猪圆环病毒2型Cap蛋白单克隆抗体的研制及其应用

以真核表达的猪圆环病毒2型（PCV2）Cap蛋白所形成的病毒样颗粒作为免疫原,按照常规方法免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞融合。经间接ELISA法和间接免疫荧光试验（IFA）筛选,总共获得了4株分泌抗PCV2 Cap蛋白单克隆抗体的杂交瘤细胞株,分别命名为7A6、3F2、3B7、1A6。4株单抗腹水效价均达到1:10⁵。IFA和免疫过氧化物酶单层实验结果显示,4株单抗均能与PCV2发生特异反应,与PCV1无交叉反应。PCV2 Cap蛋白单克隆抗体的制备,为猪圆环2型病毒抗原表位分析及其相关抗原抗体诊断试剂盒的研制奠定了基础。     

禽流感病毒单克隆抗体的制备及其抗蛋白抗原的分析

以纯化的H9N2亚型禽流感病毒为抗原,免疫BALB/c小鼠,细胞融合后,经间接ELISA和血凝抑制试验(HI)筛选,获得了8株能稳定分泌抗禽流感病毒单克隆抗体的杂交瘤细胞株。特异性试验证明,8株杂交瘤细胞株诱生小鼠腹水的特异ELISA抗体效价可达1∶3.2×103~1∶5.1×106,其中2株HI效价达212。8株单抗与H5亚型血凝素分型抗原不发生血凝,与减蛋综合征(EDS-76)病毒、传染性支气管炎病毒(IBV)、新城疫病毒(NDV)均不反应。亚类鉴定证实,除1C7单抗为IgG2b外,其他7株均为IgG1亚类。Westernblotting试验分析初步表明,8株单抗至少针对纯化病毒粒子3种不同的蛋白抗原,其中3株针对核蛋白(NP),2株针对基质蛋白M1,2株针对血凝素HA/HA1。对感染细胞的Western blotting分析结果与纯化病毒结果基本一致,其中1株未明显沉淀纯化病毒粒子蛋白的单抗可以与感染细胞的M2蛋白多肽反应。     

鸭星状病毒单克隆抗体的制备及鉴定

为了制备针对鸭星状病毒(Duck astrovirus,DAstV)衣壳蛋白的单克隆抗体,以DAstV C-NGB株为免疫原,用ORF2重组蛋白作为抗原进行筛选,获得7株稳定分泌抗DAstV ORF2蛋白单抗的杂交瘤细胞。取E5C1株和C10F6株进行鉴定,结果表明,2株单抗均为IgM,轻链均为κ型;细胞上清和小鼠腹水的效价分别为10~5和10~6以上;免疫印迹试验表明,2株单抗均能识别重组ORF2蛋白;特异性试验表明,E5C1株杂交瘤细胞的培养上清只与DAstV反应。     

猪细小病毒核衣壳VP2蛋白单克隆抗体的制备及部分特性鉴定

以纯化的PPV重组VP2蛋白免疫BALB/c小鼠,通过细胞融合技术,间接ELISA筛选和3次以上细胞克隆,获得了5株稳定分泌抗PPV VP2蛋白单克隆抗体杂交瘤细胞株,分别命名为2D5、1F11、2B4、3C8、1 H3。其染色体平均数均为87～102条,分泌抗体亚类4株为Ig M类,1株为IgG类,Western blot检测表明,5株单抗均识别猪细小病毒VP2蛋白;间接免疫荧光鉴定表明,5株单抗均与PPV全病毒发生反应;间接ELISA鉴定与其他相关病毒的交叉反应性表明,制备的5株单抗不与TEGV、PRV和PEDV反应,表明所制备的抗体与猪细小病毒具有较强的特异性反应。     

抗乙型脑炎病毒非结构蛋白NS1单克隆抗体的制备与鉴定

本研究利用原核表达的乙型脑炎病毒(JEV)SA14-14-2株非结构蛋白NS1作为免疫原.免疫8周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术进行融合,共获得4株特异性针对JEV NS1的杂交瘤细胞,分别命名为1H6、2C3、3A7、4C8,经测定1H6单抗亚类属于IgG2b,其他3株为IgG1,轻链均为K链.4株杂交瘤细胞诱生小鼠腹水效价分别达1:20 480、1:2 560、1:20 480、1:10 240,western blot证实所得杂交瘤细胞分泌的抗体均可与JEVNS1蛋白发生特异性反应,间接免疫荧光试验表明1H6、3A7、4C8 3株单抗能够识别天然的JEV NS1蛋白.本研究为进一步探究JEV NS1蛋白结构及其功能奠定了基础.     

