摘要
猫泛白细胞减少症是由猫泛白细胞减少症病毒(Feline panleukopenia virus,FPV)引起猫科动物以高热、呕吐、脱水、白细胞严重减少和出血性肠炎为主要特征的急性、高度接触性传染病。在自然条件下,FPV可感染虎、豹、狮、水貂、浣熊等多种食肉类动物,具有很高的致死率,严重威胁圈养及野生虎等大型猫科动物的养殖和保护,迫切需要建立能够同时大批量检测多种动物FPV感染的通用诊断方法。
VP2蛋白为FPV主要结构蛋白,是FPV衣壳的主要成分,暴露在衣壳蛋白表面,为FPV的主要免疫保护性抗原蛋白,能诱导机体产生中和抗体,是研究FPV诊断抗原制备的首选对象。目前,尚未见有利用原核表达系统完整表达VP2基因的报道。
本研究利用CRFK细胞对某东北某虎林园发生传染性出血性肠炎的老虎粪便进行了病毒的分离和鉴定,对分离的虎源FPV保护性抗原VP2蛋白全长基因进行克隆与遗传变异分析。在此基础上,利用原核表达载体PGEX-6P-1在BL21宿主菌中表达该基因。以表达的重组VP2蛋白作为抗原,通过建立检测家猫FPV自然感染抗体的间接ELISA方法来研究2种属来源FPV VP2蛋白的抗原特性变化及该蛋白作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。基于VP2蛋白为检测FPV优势诊断抗原特性,利用纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,以辣根过氧化物酶(HRP)标记的SPA作为广谱第二抗体,初步建立适用于2种猫科动物猫泛白细胞减少症血清抗体检测的SPA-ELISA方法。用建立的SPA-ELISA方法对临床30份猫血清样本、86份虎血清样本进行检测,同时与HI、间接ELISA方法进行比较。在此基础上,以纯化的重组VP2蛋白为免疫原,FPV全病毒作为筛选抗原,运用淋巴细胞杂交瘤技术,制备虎源FPV VP2蛋白特异性单克隆抗体。结果如下:
利用CRFK细胞从老虎粪便中成功分离出强毒株FPV-HLJ2,测得该毒株VP2基因全长为1755个核苷酸,编码584个氨基酸。序列分析表明该病毒与虎源FPV-HLJ株核苷酸同源性为100%,实为同一毒株。
利用原核表达系统实现了虎源FPVVP2基因的表达,SDS-PAGE和Western blot显示表达产物以包涵体形式存在,大小约为84 ku,并具有抗原性。其中目的蛋白58 ku, GST标签蛋白26ku。在家猫猫泛白细胞减少症间接ELISA抗体检测法的应用中,该外源表达蛋白亦显示出很好的抗原特异性,VP2蛋白上一些位点氨基酸的变异并未影响FPV抗原性,表达的重组VP2蛋白可以作为诊断抗原用于家猫FPV抗体的间接ELISA检测,初步证明该蛋白具有作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。
以纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,建立了SPA-ELISA方法,确定了检测2种猫科动物FPV抗体的SPA-ELISA最佳操作程序,批内及批间重复试验均显示变异系数小于10%。对临床30份猫血清样本、86份虎血清样本的对比检测结果显不,SPA-ELISA方法检出率较高,大于HI法的检出率,与间接ELISA法接近。三种检测方法检测猫血清的总体符合率为96.7%。在虎血清检测中,SPA-ELISA方法的阳性检出率亦高于HI方法,二者总体符合率为94.2%。
以重组VP2蛋白作为免疫抗原,用FPV全病毒作为筛选抗原,采用传统的杂交瘤技术,经过间接ELISA方法筛选和有限稀释法亚克隆,最终获得了1株可稳定分泌虎源FPVVP2蛋白特异性单克隆抗体的杂交瘤细胞株3C4。间接ELISA方法测定3C4株单抗腹水效价为1:12800,亚类鉴定为IgG2a类型,间接免疫荧光鉴定试验显示,该株单抗能使接种虎源FPV的CRFK细胞产生亮绿色荧光,而免疫印迹试验显示该株单抗未见特异性反应带。
本研究初步明确了2种属来源的FPV抗原性差异,实现了FPV VP2蛋白的初步实践应用。基于FPV VP2蛋白建立的SPA-ELISA诊断方法,解决了目前虎等猫科动物FPV抗体检测方法非常单一、不能大批量检测等问题,有望用于豹、狮、熊猫、豹猫、浣熊、水貂、猞猁等更多种类动物血清猫泛白细胞减少症病毒抗体的检测。对实现适用于更多种圈养及野生猫科动物等猫瘟热病的快速诊断、疫情监测、流行病学调查和免疫抗体效价测定具有重要意义。基于FPV VP2蛋白制备的FPV VP2蛋白特异性单克隆抗体,为FPV、CPV和MEV诊断及分子生物学研究提供一种更为敏感和特异的试剂。
Feline Panleukopenia is an acute and highly contagious viral disease caused by Feline Panleukopenia virus (FPV). The main characteristics of this disease are high fever, vomiting, clehydration, serious decrease of the white blood cells and haemorrhagic enteritis. Under the natural conditions, multiple kinds of carnivores, such as tigers, leopards, lions minks and raccoons, can be infected by FPV with a high mortality rate; and which has posed a serious threat to the feeding and protection of big cats including both domestic and wild animals of the Felinedae. Therefore, a common diagnostic method is badly needed to set up to test FPV of various animals simultaneously on a large scale.
VP2 protein is the main component of the capsid and major structure protein of FPV which is exposed to the surface the capsid protein. Furthermore, it is also the main immune protein antigen which is able to induce the body to produce antibodies. Thus, VP2 protein is the best for the research of the preparation of FPV diagnostic antigens. At the moment, there haven't researches of whole VP2 gene expression by prokaryotic expression system been reported.
