犬瘟热病毒F、H和N基因的克隆和表达
|
摘要
犬瘟热病是由犬瘟热病毒(CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副粘病毒科麻疹病毒属，为有囊膜的负链线性RNA病毒，基因组长约16kb。基因组中包括6个非重叠基因区。它们的编码基因按3′～5′端顺序依次为N-P-M-F-H-L系列。最新研究表明CDV融合蛋白(F)和血凝素(H)蛋白是宿主免疫系统的主要目的抗原，是产生中和抗体的重要抗原之一。核衣壳(N)蛋白是保守性较强的免疫原性蛋白，此外N蛋白上含有T细胞表位。H，F和N蛋白在犬瘟热的免疫预防方面起重要作用。
本研究旨在获得CDV的N，F和H蛋白基因，将其克隆后在大肠杆菌中表达，获得大量易于纯化的N，F和H蛋白并对其功能进行研究，为研制基因工程疫苗和诊断试剂等打下理论基础。
一、CDV N基因保守区序列(NC)的克隆及表达：从犬瘟热病毒Onderstepoort株中提取病毒RNA，经反转录聚合酶链反应(RT-PCR)获得犬瘟热病毒全长N基因，然后应用2对引物扩增获得2段CDV编码N蛋白保守区基因(NC1和NC2)，将此片段克隆到质粒pGEM-Teasy测序，测序结果显示NC1和NC2与CDV Onderstepoort株DNA和推导的氨基酸同源性分别为99.7％、99.3％和100％、100％，利用谷胱苷肽巯基转移酶(GST)融合表达载体pGEX-5x-3和麦芽糖结合蛋白(MBP)融合表达载体pMAl-C2构建了重组表达载体pGEX-5x-3-NC2和pMAl-C2-NC1，重组表达质粒经PCR和酶切鉴定后，将其分别转化到大肠杆菌BL21(DE3)plys中和DH5α细胞中，经IPTG诱导，pGEX-5x-3-NC2没有获得目的蛋白表达，而pMAl-C2-Nc1成功获得了目的蛋白表达。
二、CDV H基因的克隆及表达尝试：应用一对引物，经RT-PCR扩增获得CDV全长H基因，将此片段克隆到pGEM-Teasy测序，测序结果显示与CDV Onderstepoort株DNA同源性为98.8％，所编码的氨基酸同源性为98.2％。然后利用6×His标记的重组表达载体pQE-32构建了重组原核表达载体pQE-32-H，重组表达质粒经PCR和酶切鉴定后，转化到大肠杆菌M15中，经IPTG诱导，没有获得目的蛋白的表达。
三、CDV F基因的克隆和表达：应用一对引物，经RT-PCR扩增获得CDV全长F基因，以全长F基因为模板应用4对引物，通过重叠PCR(OE-PCR)扩增获得缺失疏水区的F基因(dF)。将此片段克隆到质粒pGEM-Teasy测序，测序结果显示，与CDVOnderstepoort株DNA同源性为99.2％，所编码的氨基酸同源性为98.4％。将此片段插入
中文摘要
到pQE一30质粒构建了重组pQE一30一dF原核表达质粒，重组表达质粒经PCR和酶切鉴定
后，转化到大肠杆菌M巧细胞中，经IPTG诱导，获得了目的基因的高效表达，经聚丙
烯酞胺凝胶电泳和W己stem一印迹试验，验证其表达的重组蛋白产物分子质量大小为预期
的47000。将表达产物用Ni一NTA琼脂糖进行纯化，获得较高纯度的目的蛋白，目的蛋
白进行复性后免疫小鼠，所得的抗血清与CDV感染的Vero细胞在免疫荧光试验(IFA)
中，呈细胞阳性染色。
四、CDVF蛋白的应用:以重组表达的F蛋白作为包被抗原，初步建立了检测犬瘟
热血清抗体的间接ELIsA方法。所建立的间接ELISA抗原包被浓度为125n留孔，血清
最佳稀释度为1:160。经阻断试验、重复性试验等表明该方法特异性强、重复性较好，
可用于犬瘟热血清抗体的定量和定性检测。
Canine distemper caused by canine distemper virus (CDV) is a highly infectious, frequently lethal disease with high mortality in dogs and other carnivores. CDV is belong to RNA genus morbillivirus in the paramyxoviridae family that has envelop, a single-stranded, negative sense gnome of approximately 16kb. The genome contains six non-overlapping gene regions, organized 3 N-P-M-F-H-L-5.Many studies have shown that two glycoproteins (Hemagglutinin H and Fusion F protein) of CDV are major target antigens for the host immune system. Nucleocapsid (N) protein is highly conserved immunogenicity protein. In addition, N protein contains T cell epitope. Hemagglutinin protein, Fusion protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.
