传染性支气管炎病毒纤突蛋白、核蛋白基因的克隆、表达及其在抗体检测中的初步应用
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摘要
鸡传染性支气管炎(Infectious bronchitis，IB)是由鸡传染性支气管炎病毒(Infectious bronchitis virus，IBV)引起的鸡的一种急性、高度接触性传染病。各种年龄、类型的鸡均易感。该病呈世界分布，是严重危害养鸡业的重大传染病之一。传染性支气管炎病毒属于冠状病毒科冠状病毒属，为不分节段的单股正链RNA病毒，包含至少3种结构蛋白基因：纤突蛋白(S)基因、核蛋白(N)基因、膜蛋白(M)基因。IBV的S蛋白由S1蛋白和S2蛋白两部分组成，在病毒进化中最为活跃，是最重要的保护性抗原，能刺激机体产生中和抗体，在病毒吸附细胞过程中发挥重要作用，在血清学分类、疫苗免疫防治上起决定性作用；N蛋白进化上最为保守，主要与基因组核酸的包裹、RNA的复制和细胞免疫有关，含有大量抗原决定簇，免疫原性仅次于S蛋白，能诱导机体产生大量抗体。自1931年发现该病以来，至少已有二十九种血清型被报道，并且新的血清型和变异株仍在不断出现。鉴于此，通过克隆表达具有高度保守性的N蛋白基因，并其为诊断抗原建立检测IBV抗体的ELISA法，为监测群体的抗体水平，预防和控制该病提供可靠的依据和有效的技术保障。同时克隆、表达具有型特异性的S1蛋白基因以便为下一步研究该病的基因工程疫苗奠定基础。本课题的主要研究内容包括：
1、传染性支气管炎病毒纤突蛋白S1基因和核蛋白基因的克隆与序列分析
以GenBank公布的IBV基因组的序列为依据，分别设计针对S1基因和N基因的特异性引物，通过RT-PCR法从IBV基因组中扩增出完整的S1和N片断，将扩增片断分别克隆到pMD18-T载体，经酶切和序列测定，S1、N基因片断大小分别为1685bp和1230bp；用BLAST比对发现：S1基因与GX1-98毒株同源性为98％，N基因与H52毒株LKQ3同源性为97％。
2、传染性支气管炎病毒纤突蛋白S1基因和核蛋白基因在大肠杆菌中的表达
构建了弃信号肽区S1基因表达载体pGEX-S1和N基因表达载体pGEX-NP，并成功进行了表达，对表达产物进行SDS-PAGE分析和Western-blot鉴定，结果表明S1分子量大约为80kDa；N蛋白表达于细胞质中，有两种主要产物，大小分别为80 kDa和60 kDa，并利用GST亲和层析柱进行了纯化，获得了相应纯化产物，Western-blot显示纯化产物均能与鸡IBV标准阳性血清发生特异性反应，说明有很好的免疫学活性。
3、基于重组N蛋白的IBV抗体ELISA检测方法的建立
Avian infectious bronchitis (IB) is an acute, highly contagious respiratory disease caused by Infectious bronchitis virus(IBV) to which all ages, types of chickens are susceptible. In most areas of world this disease happened as one of the most important infectious diseases which seriously hinder the development of poulty industry. IBV is the typical virus of Coronavirus in the family Coronaviridae with a single-strand positive-sense RNA genome, and include no less than three main structure proteins: Spike glycoprotein (S protein), Membrane glycoprotein (M protein) and Nucleocapsid protein (N protein). S protein is the most variable one in evolution and the most important antigen that could induce neutralizing antibody comparied with M protein and N protein. It acts essentially in the process that virus absorbs onto cell and in the serological classification of virus. It is important in vaccine immunization against IBV too. N protein is the most conservative one in evolution of IBV of which the main function is packaging nucleotide, duplicating RNA of IBV genome and activing cell-mediated immunity. From the year 1931 by now more than twenty nine serotype of IBV is reported with occurrence of new variant serotypes. For weak cross protection between different serotypes and monitoring immunological level of antibodies against IBV, a ELISA method was developed with the conservative recombinated N protein expressed in E.coli that provide dependable and available technological safeguard for controlling of IBV . And in the same time S1 gene charactered serology was cloned from genome of IBV and S1 protein was expressed in E.coli to contribute to the development of vaccine against IBV. So following researchs were explored: 1. Cloning and sequence analysis of S1 protein gene and N protein gene
According to published S1 protein gene and N protein gene sequence of IBV in Genebank, specific primers were designed and synthesized. A 1685bp fragments of S1 gene and 1700bp fragments of N gene were amplified from RNA of Infectious Bronchitis virus preservatived in our lab by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid was proved to be true by restriction-enzyme analysis and sequenced. The sequence BLAST showed that S1 gene have the most homology 98% with the strain GX1-98, N gene most 97% with the strain H52 LKQ3. 2.Expression of S1 、 N gene of IBV in E.coli
According to analysis of amino acids translated from the S1 gene, a pair of primers were designed and used to amplify the S1 gene lacked signal peptide. Then the subcloned fragment of S1 gene and the fragment of N gene were each recombinated into prokaryotic
1、传染性支气管炎病毒纤突蛋白S1基因和核蛋白基因的克隆与序列分析
以GenBank公布的IBV基因组的序列为依据，分别设计针对S1基因和N基因的特异性引物，通过RT-PCR法从IBV基因组中扩增出完整的S1和N片断，将扩增片断分别克隆到pMD18-T载体，经酶切和序列测定，S1、N基因片断大小分别为1685bp和1230bp；用BLAST比对发现：S1基因与GX1-98毒株同源性为98％，N基因与H52毒株LKQ3同源性为97％。
2、传染性支气管炎病毒纤突蛋白S1基因和核蛋白基因在大肠杆菌中的表达
构建了弃信号肽区S1基因表达载体pGEX-S1和N基因表达载体pGEX-NP，并成功进行了表达，对表达产物进行SDS-PAGE分析和Western-blot鉴定，结果表明S1分子量大约为80kDa；N蛋白表达于细胞质中，有两种主要产物，大小分别为80 kDa和60 kDa，并利用GST亲和层析柱进行了纯化，获得了相应纯化产物，Western-blot显示纯化产物均能与鸡IBV标准阳性血清发生特异性反应，说明有很好的免疫学活性。
