鸡γ干扰素基因的克隆和表达及其多克隆抗体的制备
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摘要
γ-干扰素(Interferon-γ，IFN-γ)主要由活化的T细胞及NK细胞产生，具有抗病毒作用，也具有上调MHCⅡ(major histocompatibility complex, MHC)类抗原分子的表达等作用，是影响机体细胞免疫和体液免疫反应的一类具有多种调节效应的细胞因子。
本试验根据国外已发表的序列，设计一对引物，应用逆转录-聚合酶链式反应(Reverse transcription-polymerase chain reaction，RT-PCR)技术，从刀豆素A(Concanavalin A，Con A)诱导培养24h的鸡脾淋巴细胞中扩增得到了大小约500bp的基因，将其连接到pUC19质粒上，经质粒PCR和EcoR Ⅰ、Hind Ⅲ双酶切鉴定后，筛选阳性重组克隆。序列分析表明，该基因阅读开放框架由492个核苷酸组成，与马和人IFN-γ基因序列的同源性分别为35％和32％，与鸡I型干扰素基因的同源性仅为15％，与国外报道的鸡IFN-γ基因序列完全一致。这表明鸡脾淋巴细胞受到有丝分裂原刺激后，其IFN-γ基因mRNA获得了表达。本试验成功克隆得到了鸡IFN-γ基因。
将克隆得到的鸡IFN-γ基因亚克隆到大肠杆菌原核表达载体pPROEX~(TM)HT的Pst I和KpnI位点上，构建重组表达质粒pPROEX~(TM)HT-IFN-γ。经序列分析鉴定，确证目的基因克隆到载体的预期位点。将重组质粒转化大肠杆菌DH5α，于37℃诱导培养8h，SDS-PAGE凝胶电泳表明该基因在大肠杆菌中获得了高水平表达，表达的鸡IFN-γ融合蛋白分子量约为22ku。该重组蛋白占菌体总蛋白的33％。经Western blot鉴定，确定该重组蛋白为包涵体，具有生物学活性。该包涵体被6M盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IFN-γ融合蛋白及其纯化产物经三次肌肉注射新西兰白兔，制备兔抗鸡IFN-γ多克隆抗体。琼脂扩散实验结果表明，多克隆抗体可与纯化的鸡IFN-γ重组蛋白发生特异性反应。
本次试验成功克隆、表达、纯化了鸡IFN-γ融合蛋白，并制备了兔抗鸡IFN-γ多克隆抗血清，可望用于鸡IFN-γ重组蛋白生物学特性及其应用的研究中。
Interferon- is an antiviral cytokine produced by activated T cell and NK cell. It upregulated MHC II antigen expression, influenced the cellular and humoral immune response and has pleiotropic regulatory effects on immune system.
One gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in splenocytes which was stimulated with ConA for 24 hours. The gene was cloned into pUC19 vector and identified with PCR and Eco RI + Hind III digestion. Then, one of the positive recombinant clone was sequenced and analysed. The result showed that its sequence was 35% and 32% identical to equine IFN- and human IFN- respectively, but it only shared 15% homology with chicken type I IFN. The sequence was just the same as the published chicken IFN- gene. So the chicken IFN- mRNA may be expressed in ConA-activated chicken splenocytes and the IFN- Y gene had been cloned from chicken splenocytes successfully.
The cDNA of chicken IFN- Y gene was subcloned into Pst I and Kpn I sites of pPROEX橦T vector to construct recombinant plasmid pPROEX橦T-IFN- . The recombinant plasmid was translated into E. coli DH5 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE and Western blot that a 22ku protein which was equal to chicken IFN- y protein in molecular weight was expressed in E.coli DH5 . This protein was purified and used to prepare the antibody against chicken IFN- y protein. The result showed that the antibody could react with purified recombinant chicken IFN- protein.
