鸡白细胞介素6基因的克隆及对新城疫LaSota疫苗的免疫增强作用研究
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摘要
根据GenBank中鸡白介素6(ChIL-6)基因序列(AJ309540)设计一对引物，采用RT-PCR方法从ConA诱导的四川雅安土鸡脾淋巴细胞总RNA中扩增出约600bp的基因片段，克隆到pMD18-T载体，进行序列测定和分析。结果显示：该克隆的cDNA全长603bp，包含了鸡IL-6基因的开放阅读框585bp，编码195个氨基酸残基，与GenBank上仅有的1条鸡IL-6序列(AJ309540)的同源性为100％。生物信息学分析表明，该基因编码的蛋白具有亲水性，有很强的抗原性，有3个蛋白激酶C磷酸化位点，2个酪蛋白激酶Ⅱ磷酸化位点。该基因经Xho I和Xba I双酶切后，插入同样经Xho I和Xba I双酶切真核表达载体pCI-neo中，构建出真核表达质粒pCI-IL-6，通过免疫试验和攻毒试验观察其对新城疫LaSota疫苗的免疫增强作用。
将21日龄非免疫健康雏鸡100只随机分为5组，肌肉免疫(LaSota疫苗组、pCI-IL-6+LaSota疫苗组、pCI-IL-6组、pCI-neo组和PBS组)。通过检测HI抗体、CD4~+、CD8~+和CD3~+T淋巴细胞百分含量，分析pCI-IL-6对LaSota疫苗的免疫佐剂效应。各组试验鸡在免疫接种后的不同时期，用血凝抑制试验(HI)测定NDV抗体，结果表明，pCI-IL-6和LaSota疫苗联合免疫组从第14天起就一直高于LaSota疫苗单独免疫组，在第14、21、35天时差异显著(P<0．05)，在28天时差异极显著(P<0．01)；pCI-IL-6和LaSota疫苗联合免疫组与其它试验组比较差异均极显著(P<0．01)。用流式细胞仪检测外周血代表T细胞亚群的CD4~+、CD8~+和CD3~+。CD4~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组与LaSota疫苗单独免疫组相比，在14天出现差异显著(P<0．05)，在21、28天差异极显著(P<0．01)；pCI-IL-6+LaSota疫苗与其它试验组比较均差异极显著(P<0．01)。CD8~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组和LaSota疫苗单独免疫组相比，在免疫后14天出现差异极显著(P<0．01)，第28天差异显著(P<0．05)；14天时与其它试验组比较均差异极显著(P<0．01)。CD3~+T淋巴细胞百分含量测定结果表明，LaSota疫苗联合佐剂pCI-IL-6组与单独LaSota疫苗组比较在7、28、35天差异显著(P<0．05)，在14、21天时差异极显著(P<0．01)；与其它试验组比较均差异极显著(P<0．01)。在免疫后35天进行攻毒，pCI-IL-6和LaSota疫苗联合免疫组的保护率为89．5％，而LaSota疫苗单独免疫组的保护率为76．5％。表明，pCI-IL-6能提高新城疫LaSota疫苗的免疫效果，能提高新城疫疫苗免疫的细胞和体液免疫水平及攻毒保护率。鸡IL-6真核质粒免疫佐剂效应的研究国内外尚未见报道。
Two primers were designed and synthesized according to the chicken interleukin-6 sequence published in GenBank (AJ309540). The Gallus interleukin-6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from splenocytes lymphocytes stimulated with ConA. The gene was inserted into pMD18-T vector and sequenced. The result showed that the length of this cDNA was 603bp, encoding 195 amino acids, and sharing 100% homology with the only one ChIL-6 sequnce (AJ309540) in GenBank. Bioinformatics analysis of this sequence shows that the protein of ChIL-6 was hydrophilic and highly antigenic, including three potential protein kinase C phosphor -ylation site, six potential Casein kinaseⅡphosphorylation site. The cDNA was digested with Xho /and XbaⅠand inserted into pCI-neo vector which was also digested with Xho /and Xba /to construct the eukaryotic expression vector pCI-IL-6. Then an immune experiment was designed to evaluate the immune adjuvant effect of pCI-IL-6.
