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为获得具有天然活性的猪传染性胃肠炎病毒(TGEV)重组S蛋白,制备针对猪传染性胃肠炎S蛋白A、D抗原位点的单克隆抗体及建立快速抗体检测方法,本研究将TGEV S基因A、D抗原位点经PCR扩增并克隆入p Fast Bac HBM-TOPO载体,经转座、转染后获得重组杆状病毒,并对重组杆状病毒进行间接免疫荧光(IFA)及Western blot分析,结果显示,TGEV S基因A、D抗原位点在杆状病毒中成功表达,其表达产物为TGE诊断试剂的制备、基因工程疫苗的研制奠定了基础。 相似文献
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本研究分别用巴斯德毕赤酵母、杆状病毒和大肠杆菌表达系统中表达的猪传染性胃肠炎病毒(TGEV)TH-98株纤突蛋白(S)基因5'端含有B和C抗原位点的片段,利用Dot-ELISA方法对表达蛋白进行了抗原性比较分析,初步建立了以巴斯德毕赤酵母系统表达的重组蛋白为包被抗原的TGEV Dot-ELISA检测方法。结果表明:两种真核系统中表达的重组蛋白的抗原性相对更强。抗原最佳包被量为50 ng,抗体稀释度1∶100,最适封闭液为10 mg/L牛血清白蛋白,血清反应时间为60 min;酶标二抗的工作浓度为1∶1 000,反应时间为30 min,底物在室温显色时间为5 min。本研究建立的Dot-ELISA方法对猪呼吸道冠状病毒、猪轮状病毒、猪瘟病毒和猪流行性腹泻病毒阳性血清的检测结果均为阴性,特异性良好。与Svanova TGEV/PRCV抗体诊断试剂盒比较,此方法的特异性和符合率分别为90%和87%。 相似文献
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为研究牛病毒性腹泻病毒（BVDV）E^rns基因的生物学功能，将含有牛病毒性腹泻病毒E^rns基因的质粒pMD18-T—EE^rns经BamHⅠ／HindⅢ双酶切，获得了E^rns片段，再与杆状病毒转移栽体pBlueBaeHis2A连接，构建成重组质粒。将重组质粒pBlueBaeHis2A-E^rns与Bac-N—Blue^TMDNA共转染至sf9昆虫细胞中，获得了重组病毒，经噬斑筛选纯化，感染sf9昆虫细胞进行表达。SDS-PAGE分析结果表明，表达的目的蛋白大小约30ku；Western-blotting检测表明，该蛋白具有良好的抗原性。 相似文献
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为建立一种敏感、特异、快速、高通量的猪繁殖与呼吸综合征病毒(PRRSV)抗体血清学检测方法,本研究利用原核表达技术表达了PRRSV M蛋白,将纯化后的重组M蛋白作为包被抗原建立了检测PRRSV抗体的间接ELISA方法。参照已发表的PRRSV基因组M基因序列,设计合成1对特异性引物,RT-PCR扩增了长约435 bp的M基因片段,将目的片段亚克隆至pET32a(+)表达载体中,经IPTG诱导获得了以包涵体形式表达的重组M蛋白,重组蛋白纯化后,免疫印迹检测结果表明具有良好的抗原性和特异性。以重组M纯化蛋白为包被抗原,经间接ELISA反应条件的优化,建立了检测PRRSV抗体的间接ELISA方法,该方法检测猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病病毒(PRV)、猪传染性胃肠炎病毒(TGEV)、猪圆环病毒2型(PCV2)其他6种常见猪病病原的阳性血清均为阴性;该方法批内与批间重复性试验的变异系数分别小于5%和10%;该方法与商品化ELISA试剂盒的符合率为95.3%。本研究建立的M-ELISA检测方法将为猪群免疫PRRS疫苗后抗体水平监测及PRRSV野毒感染的快速诊断与流行病学调查等提供了一种简便易行、快速、高通量的血清学抗体检测方法。 相似文献
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《黑龙江畜牧兽医》2016,(18)
为建立一种敏感、特异、高通量的猪细小病毒抗体检测方法,参照猪细小病毒(PPV)VP2蛋白的基因序列设计合成1对特异性引物,PCR扩增了VP2蛋白主要抗原区域,将目的片段克隆至p ET30a原核表达载体中,获得了以可溶形式表达的重组VP2蛋白,重组蛋白纯化后,经免疫印迹检测显示具有良好的抗原性;以纯化蛋白为包被抗原,经间接ELISA反应条件的优化,建立了检测PPV抗体的间接ELISA方法。结果表明:用该方法检测猪瘟病毒(CSFV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪圆环病毒2型(PCV-2)、猪伪狂犬病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)阳性血清,结果均为阴性;批内重复性试验的变异系数小于5%,批间重复性试验的变异系数小于10%;与血凝抑制试验(HI)方法的符合率为94.49%;应用该方法检测869份疫苗免疫猪血清样品,免疫合格率为87.92%;检测248份未免疫疫苗猪血清样品,阳性感染率为7.25%。 相似文献
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猪α干扰素具有抗病毒作用,临床上具有广阔的应用前景.为获得重组猪α干扰素,本研究应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将编码成熟猪α干扰素基因插入供体质粒pFastBac~(TM) Ⅰ多克隆位点,置于pH启动子控制下,在C端融合6个组氨酸标签以利于纯化.将重组转移栽体质粒转化DH10感受态细胞获得重组穿梭质粒rBacmid,转染对数生长期的Sf9昆虫细胞获得重组杆状病毒.重组蛋白通过间接免疫荧光、Western-blotting证明在重组杆状病毒感染的昆虫细胞中获得表达.镍亲和层析柱纯化的重组蛋白经SDS-PAGE电泳相对分子质量为19 000.通过在猪肾细胞(PK-15)上抑制猪水泡性口炎病毒(VSV)致病变作用检测其抗病毒活性为9.67×10~4 U/mL.昆虫培养上清及细胞裂解液经2~8稀释在Mare-145细胞上能够抑制猪蓝耳病病毒增殖.从而为进一步作为抗病毒药物应用于猪疫病的防治研究奠定了基础. 相似文献
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《动物医学进展》2021,42(9)
