mut~+). GS115/pPIC9K-M His~+Mut~+was induced with 1% methanol at 28℃for target protein expression. Expressed M protein of different time section was analysised by SDS-PAGE,the resultsshowed that the target protein was up to the highest yield after 3 days induced, and theproduct was about 33KD. The amount of target protein shares about 13.3% of the totalprotein which consist in the supematant fluid of yeast culture medium.
Western blotting assay showed that the expressed protein could act with the positiveserum of IBV. The protein was initially purifed and acted as the envelope antigen of ELISAdetecting, the results proved expressed protein acted well with the specific antibody andthe expressed M protein has powerful biologic activity.
Now there had no report about expressing M protein with Pichia pastoris. The expressedM protein may act as diagnostic antigen meterial of IB.The result has important theoreticaland practical value to prevention and cure of IB.
本研究根据本课题组在GenBank中注册的禽传染性支气管炎病毒中国分离株SAIBK株的M基因序列(注册号AY302742)及毕赤酵母分泌型表达载体pPIC9K的序列设计引物，通过鸡胚培养收获禽传染性支气管炎病毒，提取病毒RNA，用RT-PCR方法扩增出IBV M基因，构建了克隆载体pMD18-M，通过菌落PCR和酶切对克隆载体进行了鉴定。将克隆载体pMD18-M和酵母表达载体pPIC9K分别用限制性内切酶EcoR I和Not I进行双酶切，回收目的片段，用T4 DNA连接酶将两者连接后转化大肠杆菌，筛选得到酵母表达载体，经酶切鉴定M基因已经正确地连接到pPIC9K上。用限制性内切酶Sac I将表达质粒pPIC9K-M线性化，然后用电转化的方法导入毕赤酵母GS115中。将在MD平板上生长的转化子经过PCR鉴定、表型筛选和不同G418抗性浓度筛选后，获得整合型阳性重组菌株，命名为GS115／pPIC9K-MHis~+Mut~+。将重组菌株在1％的甲醇中进行诱导分泌表达，通过收集不同时间段的表达产物进行SDS-PAGE分析表明，在诱导72小时后目的蛋白表达量最大，表达蛋白占上清中总蛋白量的13.3％，表达蛋白的分子量约为33KD。
通过Western-blot检测显示，表达蛋白能与IBV阳性血清特异性结合。对表达蛋白进行初步纯化后，以M蛋白为包被抗原的ELISA检测结果显示表达蛋白与特异性抗体反应良好，表明表达的M蛋白生物学活性良好。目前国内外还没有利用毕赤酵母分泌型表达载体pPIC9K来表达IBV M蛋白的报道。本研究表达的IBV M蛋白可作为制备IB诊断抗原的基础材料，对IB的防治有重要理论和实用价值。
IBV is a fast and highly—touched infectious disease which has been variating frequently.It has much serological type and was difficult to prevent. IBV M protein was ratherconservative, concerned with replication of viruses it was an important immunogen gene.In addition, M gene holds a largest proportion in IBV particle and has typical feature ofmembrane protein. The characteristics of M protein provided molecular biology andimmunology base for M protein becaming IB diagnostic antigen.
A pair of M gene expression fragment PCR primers were designed according toisolated IBV's M gene sequence which was accepted by Genbank and the sequence ofPichia pastoris expression vector pPIC9K's Multiple Cloning Site(MCS). Virus wereobtained by chicken embryo cultivating and the RNA of IBV virus was extracted. M genewas amplified by RT-PCR and cloned into the vector pMD18.Then the cloned vectorpMD18-M was identified by colony-PCR and enzyme digestion. The M fragment,producing from pMD18-M by EcoR I and Not I, was ligated with the yeast expressingvector pPIC9K which was also digested by EcoR I and Not I,then the ligation wastransformed to E.coli. The results of PCR and enzyme digestion of plasmid proved thatrecombination vector was obtained (named pPIC9K-M). The expressing plasmidpPIC9K-M was linearized with Sac I, then it was transformed into Pichia pastoris GS115by electroporation. After the transformants were selected by PCR、Methanol-utilization-plus and different G418 concentrations YPD medium, the positiverecombination yeast was obtained(named GS115/ pPIC9K-M His~+Mut~+). GS115/pPIC9K-M His~+Mut~+was induced with 1% methanol at 28℃for target protein expression. Expressed M protein of different time section was analysised by SDS-PAGE,the resultsshowed that the target protein was up to the highest yield after 3 days induced, and theproduct was about 33KD. The amount of target protein shares about 13.3% of the totalprotein which consist in the supematant fluid of yeast culture medium.
Western blotting assay showed that the expressed protein could act with the positiveserum of IBV. The protein was initially purifed and acted as the envelope antigen of ELISAdetecting, the results proved expressed protein acted well with the specific antibody andthe expressed M protein has powerful biologic activity.