One hundred chickens were randomly assigned to five groups of twenty animals per treatment, including LaSota vaccine group, pCI-IL-6+LaSota vaccine group, pCI-IL-6 group, pCI-neo group and PBS control group. The immune adjuvant effect of the pCI-IL-6 to LaSota had been analyzed using the Hammaeglutination test (HI) antibody and the percentage of the CD4~+, CD8~+ and CD3~+T lymphocyte.The results shows that HI antibody of pCI-IL-6+LaSota vaccine group shows notable difference in 14,21, 35day (P<0.05) with LaSota vaccine group, shows extremely notable difference(P<0.01) in 28 days, HI antibody of pCI-IL-6+LaSota vaccine group shows extremely notable difference (P<0.01)with other treatments. The percentage of CD4~+, CD8~+ and CD3~+T lymphocyte was tested by flow cytometer technique, the results shows that the percentage of CD4~+T lymphocyte of pCI-IL-6 +LaSota vaccine group shows shows notable difference and extremely notable difference in 14 day (P<0.05) and 21, 28day (P<0.01), pCI-IL-6+LaSota vaccine group shows extremely notable difference in all days (P<0.01) with other groups. The percentage of CD8~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14 day (P<0.01) with LaSota vaccine group, shows notable difference in the 28 day (P<0.05). The percentage of CD3~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14, 21day (P<0.01) with LaSota vaccine group, shows notable difference in the 7, 28, 35day (P<0.05). Challenge test on 35th day after immunization shows that the protection ratio of co-immunization group of pCI-IL-6 and LaSota was 89.5%, while LaSota vaccine group was only 76.5%.
The results indicated that pCI-IL-6 can improve the NDV immunity level of the humoral and cellualrimmunity as well as the challenge protection rate, showing immune adjuvant function. As we know, the immune adjuvant effect of eukaryotic plasmid of the ChIL-6 to LaSota vaccine hasn't been reported now, this study provides referrence to the united application of pCI-IL-6 and NDV vaccine.
将21日龄非免疫健康雏鸡100只随机分为5组，肌肉免疫(LaSota疫苗组、pCI-IL-6+LaSota疫苗组、pCI-IL-6组、pCI-neo组和PBS组)。通过检测HI抗体、CD4~+、CD8~+和CD3~+T淋巴细胞百分含量，分析pCI-IL-6对LaSota疫苗的免疫佐剂效应。各组试验鸡在免疫接种后的不同时期，用血凝抑制试验(HI)测定NDV抗体，结果表明，pCI-IL-6和LaSota疫苗联合免疫组从第14天起就一直高于LaSota疫苗单独免疫组，在第14、21、35天时差异显著(P<0．05)，在28天时差异极显著(P<0．01)；pCI-IL-6和LaSota疫苗联合免疫组与其它试验组比较差异均极显著(P<0．01)。用流式细胞仪检测外周血代表T细胞亚群的CD4~+、CD8~+和CD3~+。CD4~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组与LaSota疫苗单独免疫组相比，在14天出现差异显著(P<0．05)，在21、28天差异极显著(P<0．01)；pCI-IL-6+LaSota疫苗与其它试验组比较均差异极显著(P<0．01)。CD8~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组和LaSota疫苗单独免疫组相比，在免疫后14天出现差异极显著(P<0．01)，第28天差异显著(P<0．05)；14天时与其它试验组比较均差异极显著(P<0．01)。CD3~+T淋巴细胞百分含量测定结果表明，LaSota疫苗联合佐剂pCI-IL-6组与单独LaSota疫苗组比较在7、28、35天差异显著(P<0．05)，在14、21天时差异极显著(P<0．01)；与其它试验组比较均差异极显著(P<0．01)。在免疫后35天进行攻毒，pCI-IL-6和LaSota疫苗联合免疫组的保护率为89．5％，而LaSota疫苗单独免疫组的保护率为76．5％。表明，pCI-IL-6能提高新城疫LaSota疫苗的免疫效果，能提高新城疫疫苗免疫的细胞和体液免疫水平及攻毒保护率。鸡IL-6真核质粒免疫佐剂效应的研究国内外尚未见报道。
Two primers were designed and synthesized according to the chicken interleukin-6 sequence published in GenBank (AJ309540). The Gallus interleukin-6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from splenocytes lymphocytes stimulated with ConA. The gene was inserted into pMD18-T vector and sequenced. The result showed that the length of this cDNA was 603bp, encoding 195 amino acids, and sharing 100% homology with the only one ChIL-6 sequnce (AJ309540) in GenBank. Bioinformatics analysis of this sequence shows that the protein of ChIL-6 was hydrophilic and highly antigenic, including three potential protein kinase C phosphor -ylation site, six potential Casein kinaseⅡphosphorylation site. The cDNA was digested with Xho /and XbaⅠand inserted into pCI-neo vector which was also digested with Xho /and Xba /to construct the eukaryotic expression vector pCI-IL-6. Then an immune experiment was designed to evaluate the immune adjuvant effect of pCI-IL-6.