FPV strain was isolated from drops of diarrhea tigers from a wildlife zoo in China and identified by culturing on the CRFK cell line. VP2 gene of FPV was cloned and analyzed. The amplified VP2 gene was subcloned into the prokaryotic expressing vector pGEX-6p-1, and then transferred into E.coli BL21(DE3) for expression under IPTG treatment. The purified recombinant VP2 protein was analyzed by Western Blot. An indirect enzyme-linked immunosorbent assay (ELISA) for target antibodies detecting was developed by the purified recombinant VP2 protein to study its characteristics of Common Diagnostic Antigen of VP2 protein. Since the purified recombinant VP2 protein is a superior diagnostic antigen, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of cats and tigers.30 clinical serumal samples of domestic cats and 86 serumal samples of tigers were tested by SPA-ELISA, HI and indirect ELISA to compare total coincidence ratio. And then, monoclonal antibody (McAb) against recombinant VP2 protein of tiger feline panleukopenia virus were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger FPV. The results are as follows:
The FPV-HLJ2 was isolated from drops of diarrhea tiger by culturing on the CRFK cell line successfully;sequencing analysis of VP2 gene showed that the full length of VP2 gene was 1755 bp and encoded 584 amino acids, and FPV-HLJ2 isolate shares 100% identity with FPV-HLJ isolate (Feline Panleukopenia Virus tiger strain), while the host tropism amino acid changes occurred in the same point.
VP2 gene was highly effectively expressed in E. coli. SDS-PAGE and Western blotting assays revealed that the fusion protein of about 84 ku was obtained from the prokaryotic expression system, including a 58 ku VP2 protein and a 26 ku GST-tag protein, and had good immunoreactivity. The indirect ELISA assay indicated a good antigenic specificity of the expressed VP2 protein and the feasibility to be used as diagnostic antigen for the diagnosis of FPV infection in cats. Some variation of sites AA on the VP2 Protein didn't affect the antigenic nature of FPV. The potential of recombinant VP2 protein to be used as Common Diagnostic Antigen.for the diagnosis of FPV infection in felids was revealed.
Based on the purified recombinant VP2 protein of tiger FPV, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of domestic cats and tigers. The assay was optimized. Variation coefficient of intraassay and interassay about SPA-ELISA were all less than 10%. The comparing detection among the 30 serumal samples of domestic cats and 86 serumal samples of tigers showed that the detection rate by SPA-ELISA was higher than HI, and the result was close to indirect ELISA. The total coincidence ratio of the three detecting methods were 96.7% by detecting 30 serum samples of cats. The coincidence ratio of the SPA-ELISA and HI was 94.2% by detecting 86 serumal samples of tigers. All these results indicated that SPA-ELISA could be a good method for serological detection of FPV antibodies in many species of felines.
Monoclonal antibody 3C4 against recombinant VP2 protein of tiger FPV were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger feline panleukopenia virus. The antibody titers of ascites for the hybridoma lines were 1:12800. The subtype of monoclonal antibodies was determined as IgG2a. Indirect immunofluorescent test indicated that the monoclonal antibody could specifically react with FPV, and it may be used as a valuable tools for diagnosis of FPV.
The difference between FPVs from two different kinds of mammals was initially known through the study, and the preliminary use of FPV VP2 Protein come true. The way of SPA-ELISA based on FPV VP2 protein, solving the problem that the present detections are too simple or can't detect a large amount of samples at one time, is likely to be used to test FPV antibodies in animals' serum such as leopards, lions, pandas, ocelots, raccoons, minks, lynx, etc. There will be great significance in realizing quick diagnosis, epidemic observation, epizootic research and detection of antibody potency. The neutralizing monoclonal antibody of FPV VP2 Protein based on FPV VP2 provides a more sensitive and different reagent for FPV, CPV and MEV diagnosis.
引文
[1]赵新泰,吴祥甫,李载平,等.貂肠炎病毒的分子克隆和结构研究.病毒学报,1991,7(3):245-240
[2]殷震,刘景华.动物病毒学.第二版.北京:科学出版社,1997:1162-1165
[3]J.R, Pattison. Parvoviruses:Medical and Biological Aspects. Virology,1990(2):1765-1784
[4]S.F. Cotmore, P. Tattersall. The Autonomously Replicating Parvoviruses of Vertebrates. ADV. Virus Res,1987(33):91-174
[5]A Steinel, CR Parrish, ME Bloom, et al. Parvovirus Infections in Wild Carnivores. J Wildl Dis, 2001,37(3):594-607
[6]K Gamoh, M Senda, Y Inoue, et al. Efficacy of an Inactivated Feline Panleucopenia Virus Vaccine Against a Canine Parvovirus Isolated from a Domestic Cat. Vet Rec,2005, 157(10):285-287
[7]K Nakamura, Y Ikeda, T Miyazawa, et al. Characterisation of Cross-reactivity of Virus Neutralising Antibodies Induced by Feline Panleukopenia Virus and Canine Parvoviruses. Res Vet Sci,2001,71(3):219-222
