NDV Sddy-02株F基因的克隆与序列分析及E.coli CpG-DNA在新城疫疫苗上的应用研究
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摘要
为探讨强毒新城疫病毒的分子生物学特性和新的疫苗免疫防制技术，本研究分离鉴定了一地方株新城疫病毒（Chicken/Shandong/Dongying/ 2002，以下简称NDV Sddy-02），对其生物学特性进行了研究，并用RT-PCR对该分离株F基因重要片段进行了克隆和序列分析。在此研究基础上，用NDV Sddy-02株和C30标准弱毒株制备了复合新城疫蜂胶灭活疫苗，同时将E.coli CpG-DNA作为佐剂以不同剂量分别加入该疫苗和C30弱毒疫苗中，对比研究E.coli CpG-DNA对此两类疫苗的免疫增强作用，为研制更为高效、安全的复合佐剂疫苗提供科学依据。本课题分三部分进行研究：
第一部分：NDV Sddy-02株的分离鉴定及生物学特性研究 对从山东东营某鸡场分离到的一株新城疫病毒（NDV Sddy-02）进行了生物学特性研究。经SPF鸡胚接种培养后，其JIF1代尿囊液血凝（HA）效价为27-8，新城疫标准阳性血清对4个单位分离毒的血凝抑制（HI）效价为28，对100 ELD50 JIF1的中和效价为1：256，表明该分离株为新城疫病毒。分离株的雏鸡脑内接种致病指数（ICPI）为1.87，静脉接种致病指数（IVPI）为2.62，最小致死量平均鸡胚死亡时间（MDT）为53.4，血凝解脱快，血凝素热稳定性差。该分离株毒力稍强于标准强毒株F48E9，在血清中和试验上也与标准株有一定差别，表明NDV Sddy-02株为发生了一定程度变异的新城疫强毒株。
第二部分：NDV Sddy-02株F基因的克隆与序列分析 本试验设计了一对特异性引物，应用RT-PCR技术扩增出NDV Sddy-02株F基因含裂解位点区363bp片段，同时进行克隆和序列测定分析。参考国内外已发表各新城疫代表株，绘制了NDV 系统发育进化树，分析其遗传变异关系。分析结果表明：NDV Sddy-02株F基因片段与ShD-1.99株、Qd-1株、Sdbz-s99株、C30株和F48E9株核苷酸序列的同源性分别为97.5%、86.5%、98.4%、84.1%
和86%，氨基酸序列同源性分别为99.2% 、92.6% 、100%、89.3%和91.7%，裂解位点区氨基酸序列组成为112R-R-Q-K-R-F117，符合强毒株裂解位点区特征，这进一步从分子水平表明了 NDV Sddy-02株为强毒株。系统发育分析得出，NDV Sddy-02株与ShD-1.99株、Sdbz-s99株在进化地位上最接近，且同为基因Ⅶ型，表明近年来山东省多为基因Ⅶ型流行。
第三部分：E.coli CpG-DNA在新城疫疫苗上的应用研究 用NDV Sddy-02株和C30标准弱毒株制备了复合新城疫蜂胶灭活疫苗；同时将用CTAB法提取的E.coli CpG-DNA作为该复合新城疫蜂胶灭活疫苗和C30弱毒疫苗的佐剂，以CpG-DNA 10μg/羽和30μg/羽两个剂量分别加入到两类疫苗中，免疫14日龄健康未免疫肉雏鸡。在免疫前、免疫后7天、14天和21天，分别进行血凝抑制(HI)试验，对比研究E.coli CpG-DNA对两类疫苗的免疫增强作用。研究结果表明：剂量10μg/羽对该复合新城疫蜂胶灭活疫苗具有免疫增强作用，但差异不显著；剂量30μg/羽无明显增强作用。两剂量10μg/羽和30μg/羽对C30弱毒疫苗均无明显免疫增强作用，具体原因有待于进一步研究。本研究初步表明E.coli CpG-DNA作为佐剂能够增强复合新城疫蜂胶灭活疫苗的免疫应答水平，这为研制新型复合佐剂疫苗奠定了基础。
In order to find the molecular biological Characteristics of virulent NDV strain and to find the preventive and remedial technology of new vaccine, one NDV strain (Chicken/Shandong/Dongying/2002, NDV Sddy-02) was isolated and identified. Its biological Characteristics were studied and the important fragment of its F gene was cloned and analyzed. On the basis of these studies, a compound proplis inactivated vaccine against ND was developed with this isolate and the C30 strain. At the same time the different doses of E.coli CpG-DNA were added into this compound proplis inactivated vaccine and C30 live vaccine to compare the results caused by E.coli CpG-DNA in these two kinds of vaccines. It is the aim that lay the foundations of producing a more efficient and more safe vaccine.