One hundred chickens were randomly assigned to five groups of twenty animals per treatment, including LaSota vaccine group, pCI-IL-6+LaSota vaccine group, pCI-IL-6 group, pCI-neo group and PBS control group. The immune adjuvant effect of the pCI-IL-6 to LaSota had been analyzed using the Hammaeglutination test (HI) antibody and the percentage of the CD4~+, CD8~+ and CD3~+T lymphocyte.The results shows that HI antibody of pCI-IL-6+LaSota vaccine group shows notable difference in 14,21, 35day (P<0.05) with LaSota vaccine group, shows extremely notable difference(P<0.01) in 28 days, HI antibody of pCI-IL-6+LaSota vaccine group shows extremely notable difference (P<0.01)with other treatments. The percentage of CD4~+, CD8~+ and CD3~+T lymphocyte was tested by flow cytometer technique, the results shows that the percentage of CD4~+T lymphocyte of pCI-IL-6 +LaSota vaccine group shows shows notable difference and extremely notable difference in 14 day (P<0.05) and 21, 28day (P<0.01), pCI-IL-6+LaSota vaccine group shows extremely notable difference in all days (P<0.01) with other groups. The percentage of CD8~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14 day (P<0.01) with LaSota vaccine group, shows notable difference in the 28 day (P<0.05). The percentage of CD3~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14, 21day (P<0.01) with LaSota vaccine group, shows notable difference in the 7, 28, 35day (P<0.05). Challenge test on 35th day after immunization shows that the protection ratio of co-immunization group of pCI-IL-6 and LaSota was 89.5%, while LaSota vaccine group was only 76.5%.
The results indicated that pCI-IL-6 can improve the NDV immunity level of the humoral and cellualrimmunity as well as the challenge protection rate, showing immune adjuvant function. As we know, the immune adjuvant effect of eukaryotic plasmid of the ChIL-6 to LaSota vaccine hasn't been reported now, this study provides referrence to the united application of pCI-IL-6 and NDV vaccine.
引文
[1]Calnek B W, John B H, Charles B W, et al. Disease of poultry (Tenth edition)[M]. Ames: Lowastate University press, 1997,541-562.
[2]刘伟忠．新城疫病毒F48E8株血凝素-神经氨酸酶基因的克隆与鉴定[J]．中国兽医科技,1997， 27(6)：16-18．
[3]陈溥言．兽医传染病学(第五版)[M]．北京：中国农业出版社,2006：345-351．
[4]Toyolda T, Sakaguchi T, Hirota H, et al. Newcastle disease virus evolution Ⅱ. Lack of generecombination in generating virulent and avirulent strains[J]. Virology, 1989,169:273-282.
[5]Ballagi-Pordany A, Wehmann E H, Herczeg J, et al. Identification and grouping of Newcastledisease virus strains by restriction site analysis of a region from the F gene[J]. Archives of virology,1996,141:243-261.
[6]Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in WesternEurope were caused by an old (Ⅵ) and a novel genotype (Ⅶ) [J]. Arch Virol, 1998,143(1):49-64.
[7]Yang Cheng-Yao, Shieh Happy K, Lin Yeou-Bang, et al. Newcastle disease virus isolated fromrecent outbreaks in Taiwan phylogenetically related to viruses (genotype Ⅶ) from recent outbreaksin Westrern Europe[J]. Avian Disease, 1999, (43): 125-130.
[8]Herczeg J, Wehmann E, Bragg R R, Two novel genetic groups (Ⅶb and Ⅷ) responsible for recentNewcastle disease outbreaks in southern Africa. One (Ⅶb) of which reached southern Europe[J].Archives of virology, 1999,144:2087-2099.