本试验根据国外已发表的序列，设计一对引物，应用逆转录-聚合酶链式反应(Reverse transcription-polymerase chain reaction，RT-PCR)技术，从刀豆素A(Concanavalin A，Con A)诱导培养24h的鸡脾淋巴细胞中扩增得到了大小约500bp的基因，将其连接到pUC19质粒上，经质粒PCR和EcoR Ⅰ、Hind Ⅲ双酶切鉴定后，筛选阳性重组克隆。序列分析表明，该基因阅读开放框架由492个核苷酸组成，与马和人IFN-γ基因序列的同源性分别为35％和32％，与鸡I型干扰素基因的同源性仅为15％，与国外报道的鸡IFN-γ基因序列完全一致。这表明鸡脾淋巴细胞受到有丝分裂原刺激后，其IFN-γ基因mRNA获得了表达。本试验成功克隆得到了鸡IFN-γ基因。
将克隆得到的鸡IFN-γ基因亚克隆到大肠杆菌原核表达载体pPROEX~(TM)HT的Pst I和KpnI位点上，构建重组表达质粒pPROEX~(TM)HT-IFN-γ。经序列分析鉴定，确证目的基因克隆到载体的预期位点。将重组质粒转化大肠杆菌DH5α，于37℃诱导培养8h，SDS-PAGE凝胶电泳表明该基因在大肠杆菌中获得了高水平表达，表达的鸡IFN-γ融合蛋白分子量约为22ku。该重组蛋白占菌体总蛋白的33％。经Western blot鉴定，确定该重组蛋白为包涵体，具有生物学活性。该包涵体被6M盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IFN-γ融合蛋白及其纯化产物经三次肌肉注射新西兰白兔，制备兔抗鸡IFN-γ多克隆抗体。琼脂扩散实验结果表明，多克隆抗体可与纯化的鸡IFN-γ重组蛋白发生特异性反应。
本次试验成功克隆、表达、纯化了鸡IFN-γ融合蛋白，并制备了兔抗鸡IFN-γ多克隆抗血清，可望用于鸡IFN-γ重组蛋白生物学特性及其应用的研究中。
Interferon- is an antiviral cytokine produced by activated T cell and NK cell. It upregulated MHC II antigen expression, influenced the cellular and humoral immune response and has pleiotropic regulatory effects on immune system.
One gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in splenocytes which was stimulated with ConA for 24 hours. The gene was cloned into pUC19 vector and identified with PCR and Eco RI + Hind III digestion. Then, one of the positive recombinant clone was sequenced and analysed. The result showed that its sequence was 35% and 32% identical to equine IFN- and human IFN- respectively, but it only shared 15% homology with chicken type I IFN. The sequence was just the same as the published chicken IFN- gene. So the chicken IFN- mRNA may be expressed in ConA-activated chicken splenocytes and the IFN- Y gene had been cloned from chicken splenocytes successfully.
The cDNA of chicken IFN- Y gene was subcloned into Pst I and Kpn I sites of pPROEX橦T vector to construct recombinant plasmid pPROEX橦T-IFN- . The recombinant plasmid was translated into E. coli DH5 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE and Western blot that a 22ku protein which was equal to chicken IFN- y protein in molecular weight was expressed in E.coli DH5 . This protein was purified and used to prepare the antibody against chicken IFN- y protein. The result showed that the antibody could react with purified recombinant chicken IFN- protein.