PartⅠ：Isolation and identification of NDV Sddy-02 strain and studies on its biological Characteristics One strain was isolated and identified from a certain chicken house in Shandong province, with its biological characteristics studied. Cultured by SPF chicken embryo, its HA is 27-8, HI is 28, ICPI is 1.87, IVPI is 2.62, MDT is 53.4, the dehaemagglutination time is fast and the haemagglutinin heat-stability is poor. By all these characteristics the isolate was confirmed to be virulent NDV strain.
PartⅡ：Molecular Cloning and Analysis of the Fusion Glycoprotein of NDV Sddy-02 Isolate A pair of primers was designed specially and synthetized. The fragment of F gene covering cleavage site region was amplified by RT-PCR, cloned and compared. Basing on published sequences of representative NDV strains, a phylogenetic tree was constructed to analyse their variation relation. Comparisons of F gene (ShD-1.99, Qd-1, Sdbz-s99, Clone30 and F48E9) showed that the homologies of nucletide sequence were
97.5%, 86.5%, 98.4%, 84.1% and 86% respectively. Their homologies in deduced amino acid sequence were 99.2%, 92.6%, 100%, 89.3% and 91.7% separately. Because the amino acid sequence of the cleavage site is made up of 112R-R-Q-K-R-F117, which match to that of all virulent NDV strains, the isolate was proved to be an virulent strain. The constructed phylogenetic tree showed that the Sddy-02, ShD-1.99 and Sdbz-s99 belonged to NDV genetype Ⅶ. From the molecular results it is further demonstrated that the ND caused by genetype Ⅶ also prevail in Shandong province.
Part Ⅲ：Applied Studies on E.coli CpG-DNA in ND Vaccines The compound proplis inactivated vaccine against ND was developed with NDV Sddy-02 strain and the C30 strain. Meanwhile the E.coli CpG-DNA extracted by CTAB method was used as a new adjuvant with dose of 10μg and 30μg added into this compound proplis inactivated vaccine and C30 live vaccine. Then 14-day-age chickens were inoculated by the two kinds of vaccines including different CpG-DNA doses respectively. At different time HI was performed to find whether the E.coli CpG-DNA has enhanced the immunostimulatory results to these two kinds of vaccines. The results indicated that the dose of 10μg had an enhanced effect to the compound proplis inactivated vaccine, while the dose of 30μg had no more effect to it. Both 10μg dose and 30μg dose were no aidant to C30 live vaccine. The detailed reasons are to be further studied in the future. It is the first study that the E.coli CpG-DNA is able to enhance the immune response in compound proplis inactivated vaccine against ND. This research laid foundations of developing new compound adjuvants in ND vaccines.