[8]PS Carman, RC Povey. Comparison of the Viral Proteins of Canine Parvovirus-2, Mink Enteritis Virus and Feline Panleukopenia Virus. Vet Microbiol,1983,8(5):423-435
[9]JD Tratschin, GK McMaster, G Kronauer, et al. Canine Parvovirus:Relationship to Wild-type and Vaccine Strains of Feline Panleukopenia Virus and Mink Enteritis Virus. J Gen Virol,1982, 61 (1):33-41
[10]Horiuchi M, Yamaguchi Y, Gojobori T, et al. Differences in the evolutionary pattern of feline panleukopenia virus, canine parvovirus.Virology,1998,249(2):440-52
[11]谢之景,夏咸柱,厄荣良,等.犬细小病毒基因型的调查.中国兽医学报,2004,24(5):421-24
[12]SHACKELTON L A, PARRISH C R, TRUYEN U, et al. High rate of viral evolution associated with the emergence of carnivore parvovirus[J]. J Virol,2005,11:379-384
[13]Parrish C R, Aquadro C F, Strassheim M L, et al. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovims.Journal of virology.1991,65(12):6544-52
[14]谢之景,夏咸柱.犬细小病毒的进化.东北师大学报,2003,35(8):82-84
[15]STEINEL A, MUNSON L, VUUREN M, et al.Genetic characterization of fline parvovirus sequences from various canivores[J]. J Gen Virol,2000,81:345-350
[16]NAKAMURA K, SAKAMOTO M, IKEDA Y, et al. Pathogenic potential of canine parvovims type 2a and 2c in domestic cats[J]. Clin Diagn Lab Iimmunol,2001,8:663-668
[17]Ikeda Y, Mochizuki M, Naito R, et al. Predominace of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats[J]. Virology,2000,278 (1):13-19
[18]Mochizuki M, Harasawa R, Nakatani H. Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan. Vet Microbiol,1993,38(1-2):1-10
[19]Osterhaus AD, van Steenis Q de Kreek P. Isolation of a virus closely related to feline panleukopenia virus from dogs with diarrhea Zentralbl Veterinarmed B,1980,27(1):11-21
[20]蔡宝祥.家畜传染病学.北京:中国农业出版社,2006:361-636
[21]侯云德.分子病毒学[M].北京:学苑出版社,1990
[22]李君阁,朱尽国.犬细小病毒和猫泛白细胞减少症病毒理化学和生物学特性的比较研究.国外兽医学-畜禽传染病,1985,5(6):58-61
[23]FW Scott. Virucidal Disinfectants and Feline Viruses. Am J Vet Res,1980,41(3):410-414
[24]蔡家利,张振兴,蔡宝祥.云豹病毒性肠炎的研究-病毒鉴定.中国兽医科技,1980(4):6-8
[25]范泉水,夏咸柱.血凝和血凝抑制试验诊断犬细小病毒性肠炎.云南畜牧兽医,1998,(3):8-10
[26]邱薇,夏咸柱,范泉水,等.犬细小病毒血凝抑制抗体和中和抗体的关系.中国兽医科技,2000,30(10):3-5
[27]A.Simpson Alan, Chandrasekar Veda, M Gail, et al. Host Range and Variability of Calcium Binding by Surface Loops in the Capsids of Canine and Feline Parvovirus. Journal of Molecular Biology,2000,300 (4):597-610
[28]Woolhouse, E.J Mark, L.H Taylor, et al. Population Biology of Multihost Pathogens. Science, 2001(5):292-298
[29]Horiuchi M, Goto H, Ishiguro N, Shinagawa M. Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses. J Gen Virol,1994,75 (Pt 6):1319-28
[30]Parker JS, Murphy WJ, Wang D, O'Brien SJ, Parrish CR. Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells. J Virol,2001, 75(8):3896-902
[31]Palermo L M, Hueffer K, Parrish C R. Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses. J Virol,2003,77(16):8915-23
[32]Spitzer A L, Parrish C R, Maxwell IH. Tropic determinant for canine parvovirus and feline panleukopenia virus functions through the capsid protein VP2. J Gen Virol,1997,78 (Pt 4):925-8
[33]Johnson RH, Siegl G, Gautschi M. Characteristics of feline panleucopaenia virus strains enabling definitive classification as parvoviruses. Arch Gesamte Virusforsch 1974;46(3-4):315-24.
[34]Lappin MR AJ, Simpson D, Jensen WA. Use of serologic tests to predict resistance to feline herpesvirus 1, feline calicivirus, and feline parvovirus infection in cats. J Am Vet Med Assoc, 2002, Jan 1,220(1):38-42
[35]Chang SF, Sgro JY, Parrish CR. Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. J Virol,1992,66(12):6858-67
[36]Truyen U, Agbandje M, Parrish CR. Characterization of the feline host range and a specific epitope of feline panleukopenia virus. Virology,1994,200(2):494-503
[37]渠川玫,徐在海,彭传贵等.细小病毒在乳猫肾细胞中的增殖动态.中国畜禽传染病,1994,(5):5-7
[38]RW Simons, WM Acree, RC Stewart, et al. A New Vaccine Strain of Feline Panleukopenia Virus Grown in Ferret Cell Culture. Vet Med Small Anim Clin,1974,69(1):40-44
[39]Strains Enabling Definitive Classification as Parvoviruses. Arch Gesamte Virusforsch,1974, 46(3-4):315-324
[40]R Bergman, B Sundquist, L Stroman. Production of a Feline Parvovirus Vaccine Using Monolayer Cell Systems in Roller Flasks and Microcarriers. Dev Biol Stand,1983,(55):77-78
[41]Cornelis lenghaus, Tham K. MUN,AND Michael J. Studdert. Feline Panleukopenia Virus Replicates in Cells in Which Cellular DNA Synthesis is Blocked[J]. Journal of virology,1985, 53(2):345-349
[42]E Rivera, KA Karlsson, R Bergman. The Propagation of Feline Panleukopenia Virus in Micro-carrier Cell Culture and Use of the Inactivated Virus in the Protection of Mink Against Viral Enteritis. Vet Microbiol,1987,13(4):371-381
[43]JR Gorham, GR Hartsough, N Sato, et al. Studies on Cell Culture Adapted Feline Panleukopenia Virus:Virus neutralization & antigenic extinction. Vet Med Small Anim Clin,1966,61(1):35-40
[44]Rhode S L, Paradis P R. Parvovims genome:Nucleotide sequence of HI and mapping of its genes by hybrid arrest translations. Virol.1983,45:173-176
[45]Astell C R, Thomson M, Merchlinsky M J, et al. The complete DNA sequence of minute virus of mice,an autonomous parvovirus. Nucleic acids Res,1984,11:999-1000.