Now there had no report about expressing M protein with Pichia pastoris. The expressedM protein may act as diagnostic antigen meterial of IB.The result has important theoreticaland practical value to prevention and cure of IB.
引文
[1] B．W．卡尔尼克主编，禽病学(第九版)[M]．北京：北京农业大学出版社，1991，407-418．
[2] 李华，杨汉春．传染性支气管炎病毒分子变异机制研究进展[J]．中国兽医杂志，1999，25(1)：46-48．
[3] 吴琦，王红宁．鸡传染性支气管炎病毒的分型研究概况[J]．中国预防兽医学报，1999，21(4)：317-320．
[4] 王红宁主编．禽传染性支气管炎综合防治[M]．北京：中国农业出版社，2000．
[5] MccaE．传染性支气管炎现状：有效的防治措施[J]．国外兽医学—畜禽传染病，1987，9(4)：39-40．
[6] King D J, Hopkins S R. Rapid serotyping of infectious bronchitis virus isolates with the hemagglutination inhibition test[J]. Avain Disease, 1984, 28:727-733.
[7] 吕爱军，胡步荣．鸡传染性支气管炎的诊断和防制研究进展[J]．养禽与禽病防治，2000，10：6-7．
[8] 吴延功．腺胃源传染性支气管炎病毒的分离、鉴定及分子流行病学．北京，中国农业大学，2002．
[9] 王红宁主编．禽呼吸系统疾病[M]．北京：中国农业出版社，2002．
[10] Schalk A. K., Hawn M. C. An apparently new respiratory disease of baby chicks, J. Am.[]].Vet. Med. Assoc., 1931., 78:413-422.
[11] J. Gelb,Jr.W.A.Nix and S.D.Gejiman,et al.Avian Disease., 1998.,42: 364-374.
[12] J. J. De Wit,D. R. Mekkes,B. Kouwenhaven. et al. Avian Pathology, 1997, 26: 105-118.
[13] Cavanagh D, Darbyshire J H, Davis P J, et al. Induction of humoral neutralizing and haemag glutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus[J]. Avian Pathology, 1984, 13:573-583.
[14] Koch G, Hartog L ,Kant A, et al. Antigenic domains on the pe-plomer protein of avian infectious bronchitis virus:correlation with biological functions[J]. J Gene Virol, 1990, 71:1929-1935.
[15] Mocker A P A, Cavanagh D, Brown T D K, et al. Monoclonal anti-bodies to the S1 spike and membrane proteins of avian infectious bronchitis coronavirus strain Mass achusetts M41 [J]. J Gene Virol, 1984, 65:2281-2286.
[16] Klumperman J.,Locker J.K.,Meijer A., et al. Coronavirus M protein accumulation in the Golgi complex beyond the site of viron budding[J]. J. Virol., 1994, 68(10): 6523-6534.
[17] 安健，田丽英，李换荣．传染性支气管炎病毒分子生物学研究进展[J]．中国兽医杂志，2001， 37(9)： 30-33．
[18] 任涛，廖明，曹伟胜等．鸡传染性支气管炎病毒中国地方分离株膜蛋白基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002：258-261．
[19] 刘胜旺，贺秀瑗，孔宪刚等．鸡传染性支气管炎病毒中国分离株M基因的分子特征[J]．中国兽医学报，2001，23(6)：404-407．
[20] 潘杰彦，陈德胜，戴亚斌等．传染性支气管炎病毒青岛腺胃分离株(SD／97／02)基质蛋白、5a、5b蛋白基因的克隆和序列分析[J]．中国兽医学报，2002，22(2)：108-110．
[21] Jia W.,Naqi S.A. Seq Jia (Jia W.,uence analysis of gene 3,gene 4,gene 5 of avian infectious bronchitis virus strain CU-T2[J]. Gene., 1997., 189(2): 189-193.
[22] Kapke P. A.,Tung F. Y.,Hogue B. G., et al. The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrance translocation and glycosylation[J]. Virology., 1988., 165(2): 367-376.
[23] Cavanagh D.,Davis P. J. Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic region of several serotypes[J]. J Gen Virol.,1988.,69: 621-629. Cavanagh D. Coronavirus IBV: Structural characterization of the spike protein[J]. J. Gen.Virol., 1983.,863-869.
[24] Ignjiatovic J.,Galli L. Structural proteins of avian infectious bronchitis virus:role in immunity and protection[J]. Adv Exp Med Biol., 1993., 342: 449-453.
[25] Cavanagh D., Davis P.J. Coronavirus IBV: removal of spike glycopolypeptide S1 by urea abolishes infectivity and heamagglutination but not attachment to cells[J]. J. Gen. Virol.,1986., 67: 1443-1448.
[26] 李德山等．中国病毒学，1992，7(4)：436-448．
[27] 王红宁，魏甬，周生等．禽传染性支气管炎病毒中国四川株S1基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002：269-271．
[28] Sadasiv, E. C.et al. J.Gen.Virol.,method.,1991., (33): 115-125.