旨在获得具有天然结构且有反应原性的猪传染性胃肠炎病毒(TGEV)受体结合域(RBD)蛋白。受体结合域是决定病毒宿主和嗜性的关键因素。根据猪传染性胃肠炎病毒纤突蛋白(S)序列(GenBank:ABC72414.1),设计、优化并合成其受体结合域(RBD)部分,亚克隆至杆状病毒pFastBac~(TM)Dual上,获得重组质粒,命名为pFBD-TGR。将该质粒转化DH10Bac感受态细胞中,筛选较大的白色菌落,获得重组杆状质粒Bacmid-TGR,转染至sf9细胞中进行拯救病毒,转染后5 d～7 d,收上清,传至第3代,应用间接免疫荧光法(IFA)和Western blot检测sf9细胞上清,进行目的蛋白表达鉴定。结果显示,重组穿梭质粒Bacmid-TGR构建成功,Western blot结果证明,杆状病毒表达了TGEV-RBD蛋白且能与Strep tag抗体和抗猪传染性胃肠炎病毒多克隆抗体发生特异性反应,间接免疫荧光结果证明,成功地表达了TGEV-RBD蛋白,为进一步开展猪传染性胃肠炎病毒抗体研究以及新型疫苗的研发奠定了基础。 相似文献
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利用RT-PCR方法扩增猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)M和N结构蛋白基因,将其分别克隆入载体FastBacTM Dual,获得转移质粒pFastBacTM Dual-M和pFastBacTM Dual-N,将重组质粒转化至DH10Bac感受态细胞,获得杆状病毒重组质粒Bacmid-M和Bacmid-N;在Cellfection作用下,将杆状病毒重组质粒转染昆虫细胞sf9,获得重组杆状病毒rBac-M和rBac-N。间接免疫荧光试验(IFA)检测表明,杆状病毒表达的M蛋白和N蛋白能够被特异性阳性血清识别,重组蛋白具有较好的反应原性。将重组杆状病毒rBac-M和rBac-N口服免疫小鼠,收集小鼠粪便检测抗TGEV sIgA抗体水平,采集血液检测血清中抗TGEV IgG。结果显示,重组杆状病毒(rBac-M和rBac-N)可诱导小鼠产生粘膜免疫和体液免疫应答。试验结果初步预示了杆状病毒经口服途径作为抗原递呈载体的可行性,也为开展TGEV口服免疫研究奠定了基础。 相似文献
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为真核表达鸡传染性支气管炎病毒(IBV)S1蛋白并鉴定其抗原性,本研究采用RT-PCR方法扩增出IBV Holte株S1基因,并将其克隆于昆虫杆状病毒转移载体pFastBacHT中,通过大肠杆茵中同源重组构建重组杆粒Bacmid-S1,将重组杆粒转染至Sf9细胞中,获得含S1基因的重组杆状病毒.将该重组杆状病毒感染Sf9细胞进行表达,经间接免疫荧光、SDS-PAGE和western blot鉴定结果表明:IBV Holte株的S1蛋白能够在Sf9中以可溶形式表达,蛋白大小约为64 ku,该蛋白具有天然蛋白的抗原性.本研究为生产检测IB的诊断试剂和研制新型IB重组亚单位疫苗奠定基础. 相似文献
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采用蔗糖密度梯度离心,纯化浓缩犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)的细胞培养物,分别设计7,17,11,10和4对引物,构建了49个基因片段的克隆。煮沸裂解法制备质粒DNA,回收PCR扩增产物,点制冠状病毒基因芯片。抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,与芯片进行杂交检测,淘汰交叉的克隆片段。结果表明:克隆CCV1,CCV2,CCV5和CCV7可特异诊断CCV,克隆FCV6,FCV7,FCV8和FCV9可特异诊断FCV,克隆FIPV2,FIPV7,FIPV8和FIPV9可特异诊断FIPV,克隆PRCV1,PRCV2和PRCV3可特异诊断PRCV,克隆TGEV3,TGEV4,TGEV5和TGEV6可特异诊断TGEV。将这些特异克隆扩增片段重新点制基因芯片,与病毒PCR产物杂交,未发现交叉现象。基因芯片检测比传统PCR敏感1000倍,可有效应用于这5种动物冠状病毒的检测与区分。 相似文献
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The use of immunofluorescence techniques for the laboratory diagnosis of transmissible gastroenteritis of swine. 总被引：1，自引：1，他引：0 
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下载免费PDF全文 Over a four year period, 74 of 250 field outbreaks of enteric disease (30%) and 110 of 440 swine (25%) were positive for transmissible gastroenteritis by immunofluorescence procedures. Of 141 swine from herds positive for transmissible gastroenteritis 110 (78%) were positive by fluorescent antibody techniques. The fastest, easiest to perform and most effective procedure was the examination of frozen sections of the jejunum from acutely ill animals by the fluorescent antibody tissue section technique. Only two herds were found to be positive by the fluorescent antibody tissue culture technique which were negative by fluorescent antibody tissue section technique. A considerable number of outbreaks, 21 of 74 (28%), of transmissible gastroenteritis were detected by immunofluorescence in swine over two weeks of age. The majority of outbreaks of transmissible gastroenteritis, 50 of 74 (68%), occurred in Missouri during the months of January through April and 63 of 74 (85%) during the months of December through May. The recurrence of the disease in a number of counties over a four-year period suggest the possibility of endemic foci. 相似文献