[46]Shade R O, Blundell M C, Cotmore S R, et al. Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. Virology, 1986,58:921-923
[47]CR Parrish. Host Range Relationships and the Evolution of Canine Parvovirus. Vet Microbiol, 1999,69(1-2):29-40
[48]MJ Studdert, JE Peterson. Some Properties of Feline Panleukopenia Virus. Arch Gesamte Virusforsch,1973,42(4):346-354
[49]徐耀先,周晓峰,刘立德.分子病毒学.武汉:湖北科学技术出版社,2000,265-269
[50]Fields B N, Knipe D M, Howley P M. Fundamental Virology.3rd edition. Philadelphia: Lippincott Willians&Wilkins Publisher,1996
[51]MA Goncalves. Adeno-associated Virus:from Defective Virus to Effective Vector. Virology Journal,2005, (2):43
[52]Wen Yuan. Studies neutralization. Doctor of canine parvovirus capsid-assembly, disassembly and antibody Dissertation of Cornell University.2000,30-31
[53]CR Parrish, CF Aquadro, ML Strassheim, et al. Rapid Antigenic-type Replacement and DNA Sequence Evolution of Canine Parvovirus. Journal of virology,1991,65:6544-6552
[54]Johnson, F. B., Blacklow, N. R., and Hoggan, M. D. Immunological reactivity of antisera prepared against the sodium dodecyl sulfate-treated structural polypeptides of adenovirus-associated virus. J Virol,1972,9:1017-1026
[55]Santaren, J. F., Ramirez, J. C., and Almendral, J. M. Protein pecies of the parvovirus minute virus of mice strain MVMp:involvement of phosphorylated VP-2 subtypes in viral momho}enesis. J Virol,1993,67:5126-5138
[56]Thllis G E, Burger L R, and Pintel D J. The minor capsid protein VP1 of the autonomous parvovirus minute virus of mice is dispensible for encapsidation of progeny single stranded DNA but is required for infectivity.J.Virol,1993,67:131-141
[57]ML Strassheim, A Gruenberg, P Veijalainen, et al. Two Dominant Neutralizing Antigenic Determinants of Canine Parvovirus are Found on the Threefold Spike of the Virus Capsid. Virology,1994,198(1):175-184
[58]Langerveld JPM, Casal JI, Cone's E, et al. Effective induction of neutralizing antibodies with the amino terminus of VP2 of canine parvovirus as a synthetic peptide. Vaccine,1994,12:1473-80
[59]JS Parker, CR Parrish. Canine Parvovirus Host Range is Determined by the Specific Conformation of an Additional Region of the Capsid. J Virol,1997,71(12):9214-9222
[60]corte's E, San Martin C, Langeveld J, et al. Topographical analysis of canine parvovirus virions and recombinant VP2 capsids. J Gen Virol,1993,74:2005-10
[61]Tsao J, M. S. Chapman, M. Agbandje, et al. The three-dimensional structure of canine parvovirus and its functional implications. Science,1991,251:1456-1464
[62]Cotmore, S.F., Tattersall, P.The autonomously replicating parvoviruses of vertebrates. ADV. Virus Res,1987,33:91-174
[63]Fields B N, Knipe D M, Howle:y P M. Lippincott Willians&Wilkins Publisher. Fundamental Virology.3rd Edition. Philadelphia:1996
[64]Christensen, J., Cotmore, S. F., and Tattersall, P. A novel cellular site-specific DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication. J Virol, 1997,71:1405-1416
[65]于亚丽,华育平,曾祥伟,等.虎源猫泛白细胞减少症病毒VPK、VP2和NS1基因的克隆 与序列分析.东北林业大学学报,2009,37(1):83-85
[66]张洪英,刘立奎,姜建宏,等.猫细小病毒NS部分基因的克隆和序列分析.中国预防兽医学报,2004,26(6):412-415
[67]Chen K.C., B.C.Shull, M. Lederman, et al. Complete nueleotide sequence and genome organization of bovine parvovirus. J.Virol,1986,60:1085-1097.
[68]Cotmore, S. F., and P. Tattersall. The autonomously replicating parvoviruses. Adv. Virus Res, 1987,33:91-174
[69]Shade, R. O., M. C. Blundell, S. F. Cotmore, et al. Nucleotide sequence and genome organization of human parvovirus B 19. isolated from the serum of a child during aplastic crisis. J. Viral,1986,58:921-936
[70]Anouja F, Wattiez R, Moussedt S et al. The cytotoxicity of parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins. J Virol.1997,71(6):4671-4678
[71]罗祖玉,施志仪,林万敏.自主性细小病毒非结构蛋白NS1对转化细胞的毒性及其调控DNA复制和转录的机制.复旦学报(自然科学版),1999,38(5):578-583
[72]Carlson J O, Lynde-Maas M K, Shen Z D. A nonstructural protein of feline panleukopenia virus:expression in Escherichia coli and detection of multiple forms in infected cells. J Virol, 1987,61(2):621-4
[73]蔡宝祥,殷震,谢三星,等.动物传染病诊断学.南京:江苏科学技术出版社,1994
[74]MJ Studdert, CM Kelly, KE Harrigan. Isolation of Panleucopaenia Virus from Lions. Vet Rec, 1973,93(6):156-158
[75]李刚.猫泛白细胞减少症病毒的分离与鉴定.病毒学报,1985,1(4):349-353
[76]蔡家利,张振兴,蔡宝祥.云豹病毒性肠炎的研究-病毒鉴定.中国兽医科技,1980,(4):6-8
[77]许树林,沈秀丽,刘鼎新.细小病毒在肉食动物中的感染流行.黑龙江畜牧兽医,1996,(10):40-43.