[9]严维巍，王永刊，田慧芳等．一株鸡副粘病毒的分子特性研究[J]．扬州大学学报(自然科学 版)，2000,3(1)：27-31．
[10]刘华雷，王永刊，严维巍等．中国部分地区新城疫病毒的分子流行病学研究[J]．扬州大学学报(自 然科学版)，2001，4(1)：35-40．
[11]吴艳涛，倪雪霞，刘其全等．我国部分地区不同动物来源新城疫病毒的分子流行病学研究[J]．病 毒学报,2002,18(3)：264-269．
[12]尹燕博，龚振华，孙承英等．35株不同来源的新城疫病毒(NDV)分离株F-糖蛋白基因序列测定 和基因型研究[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,2002,10：26-30．
[13]杨润德，梅双．新城疫病毒野毒株(HBG-1)的F蛋白部分序列同源性分析[J]．中国预防兽医学 报,2003,35(2)：188-190．
[14]田夫林，陈静，马惠玲等．10株新城疫分离F基因的克隆及遗传变异分析[J]．中国预防兽医学 报,2004,26(1)：28-31．
[15]刘文斌，崔尚金，刘立奎等．东北地区不同宿主NDV分离株的系统发育分析[J]．中国兽医杂 志,2005,4 1(7)：3-6．
[16]秦卓明，马保臣，何叶峰等．新城疫病毒HN和F基因遗传变异相关性的研究[J]．微生物学??报，2006，46(2)：227-232．
[17]Balkwill, E. R&Burke E. The cytokine network [J].Immunology Today, 1989,10:299-304.
[18]Ari. L., Lee.E et al. cytokine: coordinators of immune and inflammatory responses. Annual Reviewof Biochemistry, 59: 783-836.
[19]Peter S, Florian P, Kirsten S, et al.Cytokines of birds: conserved of functions a largely differentlook(review)[J].Journal of Interferon and Cytokine Research, 2001,21:993-1010.
[20]Issaes A,Lindanmann. Virus interference I. the interferon[J]. Prne Roy Sce B, 1957(145): 258-267.
[21]吴志光，汪明等．禽类干扰素分子分子生物学研究进展[J]．中国兽医杂志，2001，37(912)：31-33．
[22]Sekellick M J, Ferrandino A F, Hopkins DA, et al. chicken interferon gene:cloning .expressionand analysis[J].Journal of Interferon Research, 1994, 14:71-79.
[23]Schijm V E,Weining K C.Nuijten P et al.Immunoadjuvant activities of E.coli andplasmid-expressed recombinant chicken IFN-α/β、 IFN-γ and IL-1βin 1-day and 3-week-oldchickens[J], Vaccine, 2000,18:2147-2154.
[24]夏春，汪明等．惠阳胡须鸡干扰素基因分子克隆与序列分析[J]．畜牧兽医学报，2000，31(6)： 563-566．
[25]Plachy J, Weining KC, Krexnmer MJ, et al. Protective effects of type I and type II interferonstoward Rous Sarcoma Virus-induced tumors in chicken[J]. Vimlogy, 1999,256,85-91.
[26]Marcus PI, Heide VD, Sekellick MJ. Interferon action oD Avian vriruses Oral administration ofchicken interferon-a Ameliorates Newcasde Disease [J]. Journal of Interferon and CytokinesResearch, 1999,19: 881-885.
[27]Dimier IH, Ouere P, Naciri M, et al. Inhibition of Eimeria tenella development in vitro mediated bychicken macrophages and fibroblasts treated with chicken cell supernatants with IFNγ activity [J].Avian Disease, 1998,42:239-247.
[28]Lowenthal JW, Onel TE, Broadway M, et al. Coadministration of IFNγ enhancers antibodyresponses in chickens [J]. Journal of Interferon and Cytokine Research, 1998,18: 617-622.
[29]贾立军，刘秀梵等．鸡γ干扰素对H5亚型禽流感疫苗的免疫增强作用[J]．农业生物技术学 报，2004，12(4)：427-430．
[30]胡慧琼，王红宁等．山地乌骨鸡IL-2、IL-15、IL-18及pDNAIFN真核表达载体构建及其对IBV DNA疫苗免疫增强作用研究[J]．高技术通讯，2006，10(16)：1057-1062．
[31]李玉峰，吴静等．重组鸡α-干扰素对鸡外周血T淋巴细胞亚类的影响[J]．山东农业科 学，2006，4(67)：67-69．
[32]Sundick R S, Gill-Dixon C. A cloned chicken lymphokine homologous to both mammalian IL-2and IL-15 [J]. J Immunol, 1997,159(2): 720-725.
[33]Thiagarajan D, Ram G C, Bansal M P. Optimum conditions for in vitro chicken IL-2 productionand its in vivo role in Newcastle disease vaccinated chickens [J]. Veterinary Immunolgy and??Immunopathology, 1999,67(1): 79-91.
[34]Choi K D, Lilehoj H S. Role of chicken IL-2 on γδ T cells and Eimeria acervulina-induced changesin intestinal IL-2 mRNA expression and γδ T cells [J]. Vet Immunol Immunopathol, 2000, 73:309-321.
[35]Lowenthal J W, Lambrecht B, Van den Berg T P, et al. Avian cytokine-the natural approach totherapeutics [J]. Development and Comparative Immunology, 2002,24:355-365.