This reseach is amied at cloning and expressing N?F and H gene of CDV and obtaining the easily purified expression products of N?F and H gene of CDV, it would help to study of gene vaccine and diagnostic reagent.
1.Cloning and expression of CDV Nc
CDV RNA was extracted from CDV-Onderstepoort strain. The full length N gene was amplified by RT-PCR and the conserved gene of N (Nc) was amplified by PCR with two specific primers. This sequence was cloned into plasmid pGEM-Teasy. Both the DNA sequence of the cloned gene and amino acid sequence have identity of 99.7%, 99.3% and 100%, 100% with CDV Onderstepoort strain. The two Nc genes were cloned into GST fusion-expression plasmid pGEX-5x-3 and MBP fusion-expression plasmid pMAL-C2, respectively, to construct recombinant plasmid pGEX-5x-3-Nc2 and pMAL-C2- Ncl. The recombinant plasmid were verified by PCR and restriction endonuclease analysis. The recombinant plasmid pGEX-5x-3-NC2 and pMAL-C2- Ncl were transformed into the BL21(DE3)plys and DH5?respectively, and induced by IPTG. As a result, recombinant protein of pMAL-C2- Ncl was obtained, however, the pGEX-5x-3-NC2 recombinant protein could not expressed.
2.Cloning and expession of CDV H gene
The full length H gene of CDV was amplified from CDV RNA by using RT-PCR with a
pair of primers, then this fragment was cloned into pGEM-Teasy. The DNA sequence of the cloned gene and the deduced amino acid sequence showed an identity of 98.8% and 98.2% with Onderstepoort strain, respectively. The recombinant expression plasmid pQE-32-H was constructed by using pQE-32 signed with 6 X His. The recombinant expression plasmid was verified with PCR and restriction endonuclease analysis before transformed into E.coli Ml5. The plasmid was induced with IPTG, however, the intrerest protein was not obtained.
3.Cloning and expressin of CDV F gene
The full length F gene of CDV was amplified from CDV RNA by using RT-PCR with a pair of specific primers, then using the full length F gene as template, the deleted hydrophobic domains of F gene (dF) was amplified by overlap extension -PCR(OE-PCR)with four specific primers and this fragment was cloned into pGEM-Teasy and sequenced.Sequence analysis showed that the DNA sequence of cloned gene and the deduced amino acid sequence has identity of 99.1% and 98.4%,respectively.The dF fragment was cloned into pQE-30 to construct a recombinant expressing plasmid pQE-30- dF. The recombinant plasmid was verified by PCR and restriction endonuclease analysis, then it was transformed into E.coli strain Ml5 and induced by IPTG and obtained high level expression of aimed protein. The expression product dF gene was identified by SDS-PAGE and western-blotting, and found to be 47000 in size as a fusion protein. The protein was purified by Ni-NTA agarose and recovered. The purified product of dF protein was injected into the m
ice every two weeks and for four times. The antiserum was collected from the immunized mice and used for immunity fluorescence assay (IFA) with Vero monolayer infected by CDV. The positive staining was found and localized on the infected cells.