[78]苏洁,姜骞,李慕瑶,等.猫泛白细胞减少症病毒FPLV/XJ-1的分离鉴定.中国比较医学杂志,2007,17(8):56
[79]杨松涛,王立刚,戈锐,等.虎源猫泛白细胞减少症病毒的分离鉴定.兽类学报,2007,27(2):170-174
[80]邱薇,夏咸柱,范泉水,等.桂林老虎猫瘟热病毒的分离鉴定.中国预防兽医学报,2000,22(4):249-51
[81]Goto H, Horimoto M, Shimizu K, Hiraga T, Matsuoka T, Nakano T, et al. Prevalence of feline viral antibodies in random-source laboratory cats. Jikken Dobutsu,1981,30(3):283-90
[82]Addie DD, Toth S, Thompson H, Greenwood N, Jarrett JO. Detection of feline parvovirus in dying pedigree kittens. Vet Rec,1998,142(14):353-6
[83]RoelkeM E, ForresterD J, JacobsonE R, KolliasG V, ScottFW, Barr M C. Seroprevalence of infectious disease agents in free-ran-ging Florida panthers (Felis concolorcoryi). J Wild Dis, 1993,29(1):36-49
[84]Paul-Murphy J, Work T, Hunter D, McFie E, Fjelline D. Serologic survey and serum biochemical reference ranges of the free-ranging mountain lion(Felis concolor)in California. J Wildl Dis,1994,30(2):205-215
[85]刘丹,于亚丽,华育平,等.圈养东北虎FPV感染情况的初步调查与研究.野生动物杂志,2008,29(4):171-173
[86]杨松涛,周明,王铁成,等.猫科动物猫泛白细胞减少症血清抗体调查.兽类学报,2008,28(1):101-104
[87]K Berthier, M Langlais, P Auger, et al. Dynamics of a Feline Virus with Two Transmission Modes within Exponentially Growing Host Populations. Proc Biol Sci,2000,267(1457): 2049-2056
[88]De Lahunta A. Comments on Cerebellar Ataxia and Its Congenital Transmission in Cats by Feline Panleukopenia Virus. J Am Vet Med Assoc,1971,158(6)Suppl 2:901-906
[89]RM Herndon, G Margolis, L Kilham. The Synaptic Organization of the Malformed Cerebellum Induced by Perinatal Infection with the Feline Panleukopenia Virus (PLV). Ⅱ. The Purkinje Cell and its afferents. J Neuropathol Exp Neurol,1971,30(4):557-570
[90]L Kilham, G Margolis, ED Colby. Cerebellar Ataxia and Its Congenital Transmission in Cats by Feline Panleukopenia Virus. J Am Vet Med Assoc,1971,158(6)Suppl 2:888
[91]DH Percy, FW Scott, DM Albert. Retinal Dysplasia Due to Feline Panleukopenia Virus Infection. J Am Vet Med Assoc,1975,167(10):935-937
[92]Y Ikeda, J S hinozuka, T Miyazawa, et al. Apoptosis in Feline Panleukopenia Virus-infected Lymphocytes. J Virol,1998,72(8):6932-6936
[93]A Okaniwa, A Yasoshima, A Kojima, et al. Fine Structure of Epithelial Cells of Lieberkuhn's Crypts in Feline Panleukopenia. Natl Inst Anim Health Q (Tokyo),1976,16(4):167-175
[94]JH Carlson, FW Scott, JR Duncan. Feline Panleukipenia. Ⅰ. Pathogenesis in Germfree and Specific Pathogen-free cats. Vet Pathol,1977,14(1):79-88
[95]EH Fowler, MW Rohovsky. Enzyme Histochemistry of Lymphoid Tissues in Germfree Cats Inoculated with Feline Infectious Enteritis (Feline Panleukopenia) Virus. Am J Vet Res,1970, 31(11):2061-2069
[96]EH Fowler, MW Rohovsky. Enzyme Histochemistry of the Small Intestine in Germfree and Specific-pathogen-free Cats Inoculated with Feline Infectious Enteritis (feline panleukopenia) Virus. Am J Vet Res,1970,31(11):2055-2060
[97]LO Wosu. Feline Panleucopenia--in vivo Infectivity Studies. Vet Microbiol,1988,16(2):137-143
[98]W Herbst, H Krauss. Electron Microscopy in the Diagnosis of Enteritis in Cats. Tijdschr Diergeneeskd,1989,114(6):328-333
[99]A Okaniwa, A Yasoshima, K Doi, et al. Morphology of Feline Kidney Culture Cells Infected with Feline Panleukopenia Virus. Natl Inst Anim Health Q (Tokyo),1977,17(2):58-66
[100]夏咸柱,陈跃鹏,孙永祥,等.猫泛白细胞减少症病毒的分离与鉴定.中国人民解放军兽医大学学报,1986,6(2):132-135
[101]李六金,李成,姜焕宏,等.猫鼻气管炎病毒、杯状病毒、泛白细胞减少症病毒的分离及其形态学观察.中国预防兽医学报,2001,23(5):341-344
[102]黄金华,刘长坤,罗春明,等.用免疫金标电镜技术检测病毒及其抗体.中国动物检疫,1998,15(4):1-2
[103]M Mochizuki, M Horiuchi, H Hiragi, et al. Isolation of Canine Parvovirus from a Cat Manifesting Clinical Signs of Feline Panleukopenia. J Clin Microbiol,1996,34(9):2101-2105
[104]T Miyazawa, Y Ikeda, K Nakamura, et al. Isolation of Feline Parvovirus from Peripheral Blood Mononuclear Cells of Cats in Northern Vietnam. Microbiol Immunol,1999,43(6):609-612
[105]FW Scott, CK Csiza, JH Gillespie. Feline Viruses. Ⅳ. Isolation and Characterization of Feline Panleukopenia Virus in Tissue Culture and Comparison of Cytopathogenicity with Feline Picornavirus, Herpesvirus, and Reovirus. Cornell Vet,1970,60(2):165-182
[106]许树林,沈秀丽.肉食动物细小病毒特性比较及亲缘关系.黑龙江畜牧兽医,1997,2:38-40
[107]亢文华,赵凤龙,郝霖雨,等.猫泛白细胞减少症的研究进展.中国畜牧兽医,2008,35(8):112-116
[108]姜平.兽医生物制品学.北京:中国农业出版社,2001
[109]丁关娥,张振兴.用醛化猪红细胞进行猫泛白细胞减少症病毒血凝试验.中国兽医科技,1986,2:48-49
[110]CR Parrish, LE Carmichael, DF Antczak. Antigenic Relationships between Canine Parvovirus Type2, Feline Panleukopenia Virus and Mink Enteritis Virus Using Conventional Antisera and Monoclonal Antibodies, Arch Virol,1982,72(4):267-278