[29] Williams A. K.,Wang L.,Sneed L.W., et al. Comparative analysis of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronavirus[J]. Virus Reserch., 1992., 25: 213-222.
[30] Zwaagstra, K.,Zeijst B.A.M.,Kusters J.G.,et al. Rapid detection and identification of avian infectious bronchitis virus[J]. Journalof Clinical Microbiology., 1992., 30(1): 79-84.
[31] Williams A. K.,Wang L.,Sneed L.W., et al. Analysis of the nucleocapsid genes of infectious bronchitis virus[J]. Virus Reserch., 1993., 3(3):213-222.
[32] Sapats S.I.,Ashton F.,Wright P.J., et al. Novel variation in the N protein of avian infectious bronchitis virus[J]. Virology., 1996., 226(2): 412-417.
[33] 陈丽君，蔡学忠，王英等．传染性支气管炎病毒T株核衣壳蛋白(N)基因的克隆及部分序列分析[J]．上海农业学报，1999，15(1)：22-27．
[34] 周生，王红宁，李春等．禽传染性支气管炎病毒中国四川株N基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002．：256-258．
[35] 常洪涛，刘红英．鸡传染性支气管炎血清学诊断方法研究进展[J]．上海畜牧兽医通讯，2003，3：7-9．
[36] Boots A. M., Kusters J. G.,Van-Norst J.M., et. al. Localization of a T-cell epitope within the nucleocapsid protein of avian coron-avirus[J].Immunology., 1991., 74:8-13.
[37] Seo S. H.,Collisson E.W.Cytotoxic T lymphocyte responses to infectious bronchitis virus infection[J]. Adv Exp Med Biol.,1998., 440: 456-460.
[38] 刘思国，康丽娟，江国托等．鸡传染性支气管炎病毒核蛋白基因重组真核表达载体的构建[J]．中国兽医学报，2001，21(1)：14-16．
[39] Seah JN et al.,Localization of linear B-cell epitopes on infectious bronchitis virus nucleo capsid protein [J].Vet Microbiol., 2000.,75(1):6-11.
[40] Abdul. N. et al..Recombinant nucleocapsid protein is potentially An inexpensive, effective serodiagnostic reagent for infectious bronchitis virus[J]. Journal of Virological Methods., 1998., 70(1): 37-445.
[41] 刘胜旺等．鸡传染性支气管炎病毒核蛋白基因的克隆及在杆状病毒系统中的表达[J]．中国预防兽医学报，2001，(6)：403．
[42] Hongying Chen et al. Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus[J].Avian pathology., 2003.,32(5): 519-526.
[43] 王宏俊、陈福勇．利用IBV重组N蛋白建立ELISA抗体检测试剂盒的研究[J]．中国兽医杂志，2003，39(9)：3-6．
[44] 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[J]．1998，Vol．18，No．3：7-11．
[45] Veehuis M and Harder W. Microbodies. In Rose, and Harrison, JS(Eds). The yeast, Vol4,2nd Academic Press Limited, London, 1991, pp601-653.
[46] Hoop MJ, de and AB Glmport of proteins into peroxisomes and other microbodies. Biochem J. 1992; 286: 657-669.
[47] Subramani S. Ann. Protein import into peroxisomes and biogenesis of the organelle. Rev Cell Biol. 1993; 9: 445-478.
[48] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreigngenes in Pichia pastoris[J].Bio/Technology, 1993, 11(8): 905-910.
[49] Nicaud JM, Raynal A, Beyou A et al. Stabilization ofmethionine-rich protein in Saccharomyces cerevisiae: targeting of BIN protein into the peroxisome. Curr Genet. 1994; 26: 390-397.
[50] Clare J J, Scorer C A,Buchholz R G, et al. Expression of EGF and HIV envelope glycoprotein.[J]. Methods Mol. Bio, 1998, 103: 209-255.
[51] Ito H, Fukuda Y, Murata K, et al. Transformation of intact yeast cells treated with alkalications[J]. J Bacteriol, 1983, 153 (1): 163-168.
[52] 马兴元，谭建华，朱平等．毕赤酵母表达系统及其在外源蛋白生产中的优势与应用前景[J]．中国兽医杂志，2003，23(1)：98-101．
[53] Creff JM, Cereghina J L, Shi J. et al. Recombinant protein expression in Pichia pastoris[J]. Mol Biotechnol, 2000, 16 (1): 23-52.
[54] 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[J]，1996，26(4)：304-306．
[55] Clare J J, Rayment F B, Ballantine S P. et al. Hight-level expression of tetanus tox in fragment C in Pichia pastoris strains containing multiple tandem integration of the gene[J]. Bio/technology, 1991a, 9: 455—460.