3、基于重组N蛋白的IBV抗体ELISA检测方法的建立
Avian infectious bronchitis (IB) is an acute, highly contagious respiratory disease caused by Infectious bronchitis virus(IBV) to which all ages, types of chickens are susceptible. In most areas of world this disease happened as one of the most important infectious diseases which seriously hinder the development of poulty industry. IBV is the typical virus of Coronavirus in the family Coronaviridae with a single-strand positive-sense RNA genome, and include no less than three main structure proteins: Spike glycoprotein (S protein), Membrane glycoprotein (M protein) and Nucleocapsid protein (N protein). S protein is the most variable one in evolution and the most important antigen that could induce neutralizing antibody comparied with M protein and N protein. It acts essentially in the process that virus absorbs onto cell and in the serological classification of virus. It is important in vaccine immunization against IBV too. N protein is the most conservative one in evolution of IBV of which the main function is packaging nucleotide, duplicating RNA of IBV genome and activing cell-mediated immunity. From the year 1931 by now more than twenty nine serotype of IBV is reported with occurrence of new variant serotypes. For weak cross protection between different serotypes and monitoring immunological level of antibodies against IBV, a ELISA method was developed with the conservative recombinated N protein expressed in E.coli that provide dependable and available technological safeguard for controlling of IBV . And in the same time S1 gene charactered serology was cloned from genome of IBV and S1 protein was expressed in E.coli to contribute to the development of vaccine against IBV. So following researchs were explored: 1. Cloning and sequence analysis of S1 protein gene and N protein gene
According to published S1 protein gene and N protein gene sequence of IBV in Genebank, specific primers were designed and synthesized. A 1685bp fragments of S1 gene and 1700bp fragments of N gene were amplified from RNA of Infectious Bronchitis virus preservatived in our lab by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid was proved to be true by restriction-enzyme analysis and sequenced. The sequence BLAST showed that S1 gene have the most homology 98% with the strain GX1-98, N gene most 97% with the strain H52 LKQ3. 2.Expression of S1 、 N gene of IBV in E.coli
According to analysis of amino acids translated from the S1 gene, a pair of primers were designed and used to amplify the S1 gene lacked signal peptide. Then the subcloned fragment of S1 gene and the fragment of N gene were each recombinated into prokaryotic
引文
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79 Lenstra J A, Kusters J G. Koch G, et al. Antigenicity of peptomer protein of infectious bronchitis virus.Molecular Immunol.1989, 26(1):7-15
80 Li D, Cavanagh D. Coronavirus IBV induced membrane fusion occurs at near-neutral PH. Arch Virol. 1992,122: 307-316
81 Li Wang, Yuan Xu, Ellen W. Collisson. Experimental confirmation of recombination upstream of the S1 hypervariable region of infectious bronchitis virus. Virus Research. 1997, 49: 139-145
82 Lucio B, Fabricant J. Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus. Avian Dis. 1990,34: 865-870
83 Miguel B, Pharr G T and Wang C. The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Arch Virol. 2002, 147: 2047-2056
84 Minglong Z, Collisson E W. The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA. Virus Res.2000, 67(1): 31-39
85 Nagano H, Tsuchimoto M, Hohdatsu T, et al. Enzyme linked immunosorbent assay for the detection of infectious bronchitis virus antigen with monoclonal antibody. Japanese Journal of Veterinary Science. 1990, 52: 657-659
86 Navas S, Weiss S R. Murine coronavirus-induced hepatitis: JHM genetic background eliminates A59 spike-determined hepatotropism. J. Virol. 2003,77 (8): 4972- 4978
87 Ndifuna A, Waters AK, Zhou M, Collisson EW. Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus. J Virol Methods. 1998 ,70(1):37-44