禽白血病病毒衣壳蛋白单克隆抗体研制及其双夹心ELISA的建立

为制备禽白血病病毒(ALV)核衣壳蛋白p27的单克隆抗体,本研究以原核表达的融合蛋白GST-ALV p27免疫小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,通过间接免疫荧光(IFA)和间接ELISA进行筛选,制备了5株能够稳定传代并分泌抗p27单抗的杂交瘤细胞株,分别命名为5D3、4F12、5B10、1C5和3C6,亚类鉴定3C6为IgG2b,其余为IgG1.通过IFA、Western-blot及竞争抑制ELISA,表明该5株单抗与J亚群禽白血病病毒(ALV-J)呈阳性反应,而与其他常见禽源病毒无交叉反应.利用5D3和4F12单抗建立了双抗体夹心ELISA方法,经663份临床样本验证,该方法与商品化试剂盒的符合率达到95.17％,证明所研制的针对ALV p27蛋白的单抗及建立的双抗体ELISA方法具有较高的应用价值.     

猪传染性胃肠炎病毒单克隆抗体的制备与鉴定

以纯化的猪传染性胃肠炎病毒(TGEV)作为抗原免疫BALB/c小鼠,经3次免疫后,通过聚乙二醇方法进行融合,利用有限稀释法在HAT培养基上筛选杂交瘤细胞,共获得12株既能稳定生长并可以分泌特异性抗TGEV的单克隆抗体的杂交瘤细胞系,分别命名为8D2、4H4、5D6、7H10、4C3、9G1、3A2、8G1、5G6、2D7、4D6、6B10。间接ELISA、Western blot、间接免疫荧光结果表明,获得的12株单抗能特异性识别TGEV,通过间接ELISA做病原检测,显示12株单抗与猪流行性腹泻病毒(PEDV)和猪轮状病毒(PrV)不发生交叉反应。经抗体亚类鉴定,该12株单克隆抗体均为IgG2b。12株抗TGEV的单抗制备成功,为猪传染性胃肠炎病原特性研究和病原快速检测提供了物质基础。     

猪血凝性脑脊髓炎病毒N蛋白单克隆抗体制备及表位鉴定

为制备猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus, PHEV)N蛋白的单克隆抗体，本研究构建了pET-32a-N重组质粒，原核表达并纯化重组N蛋白，免疫BALB/c小鼠，细胞融合后利用间接ELISA进行筛选，获得了3株稳定分泌的阳性杂交瘤细胞株，分别命名为2C3、4F3、5C6。Western blot与间接免疫荧光试验表明，3株单抗均能与293T细胞中表达的N蛋白产生特异性反应。经测定，3株单抗的效价均为1×10⁶,重链类型均为IgG1,轻链类型均为κ。利用一系列表达的部分重叠的N截短蛋白片段，经Western blot鉴定单抗所识别的抗原表位，结果显示，²⁶⁷KPRQK²⁷¹为单抗2C3、4F3、5C6所识别的表位。本研究为PHEV临床检测方法的建立和表位疫苗的研发奠定了基础。     

猪繁殖与呼吸综合征病毒N蛋白单克隆抗体的制备及生物学特性分析

分别用纯化的猪繁殖与呼吸综合征病毒(PRRSV)和纯化的重组N蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗PRRSV N蛋白的单克隆抗体(McAb)。用纯化的PRRSV免疫小鼠,经细胞融合获得2株可分泌特异性单抗的杂交瘤细胞株,分别命名为4B8、4D8。用重组N蛋白免疫的小鼠,经细胞融合获得3株可分泌特异性McAb的抗PRRSV N蛋白的杂交瘤细胞2F3、4D5、5D11。间接ELISA检测4D8、4B8、4D5和5D11杂交瘤上清效价为1∶32~1∶512,而2F3的腹水效价为1∶12 800。单抗2F3、4B8和4D8与纯化病毒的Western blotting反应都为阳性,而4D5和5D11为阴性。IFA检测结果5株单抗都有明显的荧光,与PRRSV呈阳性反应。2F3的Ig亚型为IgM。5株单抗杂交瘤细胞连续传代至20代,分泌相应McAb的效价基本一致。本研究为PRRSV生物学诊断和方法研究提供有用工具。     