第一部分：NDV Sddy-02株的分离鉴定及生物学特性研究 对从山东东营某鸡场分离到的一株新城疫病毒（NDV Sddy-02）进行了生物学特性研究。经SPF鸡胚接种培养后，其JIF1代尿囊液血凝（HA）效价为27-8，新城疫标准阳性血清对4个单位分离毒的血凝抑制（HI）效价为28，对100 ELD50 JIF1的中和效价为1：256，表明该分离株为新城疫病毒。分离株的雏鸡脑内接种致病指数（ICPI）为1.87，静脉接种致病指数（IVPI）为2.62，最小致死量平均鸡胚死亡时间（MDT）为53.4，血凝解脱快，血凝素热稳定性差。该分离株毒力稍强于标准强毒株F48E9，在血清中和试验上也与标准株有一定差别，表明NDV Sddy-02株为发生了一定程度变异的新城疫强毒株。
第二部分：NDV Sddy-02株F基因的克隆与序列分析 本试验设计了一对特异性引物，应用RT-PCR技术扩增出NDV Sddy-02株F基因含裂解位点区363bp片段，同时进行克隆和序列测定分析。参考国内外已发表各新城疫代表株，绘制了NDV 系统发育进化树，分析其遗传变异关系。分析结果表明：NDV Sddy-02株F基因片段与ShD-1.99株、Qd-1株、Sdbz-s99株、C30株和F48E9株核苷酸序列的同源性分别为97.5%、86.5%、98.4%、84.1%
和86%，氨基酸序列同源性分别为99.2% 、92.6% 、100%、89.3%和91.7%，裂解位点区氨基酸序列组成为112R-R-Q-K-R-F117，符合强毒株裂解位点区特征，这进一步从分子水平表明了 NDV Sddy-02株为强毒株。系统发育分析得出，NDV Sddy-02株与ShD-1.99株、Sdbz-s99株在进化地位上最接近，且同为基因Ⅶ型，表明近年来山东省多为基因Ⅶ型流行。
第三部分：E.coli CpG-DNA在新城疫疫苗上的应用研究 用NDV Sddy-02株和C30标准弱毒株制备了复合新城疫蜂胶灭活疫苗；同时将用CTAB法提取的E.coli CpG-DNA作为该复合新城疫蜂胶灭活疫苗和C30弱毒疫苗的佐剂，以CpG-DNA 10μg/羽和30μg/羽两个剂量分别加入到两类疫苗中，免疫14日龄健康未免疫肉雏鸡。在免疫前、免疫后7天、14天和21天，分别进行血凝抑制(HI)试验，对比研究E.coli CpG-DNA对两类疫苗的免疫增强作用。研究结果表明：剂量10μg/羽对该复合新城疫蜂胶灭活疫苗具有免疫增强作用，但差异不显著；剂量30μg/羽无明显增强作用。两剂量10μg/羽和30μg/羽对C30弱毒疫苗均无明显免疫增强作用，具体原因有待于进一步研究。本研究初步表明E.coli CpG-DNA作为佐剂能够增强复合新城疫蜂胶灭活疫苗的免疫应答水平，这为研制新型复合佐剂疫苗奠定了基础。
In order to find the molecular biological Characteristics of virulent NDV strain and to find the preventive and remedial technology of new vaccine, one NDV strain (Chicken/Shandong/Dongying/2002, NDV Sddy-02) was isolated and identified. Its biological Characteristics were studied and the important fragment of its F gene was cloned and analyzed. On the basis of these studies, a compound proplis inactivated vaccine against ND was developed with this isolate and the C30 strain. At the same time the different doses of E.coli CpG-DNA were added into this compound proplis inactivated vaccine and C30 live vaccine to compare the results caused by E.coli CpG-DNA in these two kinds of vaccines. It is the aim that lay the foundations of producing a more efficient and more safe vaccine.
PartⅠ：Isolation and identification of NDV Sddy-02 strain and studies on its biological Characteristics One strain was isolated and identified from a certain chicken house in Shandong province, with its biological characteristics studied. Cultured by SPF chicken embryo, its HA is 27-8, HI is 28, ICPI is 1.87, IVPI is 2.62, MDT is 53.4, the dehaemagglutination time is fast and the haemagglutinin heat-stability is poor. By all these characteristics the isolate was confirmed to be virulent NDV strain.
PartⅡ：Molecular Cloning and Analysis of the Fusion Glycoprotein of NDV Sddy-02 Isolate A pair of primers was designed specially and synthetized. The fragment of F gene covering cleavage site region was amplified by RT-PCR, cloned and compared. Basing on published sequences of representative NDV strains, a phylogenetic tree was constructed to analyse their variation relation. Comparisons of F gene (ShD-1.99, Qd-1, Sdbz-s99, Clone30 and F48E9) showed that the homologies of nucletide sequence were
97.5%, 86.5%, 98.4%, 84.1% and 86% respectively. Their homologies in deduced amino acid sequence were 99.2%, 92.6%, 100%, 89.3% and 91.7% separately. Because the amino acid sequence of the cleavage site is made up of 112R-R-Q-K-R-F117, which match to that of all virulent NDV strains, the isolate was proved to be an virulent strain. The constructed phylogenetic tree showed that the Sddy-02, ShD-1.99 and Sdbz-s99 belonged to NDV genetype Ⅶ. From the molecular results it is further demonstrated that the ND caused by genetype Ⅶ also prevail in Shandong province.