88 Norio Yamamoto, Rongge Yang, Yoshiyuki Yoshinaka, Shinji Amari, et al. HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus. Biochemical and Biophysical Research Communications. 2004, 318: 719-725
89 Sadasiv E C, et al. J. Gen. Virol. 1991,(33): 115-125
90 Sang H S, Wang L, Smith R, et al. The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. J Virol.1997, 71(10): 7889-7894
91 Sapat S I, Ashton F, Wrigh P J and Ignjatovic J. Novel Variation in the N Protein of Avian Inf(?)us Bronchitis Virus. Virology. 1996, 226:412-417
92 Sc(?) A F, Hawn M C. An apparently new respiratory disease of baby chicks. J Am Vet Med Assoc. 1931,78:413-422
93 Schultze B, Enjuanes L, Cavanagh D, Herrler G N-acetylneuraminic acid plays a critical role for the haemagglutinating activity of avian infectious bronchitis virus and porcine transmissible gastroenteritis virus. Adv Exp Med Biol. 1993, 342: 305-310
94 Seah JN et al. Localization of linear B-cell epitopes on infectious bronchitis virus nucleo capsid protein. Vet Microbiol. 2000,75(1):6-11
95 Seo S H, Collisson E W. Cytotoxic T lymphocyte responses to infectious bronchitis virus infection. Adv Exp Med Biol. 1998,440: 456-460
96 Shen S, Law Y C, Liu D X. A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature. Virology .2004, 326: 288-298
97 Soe H S, Wang L, Smith R. The carboxy-terminal 120-residue polypeptide of infectious bronchitis virus from nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. J virol.1997, 71:7889-7894
98 Song C S, Lee Y J, Lee C W, Sung H W, Kim J H, Mo I P, Izumiya Y, Jang H K, Mikami T, Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus. J. Gen. Virol. 1998,79: 719-723
99 Sotou S, Sato S, Okaba, et al. Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus. Virol.1988, 6: 504-508
100 Spaan W, Cavanagh D, Horzinek M C. Coronaviruses: structure and genome expression. J Gen Virol. 1988, 69 (12): 2939-2952
101 Tomely F M, Mockett A P A, Boursnell M E G, et al. Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice. J Gen Virol. 1987, 68: 2291-2298
102 Wai J, Karaca K C, Parrish R, Naqi SA. A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch Virol. 1995, 140(2): 259-271
103 Wang C H, Hong C C, Seak J.C.H. An ELISA for antibodies against infectious bronchitis virus using an S1 spike polypeptide. Veterinary Microbiology. 2002, 85: 333-342
104 Wang X, Schnitzlein W M, Tripathy D N, et al. Construction and immunogenicity studies of recombinant fowl poxvirus containing the S 1 gene of Massachusetts 41 strain of infectious bronchitis virus. Avian Dis. 2002 ,46(4): 831-838
105 Williams A K, Wang L, Sneed L W, et al. Analysis of the nucleocapsid genes of infectious bronchitis virus.Virus Reserch. 1993,3(3):213-222
106 Yagyu K, Ohta S. Enzyme-linked immunosorbent assay for the detection of infectious bronchitis virus antigens. Japanee Journal of Veterinary Science.1987, 49: 757763
107 Yu L, Liu W, Schnizlein W M, et al. Study of protection by recombinant fowl pox virus expressing C-teminal nucleocapsid protein of infectious bronchitis virus against challenge. Avian Dis. 2003, 45(2):340-348
108 Zaagstra K A, Vander Z B, Kustrers J G. Rapid detection and identification of avian infectious bronchitis virus. J Clinical Microbiol. 1992, 30: 79-84
109 Zhou J Y, Cheng L Q, Zheng X J, Wu J X, Shang S B, Wang J Y, Chen J G Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus. Journal of Biotechnology. 2004,111: 121-130
110 Zhou M, Williams A K, Chung S I, et al. The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3'terminus of the genome. Virol. 1996, 217(1): 191-199
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71 Klumperman J, Locker J K, Meijer A, et al. Coronavirus M protein accumulation in the Golgi complex beyond the site of viron budding. J. Virol. 1994, 68(10): 6s23-6534
72 Koch G. Antigenic domains on the peptomer protein of avian infectious bronchitis virus:correlation with biological functions. J Gen Vrol. 1990,71:1923-1935
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76 Kwon H M, Jackwood M W. Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus. Virus Genes.1995, 24:313-332
77 Kwon H M. Jackwood W M, Brown T R et al. Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens. Avian Dis. 1993, 37:149-156