引文
1 龚非力主编．医学免疫学[M]．科学出版社，1998
2 金冬雁等译．分子克隆实验指南(第二版)[M]．科学出版社，1992
3 李连奋．免疫佐剂的发展概况．微生物学免疫学进展，1997，25(2)：57-61
4 林学颜、张玲主编．现代细胞与分子免疫学[M]．科学出版社
5 刘胜旺、孔宪刚、陈洪岩等．应用反转录聚合链式反应检测鸡Th1样淋巴因子mRNA表达。中国预防兽医学报，2000，22：48-52
6 柳钟勋．新型免疫佐剂在疫苗中的应用．中华微生物学和免疫学杂志，1997，17(9)：337-341
7 孙卫民、王惠琴编著．细胞因子方法学[M]．人民卫生出版社，1999
8 王明俊．免疫佐剂．兽医生物制品，1987，137
9 许剑琴．创造新世纪中兽医学[M]．北京：中国农业大学出版社，1998
10 Albina J E,Abate J A,et al,Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation [J].The Journal of Immunology,1991,147:144-148
11 Bao S,Beagley K W,Interferon-γ plays a critical role in intestinal immunity against Salmonella typhimurium infection[J].Immunology,2000,99:464-472
12 Benencia F,Courreges M C,Nitric oxide and macrophage antiviral extrinsic activity [J].Immunology,1999,98:363-370
13 Bernasconi B D,Schultz U & Staeheli P,The interferon-induced Mx protein of chicken lacks antiviral activity.J Interferon Cytokine Res.,1995,15:47-53
14 Conway G.The doubly green revolution:food for the 21st century [M].Cornell university press,New York,USA,1998.
15 Digby M R,Lowenthal J W.Cloning and expression of the chicken interferon-γ gene[J].J Interferon Res,1995,15:939-945
16 Dijkmans R,Creemers J & Billiau A,Chicken macrophage activation by interferon:do birds lack the molecular homologue of mammalian interferon-gamma [J].Vet Immunol Immunopathol,1990,26:319-332
17 Dimier I H,Inhibition of Eimeria tenella development in vitro mediated by chicken macrophages and Fibroblasts treated with chicken cell supematants with IFN-γ activity [J].Avian Dis,1998,42:239-247
18 Dimier I H,Mechanisms of the Eimeria tenella Growth Inhibitory activity induced by concanavalin A and reticuloendotheliosis virus supernatants with interferon gamma activity in chicken macrophage and fibroblasts [J].Avian Dis,1999,74:65-74
19 Djeraba A,Bernardet N,et al,Nitric oxide inhibits Marek's disease virus replication but is not the sigle decisive factor in interferon-γ-mediated viral inhibition [J].Virology,2000,277:58-65
20 Dowkins K,Gene wars:the politics of biotechnology [M].Seven stories press,New York,USA,1997.
21 Eals S A,Schindler C,Leonard D,Fu X Y,Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding protein [J].Mol Cell Biol.1992,12:3315-3324
22 Estes D M,Brown W C,et al,CD40 ligand-dependent signaling of bovine B lymphocyte development and differentiation [J].Vet.Immunol.Immunopathol,1998,63:15-20
23 Famell M B,Halavani M E,et al,In vivo biologic effects of recombinant-turkey interferon-gamma in neonatal leghorn chicks:protection against salmonella enteritidis organ invasion[J].Avian Diseases,2001,45:473-478
24 Finkelman F D,Katona I M.IFN-γ regulates the isotypes of Ig secreted during in vivo humoral immune responses [J].J Immunol,1988,140:1022-1027
25 Gulig P A,Doyle T J,et al,Systemic infection of mice by wild-type but not spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha[J].Infection and Immunity.1997,65:5191-5197
26 Heath A W,Playfair J H L,Conjugation of interferon-gamma to antigen enhances its adjuvanticity [J].Immunology,1990,71:454-456
27 Heath A H,Playfair J H L,Cytokines as immunological adjuvants [J].Vaccine,1992,10:432-434