4. Application of CDV F protein
An indirect ELISA method for measuring antibody of canine distemper virus (CDV) was established with recombinant fusion protein (F) of CDV as antige
本研究旨在获得CDV的N，F和H蛋白基因，将其克隆后在大肠杆菌中表达，获得大量易于纯化的N，F和H蛋白并对其功能进行研究，为研制基因工程疫苗和诊断试剂等打下理论基础。
一、CDV N基因保守区序列(NC)的克隆及表达：从犬瘟热病毒Onderstepoort株中提取病毒RNA，经反转录聚合酶链反应(RT-PCR)获得犬瘟热病毒全长N基因，然后应用2对引物扩增获得2段CDV编码N蛋白保守区基因(NC1和NC2)，将此片段克隆到质粒pGEM-Teasy测序，测序结果显示NC1和NC2与CDV Onderstepoort株DNA和推导的氨基酸同源性分别为99.7％、99.3％和100％、100％，利用谷胱苷肽巯基转移酶(GST)融合表达载体pGEX-5x-3和麦芽糖结合蛋白(MBP)融合表达载体pMAl-C2构建了重组表达载体pGEX-5x-3-NC2和pMAl-C2-NC1，重组表达质粒经PCR和酶切鉴定后，将其分别转化到大肠杆菌BL21(DE3)plys中和DH5α细胞中，经IPTG诱导，pGEX-5x-3-NC2没有获得目的蛋白表达，而pMAl-C2-Nc1成功获得了目的蛋白表达。
二、CDV H基因的克隆及表达尝试：应用一对引物，经RT-PCR扩增获得CDV全长H基因，将此片段克隆到pGEM-Teasy测序，测序结果显示与CDV Onderstepoort株DNA同源性为98.8％，所编码的氨基酸同源性为98.2％。然后利用6×His标记的重组表达载体pQE-32构建了重组原核表达载体pQE-32-H，重组表达质粒经PCR和酶切鉴定后，转化到大肠杆菌M15中，经IPTG诱导，没有获得目的蛋白的表达。
三、CDV F基因的克隆和表达：应用一对引物，经RT-PCR扩增获得CDV全长F基因，以全长F基因为模板应用4对引物，通过重叠PCR(OE-PCR)扩增获得缺失疏水区的F基因(dF)。将此片段克隆到质粒pGEM-Teasy测序，测序结果显示，与CDVOnderstepoort株DNA同源性为99.2％，所编码的氨基酸同源性为98.4％。将此片段插入
中文摘要
到pQE一30质粒构建了重组pQE一30一dF原核表达质粒，重组表达质粒经PCR和酶切鉴定
后，转化到大肠杆菌M巧细胞中，经IPTG诱导，获得了目的基因的高效表达，经聚丙
烯酞胺凝胶电泳和W己stem一印迹试验，验证其表达的重组蛋白产物分子质量大小为预期
的47000。将表达产物用Ni一NTA琼脂糖进行纯化，获得较高纯度的目的蛋白，目的蛋
白进行复性后免疫小鼠，所得的抗血清与CDV感染的Vero细胞在免疫荧光试验(IFA)
中，呈细胞阳性染色。
四、CDVF蛋白的应用:以重组表达的F蛋白作为包被抗原，初步建立了检测犬瘟
热血清抗体的间接ELIsA方法。所建立的间接ELISA抗原包被浓度为125n留孔，血清
最佳稀释度为1:160。经阻断试验、重复性试验等表明该方法特异性强、重复性较好，
可用于犬瘟热血清抗体的定量和定性检测。
Canine distemper caused by canine distemper virus (CDV) is a highly infectious, frequently lethal disease with high mortality in dogs and other carnivores. CDV is belong to RNA genus morbillivirus in the paramyxoviridae family that has envelop, a single-stranded, negative sense gnome of approximately 16kb. The genome contains six non-overlapping gene regions, organized 3 N-P-M-F-H-L-5.Many studies have shown that two glycoproteins (Hemagglutinin H and Fusion F protein) of CDV are major target antigens for the host immune system. Nucleocapsid (N) protein is highly conserved immunogenicity protein. In addition, N protein contains T cell epitope. Hemagglutinin protein, Fusion protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.
This reseach is amied at cloning and expressing N?F and H gene of CDV and obtaining the easily purified expression products of N?F and H gene of CDV, it would help to study of gene vaccine and diagnostic reagent.