[111]PM Veijalainen, E Neuvonen, A Niskanen, et al. Latex Agglutination Test for Detecting Feline Panleukopenia Virus, Canine Parvovirus, and Parvoviruses of Fur Animals. J Clin Microbiol, 1986,23(3):556-559
[112]S. Winston, S. Fiscus, L. Hesterberg, T. Matsushita, M. Mildbrand, J. Porter, Y. Teramoto. Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Veterinary Immunology and Immunopathology,1987,17(4):453-464
[113]刘宏伟,田克恭,渠川玫,等.犬细小病毒酶标试剂盒的研制.中国畜禽传染病,1994,(5):10-13
[114]吴石金,周建群.运用ELISA快速检测CPV抗体方法的建立与均衡性研究.微生物学杂志,2002,22(4):14-16
[115]E Rivera, KA Karlsson. A Solid-phase Fluorescent Immunoassay for Detecting Canine or Mink Enteritis Parvoviruses in Faecal Samples. Vet Microbiol,1987,15(1-2):1-9
[116]蒋红,时建东,吴小贤.猫病毒血清学检测方法的建立及初步应用.中国实验动物学杂志.1999,9(1):39-41
[117]M Mochizuki, S Konishi, M Ajiki, et al. Comparison of Feline Parvovirus Subspecific Strains Using Monoclonal Antibodies Against a Feline Panleukopenia Virus. Nippon Juigaku Zasshi, 1989,51(2):264-272
[118]钱建飞,陈溥言,蔡宝祥,等.抗猫泛白细胞减少症病毒单克隆抗体的制备及其生物学特性鉴定.中国兽医学报,2000,20(7):339-342
[119]FW Scott, CK Csiza, JH Gillespie. Feline viruses. Ⅴ. Serum-neutralization Test for Feline Panleukopenia. Cornell Vet,1970,60(2):183-191
[120]Y Ikeda, J Shinozuka, T Miyazawa, et al. Apoptosis in Feline Panleukopenia Virus-infected Llymphocytes. J Virol,1998,72(8):6932-6936
[121]RH Johnson, G Siegl, M.Gautschi. Characteristics of Feline Panleucopaenia Virus Strains Enabling Definitive Classification as Parvoviruses. Arch Gesamte Virusforsch,1974,46(3-4):315-324
[122]Chman H. Genetic applification of polymerase chain reaction [J]. Genetics,1988,120:621
[123]Erlich ⅡA, Gelfand D, Snisky JJ. Science,1991,252:1643-1651
[124]乔军,孟庆龄,夏咸柱,等.用PCR法检测东北虎感染猫细小病毒的研究.畜牧与兽医,2001,(5):14-16
[125]刘维全,范泉水,江禹,等.肉食兽细小病毒通用PCR诊断技术的建立.中国兽医学报,2001,21(3):249-251
[126]B Schunck, W Kraft, U Truyen. A Simple Touch-down Polymerase Chain Reaction for the Detection of Canine Parvovirus and Feline Panleukopenia Virus in Feces. J Virol Methods,1995, 55(3):427-433
[127]刘雪燕,黄韧,刘忠华,等.猫肾F81传代细胞中犬细小病毒原位PCR检测方法.中国实验动物学报,2001,9(3):146-149
[128]刘忠华,钟翎,贺星亮,等.用PCR技术鉴定犬细小病毒弱毒疫苗株.中国兽医学报,2003,23(5):444-446
[129]刘忠华,钟翎,黄韧,等.半套式PCR技术区别检测犬细小病毒疫苗株和强毒株的研究.中国病毒学,2003,18(4):401-403
[130]T Hirasawa, T Kaneshige, K Mikazuki. Sensitive Detection of Canine Parvovirus DNA by the Nested Polymerase Chain Reaction. Vet Microbiol,1994,41(1-2):135-145
[131]吴乃虎.基因工程原理[M].第2版,北京:高等教育出版社,1998
[132]朱平.PCR基因扩增实验操作册[M].北京:中国科学技术出版社,1992,533-539
[133]Glad C, Grubb A O. Immunocapillarymigration with enzymelabelled antibodies:rapid quantification of creactive protein in human plasm[J]. AnalBiochem,1981,116:335-340
[134]Zuk RF, Ginsberg VK, Houts T, et al. Enzyme immuno chromatography:aquantitative immunoassay requiring no instrumentation[J]. Clin Chem,1985,31:1144-1154
[135]吴冯波,侯惠仁.免疫层析分析进展[J]. Chin J Med Lab Sci,1998,21(2):122-124
[136]王中力,宋晓晖,陈西召,等.犬细小病毒免疫胶体金诊断技术的研究.中国预防兽医学报,2004,26(1):62-66
[137]J Esfandiari, B Klingeborn. A Comparative Study of a New Rapid and One-step Test for the Detection of Parvovirus in Faeces from Dogs, Cats and Mink. J Vet Med B Infect Dis Vet Public Health,2000,47(2):145-153
[138]卢锦汉,章以浩,赵铠.医学生物制品学.北京:人民卫生出版社,1995:127-128
[139]乔军,夏咸柱,杨松涛,等.犬冠状病毒S糖蛋白基因重组犬2型腺病毒的构建及其免疫原性研究.微生物学报,2005,45(4):588-592
[140]FW Scott, AF Glauberg. Aerosol Vaccination Against Feline Panleukopenia. J Am Vet Med Assoc,1975,166(2):147-149
[141]GR Sampson, FT Counter, BF Schlegel, et al. Antibody Response of Cats Vaccinated with an Inactivated Cell Culture Feline Panleukopenia Vaccine. J Am Vet Med Assoc,1972, 160(12):1619-1621
[142]FW Scott, CM Geissinger. Long-term Immunity in Cats Vaccinated with an Inactivated trivalent vaccine. Am J Vet Res,1999,60(5):652-658
[143]RV Pollock, LE Carmichael. Use of Modified Live Feline Panleukopenia Virus Vaccine to Immunize Dogs Against Canine Parvovirus. Am J Vet Res,1983,44(2):169-175
[144]M Camero, A Cavalli, G Bozzo, et al. A Severe Dual Infection by Feline Panleukopenia Virus and Feline Calicivirus in an Adult Cat. New Microbiol,2004,27(1):79-82
[145]JR Gorham, GR Hartsough, D Burger, et al. The Preliminary Use of Attenuated Feline Panleukopenia Virus to Protect Cats Against Panleukopenia and Mink Against Virus Enteritis. Cornell Vet,1965,55(4):559-566
[146]G Siegl, G Kronauer. A Plaque Assay for Feline Panleukopenia Virus. J Gen Virol,1980, 46(1):211-218
[147]Sutter G, Moss B. Non-replicating vaccinia vector efficiently expresses recombinant genes.Proc Natl Acad Sci USA,1992,89:10847-10851.