Part Ⅲ：Applied Studies on E.coli CpG-DNA in ND Vaccines The compound proplis inactivated vaccine against ND was developed with NDV Sddy-02 strain and the C30 strain. Meanwhile the E.coli CpG-DNA extracted by CTAB method was used as a new adjuvant with dose of 10μg and 30μg added into this compound proplis inactivated vaccine and C30 live vaccine. Then 14-day-age chickens were inoculated by the two kinds of vaccines including different CpG-DNA doses respectively. At different time HI was performed to find whether the E.coli CpG-DNA has enhanced the immunostimulatory results to these two kinds of vaccines. The results indicated that the dose of 10μg had an enhanced effect to the compound proplis inactivated vaccine, while the dose of 30μg had no more effect to it. Both 10μg dose and 30μg dose were no aidant to C30 live vaccine. The detailed reasons are to be further studied in the future. It is the first study that the E.coli CpG-DNA is able to enhance the immune response in compound proplis inactivated vaccine against ND. This research laid foundations of developing new compound adjuvants in ND vaccines.
引文
[1]常维山, 一株新城疫病毒强毒株的F基因序列测定研究,变异性新城疫病毒分子生物学的研究鉴定材料.
[2]王兴龙,新城疫病毒分子生物学. 动物科学与动物医学, 2001,18(3),28-30.
[3]殷震,刘景华 动物病毒学[M]第2版 北京科学出版社,1997.737-741.
[4]陆承平,最新脊椎动物病毒分类简介[J],中国兽医学报,1996,16(1):94～100.
[5]Toyoda T,Sakaguchi T,Imai K et al;Virology,1987,158:242-247.
[6]黄小波,新城疫病毒强弱毒株鉴别方法的研究进展.中国兽医科技,2002,32(10):20-21.
[7]成大荣,徐建生等,新城疫病毒的F蛋白与遗传学分群.预防兽医学进展,2001,3(1):37-39.
[8]Collins M S,Bashiruddin J B,Arch.Virol.1993,128:363-370.
[9]王兴龙,四株新城疫病毒流行株F基因的遗传变异分析.中国预防兽医学报,2002,24(2):123-125.
[10]曹殿军,李祥瑞,等(译).国外兽医学-畜禽传染病,1997,17(2):8-10.
[11]Krieg AM,et al.Nature,1995;374:546-549.
[12]Yiak,et al.Int Immunol,1999;11:2014-2016.
[13]Yi AK, Tuetken R, Redford T, et al. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.J Immunol,1998,160:4755-4761.
[14]DNA vaccine web site:http//www.genweb.com/Dnavax/dnavax.html.
[15]Oxenius A, Martinic MMA,Hengartner H, et al. CpG -containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune respones with T-cell peptide vaccines.J Virol,1999,73:4120-4126.
[16]Krieg A M,Kline J N.Immune effects and therapeutic applications of CpG motifs in bacterial DNA[J].Immunopharmacology,2000,48:303-305.
[17]Yew N S,Zhao H, Wu I H,et al.Reduced inflammatory responses to plasid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs[J].Mol Ther,2000,1(3):255-262.
[18]Filion M C, Filion B,Reader S, et al. Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and persent in a mycobacterial cell wall complex[J].Cancer Immunol Immunother,2000,49(6):325-334.
[19]Sun S, Beard C,Jaenisch R, et al.Mitogenicity of DNA from different organisms for murine B cells[J].J Immunol,1997,159:3119-3125.
[20]Lipford G B,Sparwasser T, Bauer M,et al. Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines[J].Eur J Immunol,1997,27(12):3420-3426.
[21]郭爱珍等.细菌CpG-DNA免疫刺激活性研究进展[J]. 中国兽医学报,2002,22(3):317-320.
[22]李文波等.CpG-DNA的免疫学活性及其作用机理研究进展[J]. 国外医学免疫学分册,2000,23(5):302-306.
[23]于冬冬等. CpG特征结构的免疫效应及应用研究[J]. 国外医学病毒学分册,2000,7(4):117-119.
[24]陆军等. CpG-DNA的免疫激活机理初探[J]. 常熟高专学报,2000,14(2):67-68.