[56] Hasslacher M, Schall M, Hayn M, et al. High-level in treacllular expression of hydroxynituile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expre Purif, 1997, 11:61-71.
[57] Rosenfeld SA, Nadeau D, Tirado J, et al. Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris[J]. Protein Expre Purif, 1996, 8: 476—482.
[58] 朱剑昆，许志祥，黄伟达等．人重组白细胞介素Ⅱ在毕氏酵母系统中的表达及纯化[J]．中国医学科学院学报，2001，23(2)：127-131．
[59] Goda S, Takano K, Yamagata Y, et al. Effect of extra N-termi-nalresidues on the stability and folding of human lysozyme expressed in Pichia pastoris[J]. Protein Eng, 2000, 13 (4): 299-307.
[60] Nakakubo H, Moria M, Imada T, et al. Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate [J]. Yeast, 2000, 16 (15): 1387-1396.
[61] Muslin E H, Kanikula A M, Clark S E, et al. Over expression, purification and characterization of a barley alphaglucosidase secreted by Pichia pastoris[J]. Protein Expr Purif, 2000, 18 (1): 20-26.
[62] K jeldsen T. Yeast secretory expression of insulin precursors[J]. Appl Microbiol Biotechnol, 2000, 54 (3): 277-286.
[63] Sen G C, Dighe R R. Hyper expression of biologically active human chorionic gonadotrop in using the methylotropic yeast Pichia pastoris[J]. J Mol Endocrinol, 1999, 22 (3): 273-283.
[64] 李英华，白俊杰，李新辉等．鲤鱼生长激素在毕赤酵母中的表达[(J]．中国生物化学与分子生物学报，2001，17(4)：488-491．
[65] 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[[J]. Bio/Technology, 1987, 5:479-485.
[66] Sugrue R J, Cui T, Xu Q,et al. The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris[J]. J Virol Methods, 1997, 69 ( 1-2): 159-169.
[67] Clare J J, Romanos M A, Rayment F B, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene cpies[J]. Gene, 1991b, 105: 205-212.
[68] 官孝群，王跃祥，吴良成等．血管生成抑制因子K4K5cDNA基因的克隆及其在毕赤酵母中的表达[J]．生物工程学报，2001，17(2)：126-130．
[69] 王红宁，马孟根，吴琦等．黑曲霉N25植酸酶phyA基因在毕赤酵母中高效表达的研究[J]．菌物系统，2001，20(4)：486-493．
[70] 吴玉水，任君萍，黄庆生等．乙型脑炎病毒E蛋白在毕赤酵母中的表达[[J1]．细胞与分子免疫学杂志，2003，19(2)：130-135．
[71] 覃雅丽，陈焕春，唐勇等．伪狂犬病病毒Ea株gE基因主要抗原区在毕赤酵母中的表达[J]．微生物学报，2002，42(5)：543-549．
[72] 钱平，周艳君，童光志等．猪繁殖与呼吸综合征病毒ORF6基因在毕赤酵母中的表达[J]．中国预防兽医学报，2002，24(3)：161-164．
[73] Despreaux C W, Manning R F. The dacA gene of Bacillus sthearothermophilus coding for D -alanine carboxypeptidase:clone,structure and expression in Escherichia Coli and Pichia Pastoris[[J]. Gene, 1993, 131 (1): 35-41.
[74] WangHongning. Isolation and Identification of Infectious Bronchitis Virus from Chickens in Sichuan China[J].Avian Disease (SCI收录), 1997, 41 (2): 279-282.
[75] 黄勇，王红宁，王林川等．应用RT-PCR技术扩增禽传染性支气管炎病毒S1基因的研究[J]．中国畜禽传染病，1998，20(5)：297-298．
[76] 王林川，王红宁，黄勇等．用RTI PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型[J]．病毒学报，1997，13(4)：365-368．
[77] 李春，王红宁，周生等．禽传染性支气管炎病毒S1基因玉米表达载体的构建[J]．四川畜牧兽医，2003，30(B09)：18-18．
[78] 王红宁．禽传染性支气管炎病毒S1、M、N基因真核表达质粒的构建[M]．中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集，2004，10：650-652．
[79] 周生，王红宁，胡慧琼等．禽传染性支气管炎病毒分离株SAIBK株全基因组的克隆与序列分析[M]．中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集，2004，10：656-660．
[80] 杨帆，王红宁等．禽传染性支气管炎病毒四川分离株M基因的克隆及真核表达载体的构建[J]．四川畜牧兽医，2003(增刊)：19．
[81] 扬帆．禽鸡传染性支气管炎病毒四川分离株M基因DNA免疫质粒的构建及免疫原性研究[D]．硕士学位论文．雅安：四川农业大学，2004．
[82] 余祖华，王红宁，周生等．禽传染性支气管炎病毒S1基因在毕赤酵母中表达的研究．中国畜牧兽医学会家畜传染病学分会第十一次学术研讨会论文集，2005：789-791．
[83] 陈萍，王红宁，周生等．禽传染性支气管炎病毒M基因在大肠杆菌中的表达研究．中国畜牧兽医学会家畜传染病学分会第十一次学术研讨会论文集，2005：784-785．
[84] David R, Higgins, James M. Cregg. Methods in Molecular Biology, 1998, 10: 31-15.