[148]WHO. Potential use of live viral and bacterial vectors for vaccines. Vaccine,1990,8:425-437
[149]JC Duenwald, JM Holland, JR Gorham, et al. Feline Panleukopenia:Experimental Cerebellar Hypoplasia Produced in Neonatal Ferrets with Live Virus Vaccine. Res Vet Sci,1971, 12(4):394-396
[150]JA Spencer, Burroughs R. Antibody Response of Captive Cheetahs to Modified-live Feline Virus Vaccine. J Wildl Dis,1991,27(4):578-583
[151]Gupka R K, Siber G R. Adjuvants for human vaccines-current status, problems and future prospects. Vaccine,1995,13:1263-1276
[152]Alving C R. Lipopolysaccharide, lipid A, and liposomes containing lipid A as immunologic adjuvants. Immunobiology,1993,187:430-446
[153]Reed R C, Louis-Wileman V, Cosmai E V, et al. Multiple antigen constructs (MACs):induction of sterile immunity against sporozoite stage of rodent malaria parasites,Plasmodium berghei and Plasmodium yoelii. Vaccine,1997,15:482-488
[154]Adams S E, Dawson K M, Gull K, et al. The expression of hybrid HIV:virus-like particles in yeast. Nature,1987,329:68-70
[155]MR Lappin, WA Jensen, TD Jensen, et al. Investigation of the Induction of Antibodies Against Crandell-Rees Feline Kidney Cell Lysates and Feline Renal Cell Lysates after Parenteral Administration of Vaccines Against Feline Viral Rhinotracheitis, Calicivirus, and Panleukopenia in Cats. Am J Vet Res,2005,66(3):506-511
[156]WB Clark, FG Diegmann, DK McIntosh, et al. Feline Rhinotracheitis-calici Vaccine and Feline Rhinotracheitis-calici-panleukopenia Vaccine:Field Evaluation for Safety. Vet Med Small Anim Clin,1980,75(3):415-420
[157]S Dawson, K Willoughby, RM Gaskell, et al. A Field Trial to Assess the Effect of Vaccination Against Feline Herpesvirus, Feline Calicivirus and Feline Panleucopenia Virus in 6-week-old Kittens. J Feline Med Surg,2001,3(1):17-22
[158]RC Povey. The Efficacy of Two Commercial Feline Rhinotracheitis-calicivirus-panleukopenia Vaccines. Can Vet J,1979,20(10):253-260
[159]V Franke, K Danner. Experiences with a New Cat Coryza-panleukopenia-rabies Combined Vaccine. Tierarztl Prax,1990,18(6):629-632
[160]Christensen, J., Alexandersen, S., Bloch, B., et al. Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system:a recombinant vaccine for mink enteritis parvovirus in mink[J]. Gen Virol,1994,75(Pt 1):149-155.
[161]Langeveld J, Casal J I, Osterhaus A D M, et al. First peptide vaccine providing protection against viral infection in the target animals:studies of canine parvovirus in dogs. J. Virol,1994, 68:4506-4513.
[162]Casal J I, Langeveld J, Corte's E, et al. Peptide Vaccine against Canine Parvovirus:Identification of Two Neutralization Subsites in the N Terminus of VP2 and Optimization of the Amino Acid Sequence. J Virol,1995,69(11):7274-7277.
[163]王亚君,华育平.犬瘟热病毒抗原表位TTB和犬细小病毒VP2蛋白的共表达及免疫小鼠特异性抗体的测定.兽类学报,2009,29(2):204-209
[164]杨松涛,夏咸柱,乔军,等.虎源猫瘟热病毒VP2蛋白基因在毕赤酵母中的表达.微生物学通报,2006,33(1):29-32
[165]杨帆,李淑梅,王红宁.DNA疫苗免疫机理及其在动物医学中的应用.中国兽医杂志,2005,41(1):38-40
[166]陈芹.犬细小病毒基因疫苗的研究[D].扬州:江苏扬州大学硕士学位论文.2004
[167]陈同海,孙志明,赵福广,等.犬细小病毒基因疫苗对幼貉的免疫试验.经济动物学报,2005,9(2):80-83
[168]郑颖,邱薇,李丽红,等.CDV、CPV、CAV三联DNA疫苗的实验免疫研究.第十二届全国养犬学术研讨会论文集.2007
[169]赵锴.病毒疫苗的研究现状与展望.第二军医大学学报,2002,23(8):813-15.