[36]Lillehoj HS, Min W, Choi KD, et al. Molecular, cellular, and functional characterization of chickencytokines homologous to mammalian IL-15 and IL-2 [J]. Vet Immunol Immunopathol. 2001,82(3-4):229-44.
[37]徐前明，李培英等．鸡白介素-2对柔嫩艾美耳球虫亚单位疫苗免疫增强作用的研究[J]．安徽农业 科学,2005,33(2)：290-293
[38]胡慧琼，王红宁等．鸡IL-2和IL-15基因真核表达质粒对禽流感疫苗的免疫增强作用[J]．中国兽 医科学,2006,36(10)：805-810．
[39]Kirsten Schneider, Florian Puehler. cDNA Cloning of biologically active chicken interleukin -18 [J].Journal of Interferon and Cytokine Research, 2000,20: 879-883.
[40]Degen WG, van Zuilekom HI, Scholtes NC, et al. Potentiation of humoral immune responses tovaccine antigens by recombinant chicken IL-18 (rChIL-18) [J]. Vaccine, 2005,23(33): 4212-4218
[41]韩宗玺．鸡白细胞介素18成熟蛋白的表达及其部分免疫功能研究[J]．新疆农业大学硕士论 文,2004．
[42]王振国，金宁一等．共表达H5N1、H7N1亚型AIV HA与鸡IL-18多价重组鸡痘病毒免疫保护性 研究[J]．中国病毒学,2005,20(6)：607-612．
[43]Louise S H, Andrew G D B, John W L, et al. The emerging role of avian cytokines as immunotherapcuties and vaccine adjuvants [J]. Vet Immunol Immunopathol.2002,85:119-128.
[44]金伯泉．细胞和分子免疫学(第二版)[M]．北京：科学出版社,2001,144．
[45]秦云龙，金宁一，罗坤等．中国流行株HIV-lgag和Hil-2／Hil-6核酸疫苗构建与实验免疫研究[J]． 免疫学杂志,2001,1，17(1)：5-8．
[46]郭炎，金宁一，张应玖等．共表达HIV-1与IL-6核酸疫苗质粒诱导小鼠免疫原性的研究[J]．中国 微生物学和免疫学杂志'2002,3,22：174．
[47]汪文正，金宁一，李子建等．共表达HIV-1 gag-gp120与IL-6重组鸡痘病毒和核酸疫苗的联合免疫 研究[J]．中华微生物学和免疫学杂志,2004,3,24(3)：206-208．
[48]Schneider K, Klaas R, Kaspers B, et al. Chicken interleukin-6 cDNA structure and biological properties [J]. Eur.J.Biochem, 2001,268:4200-4206.
[49]孙建和，陆苹，蒋静．鸡白介素-6(ChIL-6)基因的克隆与序列分析[J]．华中农业大学学 报,2003,22(1)：1-3.
[50]Nishimichi N, Aosasa M, Kawashima T. et a!. Generation of a mouse monoclonal antibody against??chicken interleukin-6 [J]. Hybridoma. 2005,24(2): 115-7.
[51]Nishimichi N, Aosasa M, Kawashima T. et al. Biological activity of recombinant chickeninterleukin-6 in chicken hybridoma cells [J]. Vet Immunol Immunopathol. 2005,106(1-2):97-105.
[52]Alexander B, Lgor M B. Expression of immunomodulating molecules by recombinant viruses: Canthe immunogenicity of live virus vaccines be improved ? [J]. Expert Rrv Vaccine, 2002,1:233-245.
[53]Derek T O, Mary L M. Manmohan S Recent developments in adjuvants for vaccines againstinfectious diseases [J]. Biom olecular Engineering, 2001,8:69-85.
[54]杜平．，细胞素抗癌疗法[M]．上海：上海科技出版社,1992：218-228．
[55]萨姆布鲁克J，拉塞尔D W．分子克隆实验指南[M]．黄培堂，王嘉玺，朱厚础等译．第3版．北京：科学 出版社,2002：87-93．
[56]L．戴维斯等编著．分子生物学试验技术[M]．姚志建，沈倍奋，刘亚霞等译．北京：科学出版社,1990．
[57]萨姆布鲁克J，拉塞尔D W．分子克隆实验指南[M]．黄培堂，王嘉玺，朱厚础等译．第3版．北京：科学 出版社,2002：27-29．
[58]谢振华，史小军，蔡国平.用于哺乳动物细胞转染的高纯度质粒DNA的制备[J]．中国科技论文在 线http：//www.paper.edu．cn
[59]Schmidt, EV, Christoph G, Zeller R, et al. The cytomegalovirus enhancer: a pan-active controlelement in transgenic mice. Mol. Cell [J]. Biol, 1990,10:4406-4411.