78 Lee C W. Jackwood M W. Evidence of genetic diversity generated bv recombination among avian coronavirus infectious bronchitis virus. Arch Virol. 2000, 145:2135-2148
79 Lenstra J A, Kusters J G. Koch G, et al. Antigenicity of peptomer protein of infectious bronchitis virus.Molecular Immunol.1989, 26(1):7-15
80 Li D, Cavanagh D. Coronavirus IBV induced membrane fusion occurs at near-neutral PH. Arch Virol. 1992,122: 307-316
81 Li Wang, Yuan Xu, Ellen W. Collisson. Experimental confirmation of recombination upstream of the S1 hypervariable region of infectious bronchitis virus. Virus Research. 1997, 49: 139-145
82 Lucio B, Fabricant J. Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus. Avian Dis. 1990,34: 865-870
83 Miguel B, Pharr G T and Wang C. The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Arch Virol. 2002, 147: 2047-2056
84 Minglong Z, Collisson E W. The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA. Virus Res.2000, 67(1): 31-39
85 Nagano H, Tsuchimoto M, Hohdatsu T, et al. Enzyme linked immunosorbent assay for the detection of infectious bronchitis virus antigen with monoclonal antibody. Japanese Journal of Veterinary Science. 1990, 52: 657-659
86 Navas S, Weiss S R. Murine coronavirus-induced hepatitis: JHM genetic background eliminates A59 spike-determined hepatotropism. J. Virol. 2003,77 (8): 4972- 4978
87 Ndifuna A, Waters AK, Zhou M, Collisson EW. Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus. J Virol Methods. 1998 ,70(1):37-44
88 Norio Yamamoto, Rongge Yang, Yoshiyuki Yoshinaka, Shinji Amari, et al. HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus. Biochemical and Biophysical Research Communications. 2004, 318: 719-725
89 Sadasiv E C, et al. J. Gen. Virol. 1991,(33): 115-125
90 Sang H S, Wang L, Smith R, et al. The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. J Virol.1997, 71(10): 7889-7894
91 Sapat S I, Ashton F, Wrigh P J and Ignjatovic J. Novel Variation in the N Protein of Avian Inf(?)us Bronchitis Virus. Virology. 1996, 226:412-417
92 Sc(?) A F, Hawn M C. An apparently new respiratory disease of baby chicks. J Am Vet Med Assoc. 1931,78:413-422
93 Schultze B, Enjuanes L, Cavanagh D, Herrler G N-acetylneuraminic acid plays a critical role for the haemagglutinating activity of avian infectious bronchitis virus and porcine transmissible gastroenteritis virus. Adv Exp Med Biol. 1993, 342: 305-310
94 Seah JN et al. Localization of linear B-cell epitopes on infectious bronchitis virus nucleo capsid protein. Vet Microbiol. 2000,75(1):6-11
95 Seo S H, Collisson E W. Cytotoxic T lymphocyte responses to infectious bronchitis virus infection. Adv Exp Med Biol. 1998,440: 456-460
96 Shen S, Law Y C, Liu D X. A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature. Virology .2004, 326: 288-298
97 Soe H S, Wang L, Smith R. The carboxy-terminal 120-residue polypeptide of infectious bronchitis virus from nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. J virol.1997, 71:7889-7894
98 Song C S, Lee Y J, Lee C W, Sung H W, Kim J H, Mo I P, Izumiya Y, Jang H K, Mikami T, Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus. J. Gen. Virol. 1998,79: 719-723
99 Sotou S, Sato S, Okaba, et al. Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus. Virol.1988, 6: 504-508
100 Spaan W, Cavanagh D, Horzinek M C. Coronaviruses: structure and genome expression. J Gen Virol. 1988, 69 (12): 2939-2952
101 Tomely F M, Mockett A P A, Boursnell M E G, et al. Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice. J Gen Virol. 1987, 68: 2291-2298
102 Wai J, Karaca K C, Parrish R, Naqi SA. A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch Virol. 1995, 140(2): 259-271
103 Wang C H, Hong C C, Seak J.C.H. An ELISA for antibodies against infectious bronchitis virus using an S1 spike polypeptide. Veterinary Microbiology. 2002, 85: 333-342
104 Wang X, Schnitzlein W M, Tripathy D N, et al. Construction and immunogenicity studies of recombinant fowl poxvirus containing the S 1 gene of Massachusetts 41 strain of infectious bronchitis virus. Avian Dis. 2002 ,46(4): 831-838
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