[85] Ridder, R. et al. Gneration of rabbit monoclonal antibody fragments from a combinatorial pHage display library and their production in the yeast Pichia pastoris. Bio/Technology1995, 13: 255-260.
[86] 汪家政，范明主编．蛋白质技术手册[M]．北京：科学出版社，2000，8：60-74．
[87] Hon W C, Griffith M,Chong P, et al.Extraction and isolation of antifreeze proteins from winter rye(Xecale ereate L.)leaves. Plant physiol, 1994, 104(3): 971～980.
[88] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiology Review, 2000, 24(1): 45-66.
[89] Hasslacher M, Schall M, and Hayn M. High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expr Purif, 1997, 11(1):61-71.
[90] Sreekrishna K, Brankamp R G, Kropp K E, et al. Strategies for optimal synthosis and secretion of heterologous proteins in themethty-lotrophic yeast pichia pastoris[J]. Gene, 1997, 190 (1): 55-62.
[91] Cregg J M,Cereghino J L, Shi J, et al.Recombinant protein expression in Pichia pastoris[J].Mol B iotechnol,2000,16(1): 23-52.
[92] Rogelj B,Strukelj B,Bosch D,et al.Expression, purification,and characterization of equistation in Pichia pastoris[J], protein Expr Purif,2000,19(3):329-334
[93] Scorer CA,Clare JJ et al.Rapid aelection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.BioYTechnology, 1994,12:181-184.
[94] 彭毅等，甲醇酵母表达系统，生物技术通报．2000，1：38-41．
[95] Romanos MA,Beesley JJ,et al. Recombinant Bordetella pertussis pertaction from the yeast Pichia pastoris:high-level production and immunological properties.Vaceine, 1991,9:901-906.
[96] 李晶等，甲醇酵母基因表达系统的研究进展，生物工程进展，1999，Vol．19，NO2：17—20．
[97] Gregg J M,Barringer K J,Hessler A Y, et al.Pichia pastoris as a host system for transformations[J].Mol Cell Biol,1985, 5:3376-3385.
[98] 章如安等，巴斯德毕赤酵母表达系统研究及进展，微生物学通报，2000，27(5)：371—373．
[2] 李华，杨汉春．传染性支气管炎病毒分子变异机制研究进展[J]．中国兽医杂志，1999，25(1)：46-48．
[3] 吴琦，王红宁．鸡传染性支气管炎病毒的分型研究概况[J]．中国预防兽医学报，1999，21(4)：317-320．
[4] 王红宁主编．禽传染性支气管炎综合防治[M]．北京：中国农业出版社，2000．
[5] MccaE．传染性支气管炎现状：有效的防治措施[J]．国外兽医学—畜禽传染病，1987，9(4)：39-40．
[6] King D J, Hopkins S R. Rapid serotyping of infectious bronchitis virus isolates with the hemagglutination inhibition test[J]. Avain Disease, 1984, 28:727-733.
[7] 吕爱军，胡步荣．鸡传染性支气管炎的诊断和防制研究进展[J]．养禽与禽病防治，2000，10：6-7．
[8] 吴延功．腺胃源传染性支气管炎病毒的分离、鉴定及分子流行病学．北京，中国农业大学，2002．
[9] 王红宁主编．禽呼吸系统疾病[M]．北京：中国农业出版社，2002．
[10] Schalk A. K., Hawn M. C. An apparently new respiratory disease of baby chicks, J. Am.[]].Vet. Med. Assoc., 1931., 78:413-422.
[11] J. Gelb,Jr.W.A.Nix and S.D.Gejiman,et al.Avian Disease., 1998.,42: 364-374.
[12] J. J. De Wit,D. R. Mekkes,B. Kouwenhaven. et al. Avian Pathology, 1997, 26: 105-118.
[13] Cavanagh D, Darbyshire J H, Davis P J, et al. Induction of humoral neutralizing and haemag glutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus[J]. Avian Pathology, 1984, 13:573-583.
[14] Koch G, Hartog L ,Kant A, et al. Antigenic domains on the pe-plomer protein of avian infectious bronchitis virus:correlation with biological functions[J]. J Gene Virol, 1990, 71:1929-1935.
[15] Mocker A P A, Cavanagh D, Brown T D K, et al. Monoclonal anti-bodies to the S1 spike and membrane proteins of avian infectious bronchitis coronavirus strain Mass achusetts M41 [J]. J Gene Virol, 1984, 65:2281-2286.