1.Cloning and expression of CDV Nc
CDV RNA was extracted from CDV-Onderstepoort strain. The full length N gene was amplified by RT-PCR and the conserved gene of N (Nc) was amplified by PCR with two specific primers. This sequence was cloned into plasmid pGEM-Teasy. Both the DNA sequence of the cloned gene and amino acid sequence have identity of 99.7%, 99.3% and 100%, 100% with CDV Onderstepoort strain. The two Nc genes were cloned into GST fusion-expression plasmid pGEX-5x-3 and MBP fusion-expression plasmid pMAL-C2, respectively, to construct recombinant plasmid pGEX-5x-3-Nc2 and pMAL-C2- Ncl. The recombinant plasmid were verified by PCR and restriction endonuclease analysis. The recombinant plasmid pGEX-5x-3-NC2 and pMAL-C2- Ncl were transformed into the BL21(DE3)plys and DH5?respectively, and induced by IPTG. As a result, recombinant protein of pMAL-C2- Ncl was obtained, however, the pGEX-5x-3-NC2 recombinant protein could not expressed.
2.Cloning and expession of CDV H gene
The full length H gene of CDV was amplified from CDV RNA by using RT-PCR with a
pair of primers, then this fragment was cloned into pGEM-Teasy. The DNA sequence of the cloned gene and the deduced amino acid sequence showed an identity of 98.8% and 98.2% with Onderstepoort strain, respectively. The recombinant expression plasmid pQE-32-H was constructed by using pQE-32 signed with 6 X His. The recombinant expression plasmid was verified with PCR and restriction endonuclease analysis before transformed into E.coli Ml5. The plasmid was induced with IPTG, however, the intrerest protein was not obtained.
3.Cloning and expressin of CDV F gene
The full length F gene of CDV was amplified from CDV RNA by using RT-PCR with a pair of specific primers, then using the full length F gene as template, the deleted hydrophobic domains of F gene (dF) was amplified by overlap extension -PCR(OE-PCR)with four specific primers and this fragment was cloned into pGEM-Teasy and sequenced.Sequence analysis showed that the DNA sequence of cloned gene and the deduced amino acid sequence has identity of 99.1% and 98.4%,respectively.The dF fragment was cloned into pQE-30 to construct a recombinant expressing plasmid pQE-30- dF. The recombinant plasmid was verified by PCR and restriction endonuclease analysis, then it was transformed into E.coli strain Ml5 and induced by IPTG and obtained high level expression of aimed protein. The expression product dF gene was identified by SDS-PAGE and western-blotting, and found to be 47000 in size as a fusion protein. The protein was purified by Ni-NTA agarose and recovered. The purified product of dF protein was injected into the m
ice every two weeks and for four times. The antiserum was collected from the immunized mice and used for immunity fluorescence assay (IFA) with Vero monolayer infected by CDV. The positive staining was found and localized on the infected cells.
4. Application of CDV F protein
An indirect ELISA method for measuring antibody of canine distemper virus (CDV) was established with recombinant fusion protein (F) of CDV as antige
引文
1．夏咸柱，养犬大全[M]．吉林．吉林人民出版社．1993，549～553．
2．蔡宝祥，家蓄传染病学[M]．第三版．北京．中国农业出版社．
3．从丽，闫新华，闫喜军等，狐貂犬瘟热、细小病毒性肠炎和脑(肝)炎流行病学调查[J]．特产研究，2000，2：46～47．
4．华国荫，刘鼎新，吴英平等，犬瘟热强毒的分离与培育[J]_畜禽传染病，1983，3：8～13．
5. Sidhu MS, Husar W, CooK SD, et at. Canine distemper terminal and intergenic non-protein coding nucleotide sequences: Completion of Entire CDV genome sequence. [J] Virology, 1993, 193: 66～72.