[25]李爱芹等.大肠杆菌免疫刺激DNA的克隆及其对抗体产生的影响[J].中国预防兽医报,2002,24(3):178-181.
[26]郭爱珍等. 细菌CpG-DNA对犬免疫增强效果的影响[J]. 中国兽医杂志,2001,31(12):6-9.
[27]孟松树等.CpG-DNA对鸡传染性喉气管炎病毒DNA疫苗免疫效果的影响[J].中国学,2001,34(3):342-344.
[28]陆军等. CpG-DNA的免疫刺激机理及应用[J]. 生物技术通报,1999,5:30-32.
[29]王志亮. CpG二核苷酸在DNA疫苗中的应用[J]. 国外兽医学-畜禽传染病,1998,18(3):42-44.
[30]唐桂运,武华译,禽病原分离鉴定实验室手册,第三版,北京:北京农业大学出版社,1993,140-150.
[31]蔡宝祥等,动物传染病学诊断,南京;江苏科学技术出版社,1992,177-180.
[32]B W卡尔尼克主编.禽病学.高福,刘文军主译. 北京:北京农业大学出版社,1991.428-432.
[33]贺东生等,新城疫病毒系统发育分析及强弱毒株的鉴别诊断.动物医学进展,2000,21(2):40-41.
[34]农业部.中华人民共和国兽用生物制品规程,北京;农业部,1992版,47-55.
[35]Schsper U M,Fuller F J,Ward M D,et al.Virology,1998,165:291-294. Peeters B P,de Leeuw OS,Koch G.Rescue of Newcastle disease virus from cloned Cdna:Evidence that cleavability of the Fusion protein is a major determinant for virulence [J].J Vrrol,1999,73(6):5001-5009..
[36]曹殿军等, 我国部分地区NDV的分子流行病学研究.中国预防兽医学报,2001,23(1):29-32.
[37]巴德年,当代免疫学技术与应用,第一版,北京医科大学中国协和医科大学联合出版,1998.
[38]卢锦汉,医学生物制品学,第一版,人民卫生出版社,1995.
[39]余传霖,现代医学免疫学,第一版,上海医科大学出版社,1998.
[40][美]F.奥斯伯,精编分子生物学实验技术,第一版,科学出版社,1998.
[41]Chambers P,Millar NS,etal.Molecular cloning of complementary DNA to Newscastle disease virus,and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large ptotein.Journal of General Virlogy,1986,67:475～486.
[42]曹殿军*,郭鑫,苑纯秀,闫丽辉等, 新城疫病毒V4株F基因的克隆与核苷酸序列分析.中国预防兽医学报,2000, 22(1):39～43.
[43]刘伟忠,姜焱,吴艳涛,张如宽,刘秀梵, 新城疫病毒特异性糖蛋白基因探针的制备及应用.畜牧兽医学报,1999,30(1),44～49.
[44]吴艳涛,刘秀梵,张如宽, 新城疫病毒F48E8?株融合蛋白基因序列分析.病毒学报,1999,15 (2):143～146.
[45]严维巍,王永坤等,一株鸡副粘病毒F基因重要片段的扩增、克隆与分析.中国预防兽医学报,2000,22(5):368-370.
[46]梅双双等,鸡新城疫病毒强毒株的分离及生物学特性鉴定.中国预防兽医学报,2002,24(5):368-370.
[47]范根成等,新城疫强毒株的分离与鉴定.中国兽医学报,1999,19(2):114-116.
[48]Lomniczi B, Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype(VIII)[J].Arch Virol, 1998,143:49～44.
[49]Yang C Y,Chang P C,Jyh M H,et al.Nucleotide sequence and phylogenetic analysis of Newscastle disease virus isolated from recent outbreaks in Taiwan[J].Avian Dis,1997,41:326～328.
[50]曹殿军,刘春丽,王丽林等,新城疫病毒F48E9株HN基因的克隆与酶切分析[J].中国兽医学报,1997,17(4):326～328.
[51]王红宁等,新城疫病毒强弱毒株检测方法研究进展.禽病防制,2001, 18(6):28-29.???????
[52]苑纯秀,曹殿军等新城疫病毒F48E9株基因主要功能区的核苷酸序列分析.中国预防兽医学报,1999,21(1):31～34.
[53]梁德勇等,RNA快速提取一步法的建立.中国医科大学学报,1999, 5(2):35-40.