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The effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotoxicity and resistance to transmissible gastroenteritis in neonatal piglets. 下载免费PDF全文
下载免费PDF全文 The purpose of this study was to attempt to establish spontaneous cell-mediated cytotoxicity effector activity in the intraepithelial lymphocytes of neonatal piglets by adoptive transfer of mononuclear leukocytes from an adult donor and to determine the effect of transfer on the resistance of piglets to transmissible gastroenteritis. Cytotoxicity was determined by a chromium release assay using PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. The experimental animals were inbred miniature pigs, in which a high degree of uniformity in lymphocyte defined histocompatibility complex antigens was demonstrated by the mixed lymphocyte reaction. Adoptive transfer of 8 X 10(7)-4 X 10(8) adult pig leukocytes established effector activity in eight recipient piglets, and leukocytes labelled with fluorescein isothiocyanate homed to the epithelium of the small intestine. When four recipients of 5 X 10(8) adult leukocytes were challenged with transmissible gastroenteritis virus, the onset of diarrhea was delayed for 24 h and the diarrhea was usually milder than in four untreated control piglets. It was concluded that the adoptive transfer of leukocytes with spontaneous cell-mediated cytotoxicity effector activity, which homed to the small intestinal epithelium, may have contributed to an increased resistance to transmissible gastroenteritis. 相似文献
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The postulated role of feeder swine in the perpetuation of the transmissible gastroenteritis virus. 总被引：1，自引：1，他引：0 下载免费PDF全文
下载免费PDF全文 M Morin R F Solorzano L G Morehouse L D Olson 《Canadian journal of veterinary research》1978,42(4):379-384
Clinical, immunofluorescence and histopathological observations were found to be an efficient approach for the confirmation of the diagnosis of transmissible gastroenteritis in feeder swine. Two cases are reported to exemplify how feeder swine exposed to points of concentration such as holding areas, sales barns and auctions can play an important role in the epizootiology of transmissible gastroenteritis. A third field case is reported as an example of an outbreak of transmissible gastroenteritis beginning in feeder swine and then spreading to baby pigs on the farm. All baby pigs died that were born during the acute phase of the outbreak in the feeder swine. Baby pigs born shortly after the clinical signs had abated in the herd, and from sows that had been exposed orally to virulent transmissible gastroenteritis virus and vaccinated with a commercial transmissible gastroenteritis vaccine ten days before farrowing, survived. This was explained by a combination of a decrease in the amount of virus shed in the environment and the immunity