[60]Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants [J].Cell,1981,23:175-82.
[61]Shih WL, Kuo ML, Cheuang SE, et al. Hepatitis B virus x protein inhibits transforming growthfactor beta-induced apoptosis through the activation of phosphatidy linositol 3-kinase pathway [J].Biol Chem, 2000,275(33): 25 858-25 864.
[62]Braun R P, Babuk L A, Van Draunen. Enhanced immune response to an intradermally deliveredDNA vaccine expressing a secreted from of BHV-IgD [J]. Vaccine, 1997,6:151-164.
[63]左边富．流式细胞术与生物医学[M]．沈阳：辽宁人民出版社，1996：129-422．
[64]程相朝，赵德明，吴庭才等．鸡IL-18真核表达载体的构建及其对新城疫疫苗免疫增强作用的研 究[J]．畜牧兽医学报,2005,36(5)：476-481．
[2]刘伟忠．新城疫病毒F48E8株血凝素-神经氨酸酶基因的克隆与鉴定[J]．中国兽医科技,1997， 27(6)：16-18．
[3]陈溥言．兽医传染病学(第五版)[M]．北京：中国农业出版社,2006：345-351．
[4]Toyolda T, Sakaguchi T, Hirota H, et al. Newcastle disease virus evolution Ⅱ. Lack of generecombination in generating virulent and avirulent strains[J]. Virology, 1989,169:273-282.
[5]Ballagi-Pordany A, Wehmann E H, Herczeg J, et al. Identification and grouping of Newcastledisease virus strains by restriction site analysis of a region from the F gene[J]. Archives of virology,1996,141:243-261.
[6]Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in WesternEurope were caused by an old (Ⅵ) and a novel genotype (Ⅶ) [J]. Arch Virol, 1998,143(1):49-64.
[7]Yang Cheng-Yao, Shieh Happy K, Lin Yeou-Bang, et al. Newcastle disease virus isolated fromrecent outbreaks in Taiwan phylogenetically related to viruses (genotype Ⅶ) from recent outbreaksin Westrern Europe[J]. Avian Disease, 1999, (43): 125-130.
[8]Herczeg J, Wehmann E, Bragg R R, Two novel genetic groups (Ⅶb and Ⅷ) responsible for recentNewcastle disease outbreaks in southern Africa. One (Ⅶb) of which reached southern Europe[J].Archives of virology, 1999,144:2087-2099.
[9]严维巍，王永刊，田慧芳等．一株鸡副粘病毒的分子特性研究[J]．扬州大学学报(自然科学 版)，2000,3(1)：27-31．
[10]刘华雷，王永刊，严维巍等．中国部分地区新城疫病毒的分子流行病学研究[J]．扬州大学学报(自 然科学版)，2001，4(1)：35-40．
[11]吴艳涛，倪雪霞，刘其全等．我国部分地区不同动物来源新城疫病毒的分子流行病学研究[J]．病 毒学报,2002,18(3)：264-269．
[12]尹燕博，龚振华，孙承英等．35株不同来源的新城疫病毒(NDV)分离株F-糖蛋白基因序列测定 和基因型研究[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,2002,10：26-30．
[13]杨润德，梅双．新城疫病毒野毒株(HBG-1)的F蛋白部分序列同源性分析[J]．中国预防兽医学 报,2003,35(2)：188-190．
[14]田夫林，陈静，马惠玲等．10株新城疫分离F基因的克隆及遗传变异分析[J]．中国预防兽医学 报,2004,26(1)：28-31．
[15]刘文斌，崔尚金，刘立奎等．东北地区不同宿主NDV分离株的系统发育分析[J]．中国兽医杂 志,2005,4 1(7)：3-6．
[16]秦卓明，马保臣，何叶峰等．新城疫病毒HN和F基因遗传变异相关性的研究[J]．微生物学??报，2006，46(2)：227-232．
[17]Balkwill, E. R&Burke E. The cytokine network [J].Immunology Today, 1989,10:299-304.
[18]Ari. L., Lee.E et al. cytokine: coordinators of immune and inflammatory responses. Annual Reviewof Biochemistry, 59: 783-836.
[19]Peter S, Florian P, Kirsten S, et al.Cytokines of birds: conserved of functions a largely differentlook(review)[J].Journal of Interferon and Cytokine Research, 2001,21:993-1010.