[54]梁荣,陈杰等,禽痘病毒载体在新城疫病毒重组疫苗研究的应用.动物医学进展2000 21(1):13-16
[55]Lomniczi B,Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI)and a novel genotype(VII)[J].Arch Virol ,1998,143:49-64.
[56]Yang C Y ,Chang P C ,Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis,1997,41:326-328.
[57]范纯秀等,鸡新城疫东北地区融合蛋白基因的克隆与鉴定[J].中国预防兽医学报,1999,21 (1):31-34.
[58]分子克隆,金冬雁等译,第二版,北京科学出版社.
[59]曾明等, 非甲基化CpG的免疫学功能及免疫佐剂的应用. 中国药事,2001,15(1):54-56.
[60]Marrack P,Kappler J. Subversion of the immune system by pathogens[J]. Cell,1994,76:323-332.
[61]Madaio M P, Hodder S, Schwartz R S,et al. Responsiveness of autoimmune and normal mice to nucleic acid antigens[J].J Immunol, 1984, 132:872.
[62]Sun S, Beard C, Jaenisch R, et al. Mitogenicity of DNA from different organisms for murine B cells[J]. J Immunol,1997,159:3119-3125.
[63]Krieg A M, Yi A K, Matson S,et al. CpG motifs in bacterial DNA trigger direct B-cell activation.[J].Nature, 1995,374:546-549.
[64]Toyoda T,Sakaguchi T,ImaiK,etal. Structural compari-son of the cleavage-activation site of the fusion glycopro-tein between virulent and avirulent strains of Newcastle diseasevirus.Virology,1987,158: 242-247.
[65]Schaper UM, Fuller FJ, Ward MDW, etal. Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus. Virolo-gy,1988,165:291 -295.
[66]Taylor J, EdbauerC, Rey-SenelongeA, etal. Newcastle disease virus fusion protein expressed in a fowl poxvirus recombinant confers pretection in chickens. J of Virology,1990,64:1441-1450.
[67]中国农业科学院哈尔滨兽医研究所.动物传染病学,北京:中国农业出版社,1999,433.
[68]BallasZK, RasmussenWL, KriegAM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA[J].J Immunol,1996,157(5):1840-1845.
[69]IhoS, YamamotoT, TakahashiT, etal. Oligodeoxynuc-leotides containing palindrome sequences within ternal 5-CpG-3′act direct lyonhuman NK and activatedT cells to induce IFN-γ production invitro[J]. JImmunol, 1999, 163:3642～3652.
[70]YamamotoS, YamamotoT, ShimadaS, etal. DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth[J]. MicrobiolImmunol,1992,36(9):983～987.
[71]DowSW, FradkinLG, LiggittDH, etal. Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously[J].J Immunol,1999,163(3):1552～1561.
[2]王兴龙,新城疫病毒分子生物学. 动物科学与动物医学, 2001,18(3),28-30.
[3]殷震,刘景华 动物病毒学[M]第2版 北京科学出版社,1997.737-741.
[4]陆承平,最新脊椎动物病毒分类简介[J],中国兽医学报,1996,16(1):94～100.
[5]Toyoda T,Sakaguchi T,Imai K et al;Virology,1987,158:242-247.
[6]黄小波,新城疫病毒强弱毒株鉴别方法的研究进展.中国兽医科技,2002,32(10):20-21.
[7]成大荣,徐建生等,新城疫病毒的F蛋白与遗传学分群.预防兽医学进展,2001,3(1):37-39.
[8]Collins M S,Bashiruddin J B,Arch.Virol.1993,128:363-370.
[9]王兴龙,四株新城疫病毒流行株F基因的遗传变异分析.中国预防兽医学报,2002,24(2):123-125.
[10]曹殿军,李祥瑞,等(译).国外兽医学-畜禽传染病,1997,17(2):8-10.
[11]Krieg AM,et al.Nature,1995;374:546-549.
[12]Yiak,et al.Int Immunol,1999;11:2014-2016.
[13]Yi AK, Tuetken R, Redford T, et al. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.J Immunol,1998,160:4755-4761.
[14]DNA vaccine web site:http//www.genweb.com/Dnavax/dnavax.html.
[15]Oxenius A, Martinic MMA,Hengartner H, et al. CpG -containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune respones with T-cell peptide vaccines.J Virol,1999,73:4120-4126.