28 Heller E D,Levy A M,et al,Chicken-embryo fibroblasts produce two typys of interferon upon stimulation with Newcastle disease virus [J].Vet.Immunol.Immunopathol,1997,57:289-303
29 Hung-yueh yeh,Barbara J W,et al.In vitro effects of recombinant chickens interferon-γ on immune cells[J].Interferon and Cytokine Res,1999,19:687-691
30 Hussain I,Qureshi M.A.The expression and regulation of inducible nitric oxide synthase gene differ in macrophages from chickens of different genetic background [J].Vet.Immunol.Immunopathol,1998,61:317-329
31 Issacs A and Lindanmann J.Proc.Roy.Sco.B,1957,145:258-267
32 Kaiser P,Sonnemans D,et al.Avian IFN-γ genes:sequence analysis suggests probable cross-species reactivity among galliforms[J].J Interferon and Cytokine Res,1998,18:711-719
33 Kaiser P,Wain H M and Rothweli L.Structure of the chicken interferon-γ gene and comparison to mammalian homologues [J].Gene,1998,207:25-32
34 Karupiah G,Xie Qiao-wen,et al,Inhibition of viral replication by interferon-γ-induced Nitric oxide synthase [J].Science,1993,261:1445-1448
35 Kaspers B,Lillehoj H S,et al,Chicken interferon-mediated induction of major histocompatibility complex class Ⅱ antigens on peripheral blood monocytes [J].Vet.Immunol.Immunopathol,1994,44:71-84
36 Kim Y,Son K,A nitric oxide production bioassay for interferon-γ [J].Journal of Immunological methods.1996,198:203-209
37 Kohase M,Moriya H,et al,Purification and characterization of chick interferon induced by virus [J].J gen virol.1986,67:215-218
38 Lambrecht B,Gonze M,et al,Comparison of biological activities of natural and recombinant chicken interferon-gamma [J].Vet.Immunol.Immunopatho.1999,70:257-267
39 Leong K H,Ramsay A J,et al,Selective induction of immune responses by cytokines coexpressed in recombinant fowlpox virus [J].Journal of Virology.1994,68:8125-8130
40 Levy A M,Heller E D,Effect of native chicken interferon on MDV replication [J].Acta Virological,1999,43:121-127
41 Lillehoj H S,Choi K D,Recombinant chicken interferon-gamma-mediated inhibition of Eimeria tenella development in vitro and reduction of oocyst production and body weight loss following eimeria acervulina challenge infection [J].Avian Diseases,1998,42:307-314
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2 金冬雁等译．分子克隆实验指南(第二版)[M]．科学出版社，1992
3 李连奋．免疫佐剂的发展概况．微生物学免疫学进展，1997，25(2)：57-61
4 林学颜、张玲主编．现代细胞与分子免疫学[M]．科学出版社
5 刘胜旺、孔宪刚、陈洪岩等．应用反转录聚合链式反应检测鸡Th1样淋巴因子mRNA表达。中国预防兽医学报，2000，22：48-52
6 柳钟勋．新型免疫佐剂在疫苗中的应用．中华微生物学和免疫学杂志，1997，17(9)：337-341
7 孙卫民、王惠琴编著．细胞因子方法学[M]．人民卫生出版社，1999
8 王明俊．免疫佐剂．兽医生物制品，1987，137
9 许剑琴．创造新世纪中兽医学[M]．北京：中国农业大学出版社，1998
10 Albina J E,Abate J A,et al,Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation [J].The Journal of Immunology,1991,147:144-148
11 Bao S,Beagley K W,Interferon-γ plays a critical role in intestinal immunity against Salmonella typhimurium infection[J].Immunology,2000,99:464-472
12 Benencia F,Courreges M C,Nitric oxide and macrophage antiviral extrinsic activity [J].Immunology,1999,98:363-370
13 Bernasconi B D,Schultz U & Staeheli P,The interferon-induced Mx protein of chicken lacks antiviral activity.J Interferon Cytokine Res.,1995,15:47-53