6．李金中，夏咸柱，邱薇等，小熊猫四种动物犬瘟热病毒的分离与鉴定[J]．中国兽医学报，1998，28(8)：8～10．
7．张振兴，高素兰，徐福南，等，一起中国熊猫瘟热病的实验报告[J]．畜牧与兽医，1983，4：3～9．
8．胡桂林，夏咸柱，李金中等，大熊猫犬瘟热致死性感染的首次报告[A]．第七届全国养犬学和第三届广东省小动物医学联合会学术研讨会论文集[C]．广州：广东省畜牧兽医学会小动物医学委员会，1997，172-174．
9．李金中，夏咸柱，胡桂学等，我国犬瘟热病毒的生态学调查研究[J]．畜牧兽医学报，1999，30(4)：375～339．
10．高得仪，犬猫疾病学[M]．北京：北京农业大学出版社，1991，2～3．
11．遇秀玲，田克恭，吴哪等，犬实验感染CDV的病理学研究[J]．中国畜禽传染病，1998，20(2)：76～78．
12. Stephenson C.B, Welter J, Thaker S.R, et al. Canine distemper virus (CDV) infection of ferrets as a model for testing morbillivirus vaccine strategies: NYVAC-and ALVAC-based CDV recombinants protect against symptomatic infection [J].Virol, 1997, 71: 1506～1513.
13. Stern-LB, Greenberg-M, Gershoni-JM, et al. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis [J]. Virol, 1995, 69(3): 1661～1668.
14. Hirayama-N, Senda-M, Nakashima-N, et al. Protective effects of monoclonal antibodies
against lethal canine distemper virus infection in mice [J]. Gen Virol, 1991, 72 (11): 2827～2830.
15. Iwatsuki-K, Miyashita-N, Yoshida-E, et al. The nucleotide and predicted amino acid sequence of the fusion protein of recent isolates of canine distemper virus in Japan [J].Vet-Med-Sci. 1998, 60(3): 381～5.
16. Winters K. A, Immuneglobulin class response to canine distemper virus in gnotobiotic dogs [J].Vet Immunology and Immunopathology, 1983/1984,5: 209～215.
17. Evans-S.A, Belsham-G.J, Barrett-T, et al. The role of the 5' nontranslated regions of the fusion protein mRNAs of canine distemper virus and rinderpest virus [J]. Virology. 1990, 177(1): 317～323.
18. Kovanmees J, The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus [J]. Virus Res. 1991, 19: 223～234.
19. Wild-T.F, Buck land-R, Inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein [J]. Gen Virol. 1997, 78 (1): 107～111.
20. Sheshberadaran-H, Norrby-E, McCullough-K.C, et al. The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies [J] Gen Virol. 1986, 67(7): 1381～1392.
21. Orvell-C, Sheshberadaran-H, Norrby-E, et al. Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus [J]. Gen Virol.1985, 66 (3): 443～56.
22. Taylor J, Vaccinia virus recombinants repressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge [J]. Virol. 1991, 4263～4274.
23．李金中，夏咸柱，殷震，等，犬瘟热病毒野毒株融合蛋白主要供能区基因的变异研究[J]．中国兽医学报，1996，16(6)：519～526
24. Curran M. D, The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus [J]. Gen Virol. 1991, 72: 443～417.
25. Iwatsuki K, Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs [J]. Gen Virol. 1997,
78: 373～380.
26. Bolt G, Genetic diversity of the attachment(H) protein gene of current filed isolates of canine distemper virus [J]. Gen Virol. 1997, 78: 367～372.
27. Kingsbury D.W Paramyxoviridae and their replication. In: B. N. Fields(Ed.), Field' s Virol. 1990.Raven Press, New YorK.
28. Wild T.F Mode of entry of morbilliviruses. Vet. Microbiol. 1995, 44: 267～270.
29. Mochizuki M, Hashimoto M, Hagiwara S, et al. Genotypes of canine distemper virus determined by analysis of haemagglutinin genes of recent isolates from dogs in Japan [J] Clin Microbiol 1999, 37: 2936～2942.
30. Blixenkrone-Mollor M, Svanson V, Appel, et al. Antigenic relationships between field isolates of morbilliviruses from different camivores [J]. Arch Virol. 1992, 123: 279～294.
31. Blixenkrone-Moller M, Svanson B, Have p, et al. Studies on manifestations of canine distemper virus infection in an urban dog population [J]. Vet Microbiol, 1993, 37: 163～173.