[16]Krieg A M,Kline J N.Immune effects and therapeutic applications of CpG motifs in bacterial DNA[J].Immunopharmacology,2000,48:303-305.
[17]Yew N S,Zhao H, Wu I H,et al.Reduced inflammatory responses to plasid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs[J].Mol Ther,2000,1(3):255-262.
[18]Filion M C, Filion B,Reader S, et al. Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and persent in a mycobacterial cell wall complex[J].Cancer Immunol Immunother,2000,49(6):325-334.
[19]Sun S, Beard C,Jaenisch R, et al.Mitogenicity of DNA from different organisms for murine B cells[J].J Immunol,1997,159:3119-3125.
[20]Lipford G B,Sparwasser T, Bauer M,et al. Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines[J].Eur J Immunol,1997,27(12):3420-3426.
[21]郭爱珍等.细菌CpG-DNA免疫刺激活性研究进展[J]. 中国兽医学报,2002,22(3):317-320.
[22]李文波等.CpG-DNA的免疫学活性及其作用机理研究进展[J]. 国外医学免疫学分册,2000,23(5):302-306.
[23]于冬冬等. CpG特征结构的免疫效应及应用研究[J]. 国外医学病毒学分册,2000,7(4):117-119.
[24]陆军等. CpG-DNA的免疫激活机理初探[J]. 常熟高专学报,2000,14(2):67-68.
[25]李爱芹等.大肠杆菌免疫刺激DNA的克隆及其对抗体产生的影响[J].中国预防兽医报,2002,24(3):178-181.
[26]郭爱珍等. 细菌CpG-DNA对犬免疫增强效果的影响[J]. 中国兽医杂志,2001,31(12):6-9.
[27]孟松树等.CpG-DNA对鸡传染性喉气管炎病毒DNA疫苗免疫效果的影响[J].中国学,2001,34(3):342-344.
[28]陆军等. CpG-DNA的免疫刺激机理及应用[J]. 生物技术通报,1999,5:30-32.
[29]王志亮. CpG二核苷酸在DNA疫苗中的应用[J]. 国外兽医学-畜禽传染病,1998,18(3):42-44.
[30]唐桂运,武华译,禽病原分离鉴定实验室手册,第三版,北京:北京农业大学出版社,1993,140-150.
[31]蔡宝祥等,动物传染病学诊断,南京;江苏科学技术出版社,1992,177-180.
[32]B W卡尔尼克主编.禽病学.高福,刘文军主译. 北京:北京农业大学出版社,1991.428-432.
[33]贺东生等,新城疫病毒系统发育分析及强弱毒株的鉴别诊断.动物医学进展,2000,21(2):40-41.
[34]农业部.中华人民共和国兽用生物制品规程,北京;农业部,1992版,47-55.
[35]Schsper U M,Fuller F J,Ward M D,et al.Virology,1998,165:291-294. Peeters B P,de Leeuw OS,Koch G.Rescue of Newcastle disease virus from cloned Cdna:Evidence that cleavability of the Fusion protein is a major determinant for virulence [J].J Vrrol,1999,73(6):5001-5009..
[36]曹殿军等, 我国部分地区NDV的分子流行病学研究.中国预防兽医学报,2001,23(1):29-32.
[37]巴德年,当代免疫学技术与应用,第一版,北京医科大学中国协和医科大学联合出版,1998.
[38]卢锦汉,医学生物制品学,第一版,人民卫生出版社,1995.
[39]余传霖,现代医学免疫学,第一版,上海医科大学出版社,1998.
[40][美]F.奥斯伯,精编分子生物学实验技术,第一版,科学出版社,1998.
[41]Chambers P,Millar NS,etal.Molecular cloning of complementary DNA to Newscastle disease virus,and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large ptotein.Journal of General Virlogy,1986,67:475～486.
[42]曹殿军*,郭鑫,苑纯秀,闫丽辉等, 新城疫病毒V4株F基因的克隆与核苷酸序列分析.中国预防兽医学报,2000, 22(1):39～43.
[43]刘伟忠,姜焱,吴艳涛,张如宽,刘秀梵, 新城疫病毒特异性糖蛋白基因探针的制备及应用.畜牧兽医学报,1999,30(1),44～49.