[20]Issaes A,Lindanmann. Virus interference I. the interferon[J]. Prne Roy Sce B, 1957(145): 258-267.
[21]吴志光，汪明等．禽类干扰素分子分子生物学研究进展[J]．中国兽医杂志，2001，37(912)：31-33．
[22]Sekellick M J, Ferrandino A F, Hopkins DA, et al. chicken interferon gene:cloning .expressionand analysis[J].Journal of Interferon Research, 1994, 14:71-79.
[23]Schijm V E,Weining K C.Nuijten P et al.Immunoadjuvant activities of E.coli andplasmid-expressed recombinant chicken IFN-α/β、 IFN-γ and IL-1βin 1-day and 3-week-oldchickens[J], Vaccine, 2000,18:2147-2154.
[24]夏春，汪明等．惠阳胡须鸡干扰素基因分子克隆与序列分析[J]．畜牧兽医学报，2000，31(6)： 563-566．
[25]Plachy J, Weining KC, Krexnmer MJ, et al. Protective effects of type I and type II interferonstoward Rous Sarcoma Virus-induced tumors in chicken[J]. Vimlogy, 1999,256,85-91.
[26]Marcus PI, Heide VD, Sekellick MJ. Interferon action oD Avian vriruses Oral administration ofchicken interferon-a Ameliorates Newcasde Disease [J]. Journal of Interferon and CytokinesResearch, 1999,19: 881-885.
[27]Dimier IH, Ouere P, Naciri M, et al. Inhibition of Eimeria tenella development in vitro mediated bychicken macrophages and fibroblasts treated with chicken cell supernatants with IFNγ activity [J].Avian Disease, 1998,42:239-247.
[28]Lowenthal JW, Onel TE, Broadway M, et al. Coadministration of IFNγ enhancers antibodyresponses in chickens [J]. Journal of Interferon and Cytokine Research, 1998,18: 617-622.
[29]贾立军，刘秀梵等．鸡γ干扰素对H5亚型禽流感疫苗的免疫增强作用[J]．农业生物技术学 报，2004，12(4)：427-430．
[30]胡慧琼，王红宁等．山地乌骨鸡IL-2、IL-15、IL-18及pDNAIFN真核表达载体构建及其对IBV DNA疫苗免疫增强作用研究[J]．高技术通讯，2006，10(16)：1057-1062．
[31]李玉峰，吴静等．重组鸡α-干扰素对鸡外周血T淋巴细胞亚类的影响[J]．山东农业科 学，2006，4(67)：67-69．
[32]Sundick R S, Gill-Dixon C. A cloned chicken lymphokine homologous to both mammalian IL-2and IL-15 [J]. J Immunol, 1997,159(2): 720-725.
[33]Thiagarajan D, Ram G C, Bansal M P. Optimum conditions for in vitro chicken IL-2 productionand its in vivo role in Newcastle disease vaccinated chickens [J]. Veterinary Immunolgy and??Immunopathology, 1999,67(1): 79-91.
[34]Choi K D, Lilehoj H S. Role of chicken IL-2 on γδ T cells and Eimeria acervulina-induced changesin intestinal IL-2 mRNA expression and γδ T cells [J]. Vet Immunol Immunopathol, 2000, 73:309-321.
[35]Lowenthal J W, Lambrecht B, Van den Berg T P, et al. Avian cytokine-the natural approach totherapeutics [J]. Development and Comparative Immunology, 2002,24:355-365.
[36]Lillehoj HS, Min W, Choi KD, et al. Molecular, cellular, and functional characterization of chickencytokines homologous to mammalian IL-15 and IL-2 [J]. Vet Immunol Immunopathol. 2001,82(3-4):229-44.
[37]徐前明，李培英等．鸡白介素-2对柔嫩艾美耳球虫亚单位疫苗免疫增强作用的研究[J]．安徽农业 科学,2005,33(2)：290-293
[38]胡慧琼，王红宁等．鸡IL-2和IL-15基因真核表达质粒对禽流感疫苗的免疫增强作用[J]．中国兽 医科学,2006,36(10)：805-810．
[39]Kirsten Schneider, Florian Puehler. cDNA Cloning of biologically active chicken interleukin -18 [J].Journal of Interferon and Cytokine Research, 2000,20: 879-883.