猪繁殖与呼吸综合症病毒单克隆抗体的制备和鉴定

以猪繁殖与呼吸综合征病毒（PRRSV）作为免疫原,免疫BALB／c小鼠,经间接ELISA、IPMA和IFA试验进行杂交瘤筛选,共获得2株能分泌针对PRRSV单克隆抗体（简称单抗）的杂交瘤细胞株,分别命名为3D10和4H11,3D10亚类为IgG1,4H11亚类为IgG2b,单抗腹水的间接ELISA效价均达到1．0×10^7,染色体数目介于90～110之间。2株单抗与猪细小病毒、猪伪狂犬病毒、猪圆环病毒2型均无交叉反应,IPMA和IFA结果显示单抗均能与接种于猴肾细胞（Marc145）的PRRSV发生特异性反应,证实抗PRRSV单抗具有良好的特异性和敏感性,为PRRSV抗原表位分析及相关抗原抗体诊断试剂盒的研制奠定了基础。     

用PCR法检测东北虎感染猫细小病毒的研究

根据GenBank中已发表的猫细小病毒基因组中的保守序列 ,利用Goldkey软件设计了一对能扩增 750bp片段大小的引物 ,对某动物园 1只病死东北虎的脾脏样品进行了PCR检测。结果得到了与设计大小完全相符且与标准猫细小病毒扩增产物大小一致的特异核酸带 ,同时对犬瘟热、犬腺病毒、轮状病毒的核酸扩增结果均为阴性。敏感性试验表明 ,此法可检出血凝价为 1 2 8×脾脏匀浆液上清 1 0 - 5倍稀释的模板 ,远高于血凝试验的敏感性 ,为虎、狮、熊猫等野生动物细小病毒病的快速诊断提供了一个有效的方法     

Serologic evaluation of vaccinated American river otters

The Oklahoma Department of Wildlife Conservation acquired 20 American river otters (Lutra canadensis) between 1984 and 1985 for reintroduction into Oklahoma waterways. In 1985, 10 otters were evaluated for serum antibody titers after vaccination with canine distemper virus, canine adenovirus type 2, canine parvovirus (CPV), feline panleukopenia virus (FPV), feline rhinotracheitis virus (FRV), and feline calicivirus. Prevaccination serum-virus neutralization (SVN) antibody to feline rhinotracheitis virus was found in 2 otters and to feline calicivirus in 1 otter. Using an indirect fluorescent antibody (IFA) assay, prevaccination antibody to CPV and FPV was found in 2 otters. A significant increase in SVN antibody titers was found after vaccination of otters with canine adenovirus type 2 (6 of 8 animals) and feline calicivirus (1 of 8 animals). One of 8 otters developed significant antibody titers to CPV and FPV, as measured by IFA assay. Otters did not develop SVN antibody titers to canine distemper virus after vaccination. Antigens of feline leukemia virus, using ELISA, or antibodies to feline infectious peritonitis, using IFA assay, were not found in the 20 otters.     

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

用 PEG60 0 0沉淀和蔗糖密度梯度离心从细胞培养物中纯化猫泛白细胞减少症病毒 ( FPV) ,以纯化FPV免疫 BALB/c小鼠 ,运用淋巴细胞杂交瘤技术 ,获得了 4株抗 FPV的特异性单克隆抗体 ( Mc Ab)。其腹水 Mc Ab的 ELISA效价在 1 0 - 4～ 1 0 - 5之间 ,其中 1株具有血凝抑制能力。经 ELISA阻断试验及 ELISA交叉反应性试验测定 ,这 4株 Mc Ab可与 FPV、犬细小病毒 ( CPV)和水貂肠炎病毒 ( MEV)呈特异性反应 ,因此可作为检测 FPV、CPV和 MEV共同抗原的通用试剂     

Feline panleukopenia virus, feline herpesvirus-1, and feline calicivirus antibody responses in seronegative specific pathogen-free cats after a single administration of two different modified live FVRCP vaccines

Two groups of feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus-1 (FHV-1) seronegative cats (five cats per group) were administered one of two modified live feline viral rhinotracheitis, calicivirus, and panleukopenia virus (FVRCP) vaccines and the serological responses to each agent were followed over 28 days. While all cats developed detectable FPV and FCV antibody titers; only two cats developed detectable FHV-1 antibody titers using the criteria described by the testing laboratory. For FPV and FHV-1, there were no differences in seroconversion rates between the cats that were administered the intranasal (IN) FVRCP vaccine and the cats that were administered the parenteral FVRCP vaccine on any day post-inoculation. For FCV, the cats that were administered the IN FVRCP vaccine were more likely to seroconvert on days 10 and 14 when compared to cats that were administered the parenteral FVRCP vaccine.     

Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

ABSTRACT: Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts.     

猫泛白细胞减少症病毒的分离与鉴定

应用猫肾传代细胞(FK),采用同步接毒的方法,从8份狮、虎、豹病料中分得6株病毒,经与标准病毒作交叉免疫电镜等系统鉴定,证明与猫泛白细胞减少症病毒的特性基本相符,从而首次证实在我国动物园狮、虎、豹等猫科动物中存在猫泛白细胞减少症。     

猫泛白细胞减少症病毒的分离与鉴定

从临床表现有体温升高、呕吐、血样腹泻、脱水等症状的疑似猫泛白细胞减少症感染的病例采取粪样28份。从粪便样品中成功分离获得了7株猫泛白细胞减少症病毒（FPV）：JX-1、JX-2、JX-3、JX-4、JX-5、JX-6和JX-7;应用F81细胞增毒,盲传至3代时在F81细胞上产生细胞病变(脱落、变形、游离等);核酸型鉴定证明,FPV毒株的代谢可被5-IUDR所抑制,其核酸属于DNA型;所分离的病毒培养物能凝集猪的红细胞（凝集效价达2⁶～2⁸）,并能被标准FPV阳性血清所抑制;电镜观察病毒粒子外观呈圆形或六边形,直径20～30 nm;该病毒耐酸、耐热、耐乙醚;动物致病性试验,经口感染分离细胞培养毒1 ml,试验组幼猫第7 d发病,采集病猫粪样做HA试验为阳性反应,做HI试验,其凝集猪红细胞的能力被抑制。     

禽脑脊髓炎病毒单克隆抗体的制备、鉴定与初步应用

为获得禽脑脊髓炎病毒(Avian Encephalomyelitis virus,AEV)VP1蛋白的单克隆抗体,通过原核表达AEV VP1蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得2株杂交瘤细胞株,命名为4#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示：制备的株单克隆抗体亚型分别为IgG2b、IgG2a,Western-blot和IFA试验结果表明单克隆抗体均能与AEV发生特异性反应而与其他禽病常见病毒均无交叉反应。运用建立的IFA对单抗进行了初步运用,在外源病毒检测方面与经典方法符合率高。本研究成功制备了AEV单克隆抗体,为进一步建立AEV检测方法和深入研究AEV的生物学特性奠定了基础。     

Detection of protective antibody titers against feline panleukopenia virus, feline herpesvirus-1, and feline calicivirus in shelter cats using a point-of-care ELISA

Serum antibody titers are a useful measurement of protection against infection (feline panleukopenia virus [FPV]) or clinical disease (feline herpesvirus-1 [FHV] and feline calicivirus [FCV]), and their determination has been recommended as part of disease outbreak management in animal shelters. The objective of this study was to determine the sensitivity, specificity, and inter-observer and inter-assay agreement of two semi-quantitative point-of-care assays for the detection of protective antibody titers (PAT) against FPV, FHV and FCV in shelter cats. Low sensitivity for FPV antibodies (28%) rendered a canine point-of-care assay inappropriate for use in cats. The feline point-of-care assay also had low sensitivity (49%) and low negative predictive value (74%) for FPV PAT detection, but was highly accurate in the assessment of FHV and FCV PAT. Improvements in accuracy and repeatability of FPV PAT determination could make this tool a valuable component of a disease outbreak response in animal shelters.     

Comparisons of feline panleukopenia virus, canine parvovirus, raccoon parvovirus, and mink enteritis virus and their pathogenicity for mink and ferrets

Parvoviruses from mink (mink enteritis virus [MEV]), cats (feline panleukopenia virus [FPV]), raccoons (raccoon parvovirus [RPV]), and dogs (canine parvovirus [CPV]) were compared. Restriction enzyme analysis of the viral replicative-form DNA revealed no consistent differences between FPV and RPV isolates, but CPV and MEV isolates could be distinguished readily from other virus types. Feline panleukopenia virus, RPV, and MEV, but not CPV, replicated to high titers in mink. However, on the first passage, disease and microscopic lesions were observed only in mink inoculated with MEV. Feline panleukopenia virus and RPV isolates replicated in ferrets, but disease or microscopic lesions were not observed. Feline panleukopenia virus and RPV isolates could be passaged repeatedly in mink and ferrets. Virulence of FPV and RPV isolates was low compared with that of MEV, and only a single mink inoculated with FPV or with RPV developed clinical disease on the sixth passage of virus.