[16] Klumperman J.,Locker J.K.,Meijer A., et al. Coronavirus M protein accumulation in the Golgi complex beyond the site of viron budding[J]. J. Virol., 1994, 68(10): 6523-6534.
[17] 安健，田丽英，李换荣．传染性支气管炎病毒分子生物学研究进展[J]．中国兽医杂志，2001， 37(9)： 30-33．
[18] 任涛，廖明，曹伟胜等．鸡传染性支气管炎病毒中国地方分离株膜蛋白基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002：258-261．
[19] 刘胜旺，贺秀瑗，孔宪刚等．鸡传染性支气管炎病毒中国分离株M基因的分子特征[J]．中国兽医学报，2001，23(6)：404-407．
[20] 潘杰彦，陈德胜，戴亚斌等．传染性支气管炎病毒青岛腺胃分离株(SD／97／02)基质蛋白、5a、5b蛋白基因的克隆和序列分析[J]．中国兽医学报，2002，22(2)：108-110．
[21] Jia W.,Naqi S.A. Seq Jia (Jia W.,uence analysis of gene 3,gene 4,gene 5 of avian infectious bronchitis virus strain CU-T2[J]. Gene., 1997., 189(2): 189-193.
[22] Kapke P. A.,Tung F. Y.,Hogue B. G., et al. The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrance translocation and glycosylation[J]. Virology., 1988., 165(2): 367-376.
[23] Cavanagh D.,Davis P. J. Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic region of several serotypes[J]. J Gen Virol.,1988.,69: 621-629. Cavanagh D. Coronavirus IBV: Structural characterization of the spike protein[J]. J. Gen.Virol., 1983.,863-869.
[24] Ignjiatovic J.,Galli L. Structural proteins of avian infectious bronchitis virus:role in immunity and protection[J]. Adv Exp Med Biol., 1993., 342: 449-453.
[25] Cavanagh D., Davis P.J. Coronavirus IBV: removal of spike glycopolypeptide S1 by urea abolishes infectivity and heamagglutination but not attachment to cells[J]. J. Gen. Virol.,1986., 67: 1443-1448.
[26] 李德山等．中国病毒学，1992，7(4)：436-448．
[27] 王红宁，魏甬，周生等．禽传染性支气管炎病毒中国四川株S1基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002：269-271．
[28] Sadasiv, E. C.et al. J.Gen.Virol.,method.,1991., (33): 115-125.
[29] Williams A. K.,Wang L.,Sneed L.W., et al. Comparative analysis of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronavirus[J]. Virus Reserch., 1992., 25: 213-222.
[30] Zwaagstra, K.,Zeijst B.A.M.,Kusters J.G.,et al. Rapid detection and identification of avian infectious bronchitis virus[J]. Journalof Clinical Microbiology., 1992., 30(1): 79-84.
[31] Williams A. K.,Wang L.,Sneed L.W., et al. Analysis of the nucleocapsid genes of infectious bronchitis virus[J]. Virus Reserch., 1993., 3(3):213-222.
[32] Sapats S.I.,Ashton F.,Wright P.J., et al. Novel variation in the N protein of avian infectious bronchitis virus[J]. Virology., 1996., 226(2): 412-417.
[33] 陈丽君，蔡学忠，王英等．传染性支气管炎病毒T株核衣壳蛋白(N)基因的克隆及部分序列分析[J]．上海农业学报，1999，15(1)：22-27．
[34] 周生，王红宁，李春等．禽传染性支气管炎病毒中国四川株N基因的克隆与序列分析[A]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C]．成都，2002．：256-258．
[35] 常洪涛，刘红英．鸡传染性支气管炎血清学诊断方法研究进展[J]．上海畜牧兽医通讯，2003，3：7-9．
[36] Boots A. M., Kusters J. G.,Van-Norst J.M., et. al. Localization of a T-cell epitope within the nucleocapsid protein of avian coron-avirus[J].Immunology., 1991., 74:8-13.
[37] Seo S. H.,Collisson E.W.Cytotoxic T lymphocyte responses to infectious bronchitis virus infection[J]. Adv Exp Med Biol.,1998., 440: 456-460.
[38] 刘思国，康丽娟，江国托等．鸡传染性支气管炎病毒核蛋白基因重组真核表达载体的构建[J]．中国兽医学报，2001，21(1)：14-16．
[39] Seah JN et al.,Localization of linear B-cell epitopes on infectious bronchitis virus nucleo capsid protein [J].Vet Microbiol., 2000.,75(1):6-11.
[40] Abdul. N. et al..Recombinant nucleocapsid protein is potentially An inexpensive, effective serodiagnostic reagent for infectious bronchitis virus[J]. Journal of Virological Methods., 1998., 70(1): 37-445.