32. Iwatsuki K, Tokiyoshi S, Hirayama N, et al. Antigenic differences in the H proteins of canine distemper viruses [J]. Vet Microbiol, 2000, 71: 281～286.
33. Iwatsuki K, Miyashita N, Yoshida E, et al. Molecular and phylogenic analyses of the haemagglutinin (H) proteins of isolates of distemper virus from naturally infected dogs [J]. Gen Virol, 1997, 78: 373～380.
34. Yoshida E, Shin Y, Iwatsuki K, et al. Epitopes and nuclear localization analyses of canine distemper virus nucleocapsid protein by expression of its deletion mutants [J]. Vet Microbiol, 1999, 66: 313～320.
35. Beauverger P, Buckland R, Wild T F. Measles virus antigens induce both type-specific and canine ditemper virus cross-reactive cytotoxic Tlymphocytein epitope [J]. Gen Virol, 1993,74: 2357～2363.
36．李昌文，张洪英，刘怀然等，犬瘟热病毒A株N蛋白基因编码区的克隆与序列分析[J]．动物医学进展，2003，24(6)：93～95．
37. Pascal Cherpillod, Andrea Tipold Monika Griot-Wenk, etal. DNA vaccine encoding nucleocapsid and surface proteins of type canine distemper virus protects its natural host against distemper [J]. Vaccine, 2000, 18: 2927～2936.
38．范宝昌，我国登革病毒全长cDNA克隆的构建及其转录体感染性研究[D]．2002．
39. Cherpillod P, Beck K, Zurbriggen A, etal. Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17 [J]. Virology, 1999, 73: 2263～2269.
40．萨姆布鲁J，里奇EF，曼尼阿蒂斯T等，分子克隆操作指南[M]．第二版．北京：科学出版社，1996．
41．姜泊，张亚历，周殿元等，分子生物学常用实验方法[M]．第一版．北京：人民军医出版社，1996．
42．候利华，丙型肝炎病毒NS2和NS5基因的克隆和表达[D]．1999．
43．孙树汉，基因工程原理与方法[M]．第一版．北京：人民军医出版社，2001．
44．温进坤，韩梅，医学分子生物学[M]．第二版．科学出版社，2002．
45．徐向明，吉荣，崔治中等，犬瘟热病毒OP株囊膜糖蛋白F基因片段表达产物的免疫原性[J]．中国兽医学报，2003，23(3)：231-233．
46. Yoshida E, Iwatsuki K, Miyashita N, etal. Molecular analysis of the nucleocapsid protein of recent isolates of canine distemper virus in Japan [J]. Vet Microbiol, 1998, 59: 237～244.
47．耿志贤，田克恭，李钟铎等，犬瘟热病毒的分子生物学研究进展[J]．中国兽医杂志，1997，23(2)45-46．
48. Stettler M, Nucleotide and deduced amino acid sequence of the nucleocapsid protein of the virulent A75/17-CDV strain of canine distemper virus [J]. Vet Microbiol, 1995, 44: 211～217.
49. Sparwasser T, Koch E S, Vebulas R M, etal.Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells [J]. Eru. J. Immunol, 1997, 28: 2045～2051.
50．吴小闲，医学实验动物监测手册[M]．卫生部医学实验动物管理委员会，1992，6：89-90．
51．丁家波，崔治中，韦平等，马立克氏病病毒广西株G2囊膜糖蛋白gI基因的克隆和表达[J]．中国兽医学报，2001，21(2)：109～112．
52. Messling V, Harder TC, Moennig V, et al. Rapid and sensitive detection of immnoglobulin M (M) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. J Clin Microbiol, 1999, 37: 1049-1056.
2．蔡宝祥，家蓄传染病学[M]．第三版．北京．中国农业出版社．
3．从丽，闫新华，闫喜军等，狐貂犬瘟热、细小病毒性肠炎和脑(肝)炎流行病学调查[J]．特产研究，2000，2：46～47．
4．华国荫，刘鼎新，吴英平等，犬瘟热强毒的分离与培育[J]_畜禽传染病，1983，3：8～13．
5. Sidhu MS, Husar W, CooK SD, et at. Canine distemper terminal and intergenic non-protein coding nucleotide sequences: Completion of Entire CDV genome sequence. [J] Virology, 1993, 193: 66～72.