[40]Degen WG, van Zuilekom HI, Scholtes NC, et al. Potentiation of humoral immune responses tovaccine antigens by recombinant chicken IL-18 (rChIL-18) [J]. Vaccine, 2005,23(33): 4212-4218
[41]韩宗玺．鸡白细胞介素18成熟蛋白的表达及其部分免疫功能研究[J]．新疆农业大学硕士论 文,2004．
[42]王振国，金宁一等．共表达H5N1、H7N1亚型AIV HA与鸡IL-18多价重组鸡痘病毒免疫保护性 研究[J]．中国病毒学,2005,20(6)：607-612．
[43]Louise S H, Andrew G D B, John W L, et al. The emerging role of avian cytokines as immunotherapcuties and vaccine adjuvants [J]. Vet Immunol Immunopathol.2002,85:119-128.
[44]金伯泉．细胞和分子免疫学(第二版)[M]．北京：科学出版社,2001,144．
[45]秦云龙，金宁一，罗坤等．中国流行株HIV-lgag和Hil-2／Hil-6核酸疫苗构建与实验免疫研究[J]． 免疫学杂志,2001,1，17(1)：5-8．
[46]郭炎，金宁一，张应玖等．共表达HIV-1与IL-6核酸疫苗质粒诱导小鼠免疫原性的研究[J]．中国 微生物学和免疫学杂志'2002,3,22：174．
[47]汪文正，金宁一，李子建等．共表达HIV-1 gag-gp120与IL-6重组鸡痘病毒和核酸疫苗的联合免疫 研究[J]．中华微生物学和免疫学杂志,2004,3,24(3)：206-208．
[48]Schneider K, Klaas R, Kaspers B, et al. Chicken interleukin-6 cDNA structure and biological properties [J]. Eur.J.Biochem, 2001,268:4200-4206.
[49]孙建和，陆苹，蒋静．鸡白介素-6(ChIL-6)基因的克隆与序列分析[J]．华中农业大学学 报,2003,22(1)：1-3.
[50]Nishimichi N, Aosasa M, Kawashima T. et a!. Generation of a mouse monoclonal antibody against??chicken interleukin-6 [J]. Hybridoma. 2005,24(2): 115-7.
[51]Nishimichi N, Aosasa M, Kawashima T. et al. Biological activity of recombinant chickeninterleukin-6 in chicken hybridoma cells [J]. Vet Immunol Immunopathol. 2005,106(1-2):97-105.
[52]Alexander B, Lgor M B. Expression of immunomodulating molecules by recombinant viruses: Canthe immunogenicity of live virus vaccines be improved ? [J]. Expert Rrv Vaccine, 2002,1:233-245.
[53]Derek T O, Mary L M. Manmohan S Recent developments in adjuvants for vaccines againstinfectious diseases [J]. Biom olecular Engineering, 2001,8:69-85.
[54]杜平．，细胞素抗癌疗法[M]．上海：上海科技出版社,1992：218-228．
[55]萨姆布鲁克J，拉塞尔D W．分子克隆实验指南[M]．黄培堂，王嘉玺，朱厚础等译．第3版．北京：科学 出版社,2002：87-93．
[56]L．戴维斯等编著．分子生物学试验技术[M]．姚志建，沈倍奋，刘亚霞等译．北京：科学出版社,1990．
[57]萨姆布鲁克J，拉塞尔D W．分子克隆实验指南[M]．黄培堂，王嘉玺，朱厚础等译．第3版．北京：科学 出版社,2002：27-29．
[58]谢振华，史小军，蔡国平.用于哺乳动物细胞转染的高纯度质粒DNA的制备[J]．中国科技论文在 线http：//www.paper.edu．cn
[59]Schmidt, EV, Christoph G, Zeller R, et al. The cytomegalovirus enhancer: a pan-active controlelement in transgenic mice. Mol. Cell [J]. Biol, 1990,10:4406-4411.
[60]Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants [J].Cell,1981,23:175-82.
[61]Shih WL, Kuo ML, Cheuang SE, et al. Hepatitis B virus x protein inhibits transforming growthfactor beta-induced apoptosis through the activation of phosphatidy linositol 3-kinase pathway [J].Biol Chem, 2000,275(33): 25 858-25 864.
[62]Braun R P, Babuk L A, Van Draunen. Enhanced immune response to an intradermally deliveredDNA vaccine expressing a secreted from of BHV-IgD [J]. Vaccine, 1997,6:151-164.
[63]左边富．流式细胞术与生物医学[M]．沈阳：辽宁人民出版社，1996：129-422．
[64]程相朝，赵德明，吴庭才等．鸡IL-18真核表达载体的构建及其对新城疫疫苗免疫增强作用的研 究[J]．畜牧兽医学报,2005,36(5)：476-481．