[44]吴艳涛,刘秀梵,张如宽, 新城疫病毒F48E8?株融合蛋白基因序列分析.病毒学报,1999,15 (2):143～146.
[45]严维巍,王永坤等,一株鸡副粘病毒F基因重要片段的扩增、克隆与分析.中国预防兽医学报,2000,22(5):368-370.
[46]梅双双等,鸡新城疫病毒强毒株的分离及生物学特性鉴定.中国预防兽医学报,2002,24(5):368-370.
[47]范根成等,新城疫强毒株的分离与鉴定.中国兽医学报,1999,19(2):114-116.
[48]Lomniczi B, Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype(VIII)[J].Arch Virol, 1998,143:49～44.
[49]Yang C Y,Chang P C,Jyh M H,et al.Nucleotide sequence and phylogenetic analysis of Newscastle disease virus isolated from recent outbreaks in Taiwan[J].Avian Dis,1997,41:326～328.
[50]曹殿军,刘春丽,王丽林等,新城疫病毒F48E9株HN基因的克隆与酶切分析[J].中国兽医学报,1997,17(4):326～328.
[51]王红宁等,新城疫病毒强弱毒株检测方法研究进展.禽病防制,2001, 18(6):28-29.???????
[52]苑纯秀,曹殿军等新城疫病毒F48E9株基因主要功能区的核苷酸序列分析.中国预防兽医学报,1999,21(1):31～34.
[53]梁德勇等,RNA快速提取一步法的建立.中国医科大学学报,1999, 5(2):35-40.
[54]梁荣,陈杰等,禽痘病毒载体在新城疫病毒重组疫苗研究的应用.动物医学进展2000 21(1):13-16
[55]Lomniczi B,Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI)and a novel genotype(VII)[J].Arch Virol ,1998,143:49-64.
[56]Yang C Y ,Chang P C ,Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis,1997,41:326-328.
[57]范纯秀等,鸡新城疫东北地区融合蛋白基因的克隆与鉴定[J].中国预防兽医学报,1999,21 (1):31-34.
[58]分子克隆,金冬雁等译,第二版,北京科学出版社.
[59]曾明等, 非甲基化CpG的免疫学功能及免疫佐剂的应用. 中国药事,2001,15(1):54-56.
[60]Marrack P,Kappler J. Subversion of the immune system by pathogens[J]. Cell,1994,76:323-332.
[61]Madaio M P, Hodder S, Schwartz R S,et al. Responsiveness of autoimmune and normal mice to nucleic acid antigens[J].J Immunol, 1984, 132:872.
[62]Sun S, Beard C, Jaenisch R, et al. Mitogenicity of DNA from different organisms for murine B cells[J]. J Immunol,1997,159:3119-3125.
[63]Krieg A M, Yi A K, Matson S,et al. CpG motifs in bacterial DNA trigger direct B-cell activation.[J].Nature, 1995,374:546-549.
[64]Toyoda T,Sakaguchi T,ImaiK,etal. Structural compari-son of the cleavage-activation site of the fusion glycopro-tein between virulent and avirulent strains of Newcastle diseasevirus.Virology,1987,158: 242-247.
[65]Schaper UM, Fuller FJ, Ward MDW, etal. Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus. Virolo-gy,1988,165:291 -295.
[66]Taylor J, EdbauerC, Rey-SenelongeA, etal. Newcastle disease virus fusion protein expressed in a fowl poxvirus recombinant confers pretection in chickens. J of Virology,1990,64:1441-1450.
[67]中国农业科学院哈尔滨兽医研究所.动物传染病学,北京:中国农业出版社,1999,433.
[68]BallasZK, RasmussenWL, KriegAM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA[J].J Immunol,1996,157(5):1840-1845.
[69]IhoS, YamamotoT, TakahashiT, etal. Oligodeoxynuc-leotides containing palindrome sequences within ternal 5-CpG-3′act direct lyonhuman NK and activatedT cells to induce IFN-γ production invitro[J]. JImmunol, 1999, 163:3642～3652.
[70]YamamotoS, YamamotoT, ShimadaS, etal. DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth[J]. MicrobiolImmunol,1992,36(9):983～987.
[71]DowSW, FradkinLG, LiggittDH, etal. Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously[J].J Immunol,1999,163(3):1552～1561.