6．李金中，夏咸柱，邱薇等，小熊猫四种动物犬瘟热病毒的分离与鉴定[J]．中国兽医学报，1998，28(8)：8～10．
7．张振兴，高素兰，徐福南，等，一起中国熊猫瘟热病的实验报告[J]．畜牧与兽医，1983，4：3～9．
8．胡桂林，夏咸柱，李金中等，大熊猫犬瘟热致死性感染的首次报告[A]．第七届全国养犬学和第三届广东省小动物医学联合会学术研讨会论文集[C]．广州：广东省畜牧兽医学会小动物医学委员会，1997，172-174．
9．李金中，夏咸柱，胡桂学等，我国犬瘟热病毒的生态学调查研究[J]．畜牧兽医学报，1999，30(4)：375～339．
10．高得仪，犬猫疾病学[M]．北京：北京农业大学出版社，1991，2～3．
11．遇秀玲，田克恭，吴哪等，犬实验感染CDV的病理学研究[J]．中国畜禽传染病，1998，20(2)：76～78．
12. Stephenson C.B, Welter J, Thaker S.R, et al. Canine distemper virus (CDV) infection of ferrets as a model for testing morbillivirus vaccine strategies: NYVAC-and ALVAC-based CDV recombinants protect against symptomatic infection [J].Virol, 1997, 71: 1506～1513.
13. Stern-LB, Greenberg-M, Gershoni-JM, et al. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis [J]. Virol, 1995, 69(3): 1661～1668.
14. Hirayama-N, Senda-M, Nakashima-N, et al. Protective effects of monoclonal antibodies
against lethal canine distemper virus infection in mice [J]. Gen Virol, 1991, 72 (11): 2827～2830.
15. Iwatsuki-K, Miyashita-N, Yoshida-E, et al. The nucleotide and predicted amino acid sequence of the fusion protein of recent isolates of canine distemper virus in Japan [J].Vet-Med-Sci. 1998, 60(3): 381～5.
16. Winters K. A, Immuneglobulin class response to canine distemper virus in gnotobiotic dogs [J].Vet Immunology and Immunopathology, 1983/1984,5: 209～215.
17. Evans-S.A, Belsham-G.J, Barrett-T, et al. The role of the 5' nontranslated regions of the fusion protein mRNAs of canine distemper virus and rinderpest virus [J]. Virology. 1990, 177(1): 317～323.
18. Kovanmees J, The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus [J]. Virus Res. 1991, 19: 223～234.
19. Wild-T.F, Buck land-R, Inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein [J]. Gen Virol. 1997, 78 (1): 107～111.
20. Sheshberadaran-H, Norrby-E, McCullough-K.C, et al. The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies [J] Gen Virol. 1986, 67(7): 1381～1392.
21. Orvell-C, Sheshberadaran-H, Norrby-E, et al. Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus [J]. Gen Virol.1985, 66 (3): 443～56.
22. Taylor J, Vaccinia virus recombinants repressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge [J]. Virol. 1991, 4263～4274.
23．李金中，夏咸柱，殷震，等，犬瘟热病毒野毒株融合蛋白主要供能区基因的变异研究[J]．中国兽医学报，1996，16(6)：519～526
24. Curran M. D, The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus [J]. Gen Virol. 1991, 72: 443～417.
25. Iwatsuki K, Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs [J]. Gen Virol. 1997,
78: 373～380.
26. Bolt G, Genetic diversity of the attachment(H) protein gene of current filed isolates of canine distemper virus [J]. Gen Virol. 1997, 78: 367～372.
27. Kingsbury D.W Paramyxoviridae and their replication. In: B. N. Fields(Ed.), Field' s Virol. 1990.Raven Press, New YorK.
28. Wild T.F Mode of entry of morbilliviruses. Vet. Microbiol. 1995, 44: 267～270.
29. Mochizuki M, Hashimoto M, Hagiwara S, et al. Genotypes of canine distemper virus determined by analysis of haemagglutinin genes of recent isolates from dogs in Japan [J] Clin Microbiol 1999, 37: 2936～2942.