14 Conway G.The doubly green revolution:food for the 21st century [M].Cornell university press,New York,USA,1998.
15 Digby M R,Lowenthal J W.Cloning and expression of the chicken interferon-γ gene[J].J Interferon Res,1995,15:939-945
16 Dijkmans R,Creemers J & Billiau A,Chicken macrophage activation by interferon:do birds lack the molecular homologue of mammalian interferon-gamma [J].Vet Immunol Immunopathol,1990,26:319-332
17 Dimier I H,Inhibition of Eimeria tenella development in vitro mediated by chicken macrophages and Fibroblasts treated with chicken cell supematants with IFN-γ activity [J].Avian Dis,1998,42:239-247
18 Dimier I H,Mechanisms of the Eimeria tenella Growth Inhibitory activity induced by concanavalin A and reticuloendotheliosis virus supernatants with interferon gamma activity in chicken macrophage and fibroblasts [J].Avian Dis,1999,74:65-74
19 Djeraba A,Bernardet N,et al,Nitric oxide inhibits Marek's disease virus replication but is not the sigle decisive factor in interferon-γ-mediated viral inhibition [J].Virology,2000,277:58-65
20 Dowkins K,Gene wars:the politics of biotechnology [M].Seven stories press,New York,USA,1997.
21 Eals S A,Schindler C,Leonard D,Fu X Y,Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding protein [J].Mol Cell Biol.1992,12:3315-3324
22 Estes D M,Brown W C,et al,CD40 ligand-dependent signaling of bovine B lymphocyte development and differentiation [J].Vet.Immunol.Immunopathol,1998,63:15-20
23 Famell M B,Halavani M E,et al,In vivo biologic effects of recombinant-turkey interferon-gamma in neonatal leghorn chicks:protection against salmonella enteritidis organ invasion[J].Avian Diseases,2001,45:473-478
24 Finkelman F D,Katona I M.IFN-γ regulates the isotypes of Ig secreted during in vivo humoral immune responses [J].J Immunol,1988,140:1022-1027
25 Gulig P A,Doyle T J,et al,Systemic infection of mice by wild-type but not spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha[J].Infection and Immunity.1997,65:5191-5197
26 Heath A W,Playfair J H L,Conjugation of interferon-gamma to antigen enhances its adjuvanticity [J].Immunology,1990,71:454-456
27 Heath A H,Playfair J H L,Cytokines as immunological adjuvants [J].Vaccine,1992,10:432-434
28 Heller E D,Levy A M,et al,Chicken-embryo fibroblasts produce two typys of interferon upon stimulation with Newcastle disease virus [J].Vet.Immunol.Immunopathol,1997,57:289-303
29 Hung-yueh yeh,Barbara J W,et al.In vitro effects of recombinant chickens interferon-γ on immune cells[J].Interferon and Cytokine Res,1999,19:687-691
30 Hussain I,Qureshi M.A.The expression and regulation of inducible nitric oxide synthase gene differ in macrophages from chickens of different genetic background [J].Vet.Immunol.Immunopathol,1998,61:317-329
31 Issacs A and Lindanmann J.Proc.Roy.Sco.B,1957,145:258-267
32 Kaiser P,Sonnemans D,et al.Avian IFN-γ genes:sequence analysis suggests probable cross-species reactivity among galliforms[J].J Interferon and Cytokine Res,1998,18:711-719
33 Kaiser P,Wain H M and Rothweli L.Structure of the chicken interferon-γ gene and comparison to mammalian homologues [J].Gene,1998,207:25-32
34 Karupiah G,Xie Qiao-wen,et al,Inhibition of viral replication by interferon-γ-induced Nitric oxide synthase [J].Science,1993,261:1445-1448
35 Kaspers B,Lillehoj H S,et al,Chicken interferon-mediated induction of major histocompatibility complex class Ⅱ antigens on peripheral blood monocytes [J].Vet.Immunol.Immunopathol,1994,44:71-84
36 Kim Y,Son K,A nitric oxide production bioassay for interferon-γ [J].Journal of Immunological methods.1996,198:203-209
37 Kohase M,Moriya H,et al,Purification and characterization of chick interferon induced by virus [J].J gen virol.1986,67:215-218
38 Lambrecht B,Gonze M,et al,Comparison of biological activities of natural and recombinant chicken interferon-gamma [J].Vet.Immunol.Immunopatho.1999,70:257-267
39 Leong K H,Ramsay A J,et al,Selective induction of immune responses by cytokines coexpressed in recombinant fowlpox virus [J].Journal of Virology.1994,68:8125-8130
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