[170]L Hu, JJ Esposito, FW Scott. Raccoon Poxvirus Feline Panleukopenia Virus VP2 Recombinant Protects Cats Against FPV Challenge. Virology,1996,218(1):248-252.
[171]L Hu, C Ngichabe, CV Trimarchi, et al. Raccoon Poxvirus Live Recombinant Feline Panleukopenia Virus VP2 and Rabies Virus Glycoprotein Bivalent Vaccine. Vaccine,1997, 15(12-13):1466-1472
[172]Spitzer AL, Maxwell F, Corsini J,et al. Species specificity for transduction of cultured cells by a recombinant LuIII rodent parvovirus genome encapsidated by canine parvovirus or feline panleukopenia virus[J]. Gen Virol,1996,77(Pt 8):1787-1792
[173]谢之景,夏咸柱,扈荣良,等.表达犬细小病毒VP2蛋白重组犬2型腺病毒的构建及鉴定.病毒学报,2006,(3):461-464
[174]杨松涛,夏咸柱,乔军,等.表达猫瘟热病毒VP2蛋白重组腺病毒的构建及其免疫原性研究.中国病毒学,2005,20(6):637-641
[175]徐志凯.实用单克隆抗体技术[M].陕西:科学技术出版社,1992,75-78
[176]Azetaka M, Hirasawa T, konishi S,et al. Lymphoid cell line[J]. Vet.Sci,1981. Jpn. J:243-255
[177]王彦红,杨跃飞,殷俊,等.抗犬细小病毒的单克隆抗体的制备.江苏农业研究,2001,22(3):65-67
[178]谢红玲,崔尚金,崔玉东,等.抗猪细小病毒单克隆抗体的制备.中国预防兽医学报,2009,31(10):813-815
[179]程言信.犬细小病毒VP2的原核高效表达及特异单克隆抗体的研制.扬州大学硕士学位论文.2007
[180]Burtonboy G, Bazin H, Delferriere N. Rat hybridoma antibodies against canine parvovirus.Arch Virol,1982,71(4):291-302
[181]Horiuchi M, Mochizuki M, Ishiguro N, Nagasawa H, et al. mapping of a monoclonal antibody specific to feline panleukopenia virus and mink enteritis virus[J]. J Vet Med Sci,1997, 59(2):133-6
[182]Nakamura M, Nakamura K, Miyazawa T, et al. Monoclonal antibodies that distinguish antigenic variants of canine parvovirus. Clin Diagn Lab Immunol,2003,10(6):1085-1089
[183]Nelson CD, Palermo LM, Hafenstein SL, et al. Different mechanisms of antibody mediated neutralization of parvoviruses revealed using the Fab fragments of monoclonal antibodies. Virology,2007,361(2):283-293
[184]Teramoto YA, Mildbrand M M, Carlson J, et al. Comparison of enzyme-linked immunosorbent assay, DNA hybridization, hemagglutination, and electron microscopy for detection of canine parvovirus infections. J Clin Microbiol,1984,20(3):373-378
[185]Mildbrand M M, Teramoto Y A, Collins JK, et al. Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay. Am J Vet Res,1984,45(11):2281-2284.
[186]Rimmelzwaan G F, Juntti N, Klingeborn B, et al. Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections. Vet Q,1990,12(1):14-20.
[187]王克坚,吴威,聂金珍,等.抗水貂肠炎细小病毒单克隆抗体的建立.中国兽医学报,1996,16(1):48-49
[188]王建科,程世鹏,闫喜军,等.抗水貂肠炎病毒单克隆抗体的制备及其生物学特性鉴定.中国兽医科学,2009,39(03):227-232
[189李成,姜绍德,张坦.应用离子捕捉电镜技术对兔瘟病毒的检测[J].中国兽医杂志,1988,8
[190]常国权,杨盛华.用离子捕捉电镜技术快速检测犬瘟热病毒[J].内蒙古畜牧科学,1994,(02):33-34
[191]CR Parrish. Host Range Relationships and the Evolution of Canine Parvovirus. Vet Microbiol, 1999,69(1-2):29-40
[192]李成,谷守林,郝桂玉,等.应用离子捕捉电镜技术对鸡粪便中病毒的快速鉴定.电子显微学报,1988,3:56
[193]李育阳.基因表达技术.北京:科学出版社,2001,2
[194]Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E.Coli. Curr Opin Biotechnol,1998,9(5):497-501
[195]Gribskov M, Burgess RR. Overexpression and purification of the sigma subunit of E.coil RNA polymerase. Gene,1983,26:109
[196]赵波,王强,牛李丽,等.金钱豹猫瘟热疫苗免疫后血清抗体水平的检测.动物医学进展,2006,27(3):69-71
[197]张东林,惠煜,周艳琴,等.检测多种动物弓形虫抗体的SPA-ELISA方法的建立及初步应用.中国兽医学报,2009,29(9):1163-1166
[198]Eliasson M R, Andersson A, Olsson H. Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG [J]. Immunology, 1988,142:575-581
[199]Kelly P J M, Tagwira L, Matthewman P R. Reactions of sera from laboratory,domestic and wild animals in Africa with protein A and a recombinant chimeric protein AG [J]. Comp Immunol Microbiol Infect Dis,1993,16:299-305
[200]钱建飞,钟声.基因工程技术在畜牧兽医上的应用及展望.畜牧与兽医,1999,31(1):41-44.
[201]姜勋平主编.基因免疫的原理和方法.北京:科学出版社,2004:103-126
[202]单振振.抗猫细小病毒单克隆抗体的制备及病毒检测方法的建立.吉林大学硕士学位论文.2009