30. Blixenkrone-Mollor M, Svanson V, Appel, et al. Antigenic relationships between field isolates of morbilliviruses from different camivores [J]. Arch Virol. 1992, 123: 279～294.
31. Blixenkrone-Moller M, Svanson B, Have p, et al. Studies on manifestations of canine distemper virus infection in an urban dog population [J]. Vet Microbiol, 1993, 37: 163～173.
32. Iwatsuki K, Tokiyoshi S, Hirayama N, et al. Antigenic differences in the H proteins of canine distemper viruses [J]. Vet Microbiol, 2000, 71: 281～286.
33. Iwatsuki K, Miyashita N, Yoshida E, et al. Molecular and phylogenic analyses of the haemagglutinin (H) proteins of isolates of distemper virus from naturally infected dogs [J]. Gen Virol, 1997, 78: 373～380.
34. Yoshida E, Shin Y, Iwatsuki K, et al. Epitopes and nuclear localization analyses of canine distemper virus nucleocapsid protein by expression of its deletion mutants [J]. Vet Microbiol, 1999, 66: 313～320.
35. Beauverger P, Buckland R, Wild T F. Measles virus antigens induce both type-specific and canine ditemper virus cross-reactive cytotoxic Tlymphocytein epitope [J]. Gen Virol, 1993,74: 2357～2363.
36．李昌文，张洪英，刘怀然等，犬瘟热病毒A株N蛋白基因编码区的克隆与序列分析[J]．动物医学进展，2003，24(6)：93～95．
37. Pascal Cherpillod, Andrea Tipold Monika Griot-Wenk, etal. DNA vaccine encoding nucleocapsid and surface proteins of type canine distemper virus protects its natural host against distemper [J]. Vaccine, 2000, 18: 2927～2936.
38．范宝昌，我国登革病毒全长cDNA克隆的构建及其转录体感染性研究[D]．2002．
39. Cherpillod P, Beck K, Zurbriggen A, etal. Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17 [J]. Virology, 1999, 73: 2263～2269.
40．萨姆布鲁J，里奇EF，曼尼阿蒂斯T等，分子克隆操作指南[M]．第二版．北京：科学出版社，1996．
41．姜泊，张亚历，周殿元等，分子生物学常用实验方法[M]．第一版．北京：人民军医出版社，1996．
42．候利华，丙型肝炎病毒NS2和NS5基因的克隆和表达[D]．1999．
43．孙树汉，基因工程原理与方法[M]．第一版．北京：人民军医出版社，2001．
44．温进坤，韩梅，医学分子生物学[M]．第二版．科学出版社，2002．
45．徐向明，吉荣，崔治中等，犬瘟热病毒OP株囊膜糖蛋白F基因片段表达产物的免疫原性[J]．中国兽医学报，2003，23(3)：231-233．
46. Yoshida E, Iwatsuki K, Miyashita N, etal. Molecular analysis of the nucleocapsid protein of recent isolates of canine distemper virus in Japan [J]. Vet Microbiol, 1998, 59: 237～244.
47．耿志贤，田克恭，李钟铎等，犬瘟热病毒的分子生物学研究进展[J]．中国兽医杂志，1997，23(2)45-46．
48. Stettler M, Nucleotide and deduced amino acid sequence of the nucleocapsid protein of the virulent A75/17-CDV strain of canine distemper virus [J]. Vet Microbiol, 1995, 44: 211～217.
49. Sparwasser T, Koch E S, Vebulas R M, etal.Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells [J]. Eru. J. Immunol, 1997, 28: 2045～2051.
50．吴小闲，医学实验动物监测手册[M]．卫生部医学实验动物管理委员会，1992，6：89-90．
51．丁家波，崔治中，韦平等，马立克氏病病毒广西株G2囊膜糖蛋白gI基因的克隆和表达[J]．中国兽医学报，2001，21(2)：109～112．
52. Messling V, Harder TC, Moennig V, et al. Rapid and sensitive detection of immnoglobulin M (M) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. J Clin Microbiol, 1999, 37: 1049-1056.