[41] 刘胜旺等．鸡传染性支气管炎病毒核蛋白基因的克隆及在杆状病毒系统中的表达[J]．中国预防兽医学报，2001，(6)：403．
[42] Hongying Chen et al. Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus[J].Avian pathology., 2003.,32(5): 519-526.
[43] 王宏俊、陈福勇．利用IBV重组N蛋白建立ELISA抗体检测试剂盒的研究[J]．中国兽医杂志，2003，39(9)：3-6．
[44] 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[J]．1998，Vol．18，No．3：7-11．
[45] Veehuis M and Harder W. Microbodies. In Rose, and Harrison, JS(Eds). The yeast, Vol4,2nd Academic Press Limited, London, 1991, pp601-653.
[46] Hoop MJ, de and AB Glmport of proteins into peroxisomes and other microbodies. Biochem J. 1992; 286: 657-669.
[47] Subramani S. Ann. Protein import into peroxisomes and biogenesis of the organelle. Rev Cell Biol. 1993; 9: 445-478.
[48] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreigngenes in Pichia pastoris[J].Bio/Technology, 1993, 11(8): 905-910.
[49] Nicaud JM, Raynal A, Beyou A et al. Stabilization ofmethionine-rich protein in Saccharomyces cerevisiae: targeting of BIN protein into the peroxisome. Curr Genet. 1994; 26: 390-397.
[50] Clare J J, Scorer C A,Buchholz R G, et al. Expression of EGF and HIV envelope glycoprotein.[J]. Methods Mol. Bio, 1998, 103: 209-255.
[51] Ito H, Fukuda Y, Murata K, et al. Transformation of intact yeast cells treated with alkalications[J]. J Bacteriol, 1983, 153 (1): 163-168.
[52] 马兴元，谭建华，朱平等．毕赤酵母表达系统及其在外源蛋白生产中的优势与应用前景[J]．中国兽医杂志，2003，23(1)：98-101．
[53] Creff JM, Cereghina J L, Shi J. et al. Recombinant protein expression in Pichia pastoris[J]. Mol Biotechnol, 2000, 16 (1): 23-52.
[54] 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[J]，1996，26(4)：304-306．
[55] Clare J J, Rayment F B, Ballantine S P. et al. Hight-level expression of tetanus tox in fragment C in Pichia pastoris strains containing multiple tandem integration of the gene[J]. Bio/technology, 1991a, 9: 455—460.
[56] Hasslacher M, Schall M, Hayn M, et al. High-level in treacllular expression of hydroxynituile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expre Purif, 1997, 11:61-71.
[57] Rosenfeld SA, Nadeau D, Tirado J, et al. Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris[J]. Protein Expre Purif, 1996, 8: 476—482.
[58] 朱剑昆，许志祥，黄伟达等．人重组白细胞介素Ⅱ在毕氏酵母系统中的表达及纯化[J]．中国医学科学院学报，2001，23(2)：127-131．
[59] Goda S, Takano K, Yamagata Y, et al. Effect of extra N-termi-nalresidues on the stability and folding of human lysozyme expressed in Pichia pastoris[J]. Protein Eng, 2000, 13 (4): 299-307.
[60] Nakakubo H, Moria M, Imada T, et al. Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate [J]. Yeast, 2000, 16 (15): 1387-1396.
[61] Muslin E H, Kanikula A M, Clark S E, et al. Over expression, purification and characterization of a barley alphaglucosidase secreted by Pichia pastoris[J]. Protein Expr Purif, 2000, 18 (1): 20-26.
[62] K jeldsen T. Yeast secretory expression of insulin precursors[J]. Appl Microbiol Biotechnol, 2000, 54 (3): 277-286.
[63] Sen G C, Dighe R R. Hyper expression of biologically active human chorionic gonadotrop in using the methylotropic yeast Pichia pastoris[J]. J Mol Endocrinol, 1999, 22 (3): 273-283.
[64] 李英华，白俊杰，李新辉等．鲤鱼生长激素在毕赤酵母中的表达[(J]．中国生物化学与分子生物学报，2001，17(4)：488-491．
[65] 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[[J]. Bio/Technology, 1987, 5:479-485.
[66] Sugrue R J, Cui T, Xu Q,et al. The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris[J]. J Virol Methods, 1997, 69 ( 1-2): 159-169.
[67] Clare J J, Romanos M A, Rayment F B, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene cpies[J]. Gene, 1991b, 105: 205-212.
[68] 官孝群，王跃祥，吴良成等．血管生成抑制因子K4K5cDNA基因的克隆及其在毕赤酵母中的表达[J]．生物工程学报，2001，17(2)：126-130．
[69] 王红宁，马孟根，吴琦等．黑曲霉N25植酸酶phyA基因在毕赤酵母中高效表达的研究[J]．菌物系统，2001，20(4)：486-493．
[70] 吴玉水，任君萍，黄庆生等．乙型脑炎病毒E蛋白在毕赤酵母中的表达[[J1]．细胞与分子免疫学杂志，2003，19(2)：130-135．
[71] 覃雅丽，陈焕春，唐勇等．伪狂犬病病毒Ea株gE基因主要抗原区在毕赤酵母中的表达[J]．微生物学报，2002，42(5)：543-549．
[72] 钱平，周艳君，童光志等．猪繁殖与呼吸综合征病毒ORF6基因在毕赤酵母中的表达[J]．中国预防兽医学报，2002，24(3)：161-164．
[73] Despreaux C W, Manning R F. The dacA gene of Bacillus sthearothermophilus coding for D -alanine carboxypeptidase:clone,structure and expression in Escherichia Coli and Pichia Pastoris[[J]. Gene, 1993, 131 (1): 35-41.
[74] WangHongning. Isolation and Identification of Infectious Bronchitis Virus from Chickens in Sichuan China[J].Avian Disease (SCI收录), 1997, 41 (2): 279-282.
[75] 黄勇，王红宁，王林川等．应用RT-PCR技术扩增禽传染性支气管炎病毒S1基因的研究[J]．中国畜禽传染病，1998，20(5)：297-298．
[76] 王林川，王红宁，黄勇等．用RTI PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型[J]．病毒学报，1997，13(4)：365-368．
[77] 李春，王红宁，周生等．禽传染性支气管炎病毒S1基因玉米表达载体的构建[J]．四川畜牧兽医，2003，30(B09)：18-18．
[78] 王红宁．禽传染性支气管炎病毒S1、M、N基因真核表达质粒的构建[M]．中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集，2004，10：650-652．
[79] 周生，王红宁，胡慧琼等．禽传染性支气管炎病毒分离株SAIBK株全基因组的克隆与序列分析[M]．中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集，2004，10：656-660．
[80] 杨帆，王红宁等．禽传染性支气管炎病毒四川分离株M基因的克隆及真核表达载体的构建[J]．四川畜牧兽医，2003(增刊)：19．
[81] 扬帆．禽鸡传染性支气管炎病毒四川分离株M基因DNA免疫质粒的构建及免疫原性研究[D]．硕士学位论文．雅安：四川农业大学，2004．
[82] 余祖华，王红宁，周生等．禽传染性支气管炎病毒S1基因在毕赤酵母中表达的研究．中国畜牧兽医学会家畜传染病学分会第十一次学术研讨会论文集，2005：789-791．
[83] 陈萍，王红宁，周生等．禽传染性支气管炎病毒M基因在大肠杆菌中的表达研究．中国畜牧兽医学会家畜传染病学分会第十一次学术研讨会论文集，2005：784-785．
[84] David R, Higgins, James M. Cregg. Methods in Molecular Biology, 1998, 10: 31-15.
[85] Ridder, R. et al. Gneration of rabbit monoclonal antibody fragments from a combinatorial pHage display library and their production in the yeast Pichia pastoris. Bio/Technology1995, 13: 255-260.
[86] 汪家政，范明主编．蛋白质技术手册[M]．北京：科学出版社，2000，8：60-74．
[87] Hon W C, Griffith M,Chong P, et al.Extraction and isolation of antifreeze proteins from winter rye(Xecale ereate L.)leaves. Plant physiol, 1994, 104(3): 971～980.
[88] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiology Review, 2000, 24(1): 45-66.
[89] Hasslacher M, Schall M, and Hayn M. High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expr Purif, 1997, 11(1):61-71.
[90] Sreekrishna K, Brankamp R G, Kropp K E, et al. Strategies for optimal synthosis and secretion of heterologous proteins in themethty-lotrophic yeast pichia pastoris[J]. Gene, 1997, 190 (1): 55-62.
[91] Cregg J M,Cereghino J L, Shi J, et al.Recombinant protein expression in Pichia pastoris[J].Mol B iotechnol,2000,16(1): 23-52.
[92] Rogelj B,Strukelj B,Bosch D,et al.Expression, purification,and characterization of equistation in Pichia pastoris[J], protein Expr Purif,2000,19(3):329-334
[93] Scorer CA,Clare JJ et al.Rapid aelection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.BioYTechnology, 1994,12:181-184.
[94] 彭毅等，甲醇酵母表达系统，生物技术通报．2000，1：38-41．
[95] Romanos MA,Beesley JJ,et al. Recombinant Bordetella pertussis pertaction from the yeast Pichia pastoris:high-level production and immunological properties.Vaceine, 1991,9:901-906.
[96] 李晶等，甲醇酵母基因表达系统的研究进展，生物工程进展，1999，Vol．19，NO2：17—20．
[97] Gregg J M,Barringer K J,Hessler A Y, et al.Pichia pastoris as a host system for transformations[J].Mol Cell Biol,1985, 5:3376-3385.
[98] 章如安等，巴斯德毕赤酵母表达系统研究及进展，微生物学通报，2000，27(5)：371—373．