induced in the sows. These observations of field outbreaks of transmissible gastroenteritis combined with recently reported experimental studies lend strong support to the hypothesis of a reservoir for transmissible gastroenteritis virus in feeder pigs. This reservoir would be based principally on the transmission of the virus on a continuous basis from the feces of recently infected pigs to susceptible pigs. Clinical signs of transmissible gastroenteritis in such pigs are difficult to recognize or absent and this contributes to the importance of the reservoir in the field. 相似文献
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An outbreak of diarrhea in piglets caused by a coronavirus antigenically distinct from transmissible gastroenteritis virus 总被引：5，自引：2，他引：3
Dea S Vaillancourt J Elazhary Y Martineau GP 《The Canadian veterinary journal. La revue veterinaire canadienne》1985,26(3):108-111
Coronavirus-like particles were visualized by electron microscopy in the intestinal contents of piglets during a diarrheal outbreak on a Quebec pig farm. The precipitating antigens of transmissible gastroenteritis virus were not detected in the intestinal contents of diarrheic animals by counter-immunoelectrophoresis. Insignificant antibody titers against transmissible gastroenteritis virus were demonstrated in the sera of convalescent pigs by indirect immunofluorescence and these sera did not react with transmissible gastroenteritis virus when tested by immunoelectron microscopy. The causative agent could not be isolated in cell cultures. It was concluded that a coronavirus antigenically distinct from transmissible gastroenteritis virus was responsible for the enteric problems observed on this farm. The outbreak was controlled after oral inoculation of adult pigs with infected intestinal contents. 相似文献
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Recombinant porcine interferon gamma (rPoIFN gamma) induced a dose-dependent inhibition of the cytopathic effect produced by vesicular stomatitis virus (VSV) challenge of both homologous and heterologous (bovine) cell lines. In addition, an antiviral effect of rPoIFN gamma was demonstrable against the coronavirus transmissible gastroenteritis virus (TGEV) infection of porcine epithelial cells and of pulmonary macrophages. A rabbit anti-PoIFN gamma antiserum was prepared and shown to specifically neutralize the antiviral effects of natural and recombinant porcine IFN gamma preparations. This antiserum could also neutralize recombinant bovine IFN gamma but not recombinant human IFN gamma. These results suggest antigenic homology of porcine and bovine IFN gamma but antigenic differences between these molecules and human IFN gamma. 相似文献
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M T Jensen L J Kemeny S S Stone A E Ritchie 《American journal of veterinary research》1980,41(1):136-139
Porcine colostral immunoglobulin (Ig)G and IgA, isolated from transmissible gastroenteritis virus-infected sows, were compared by direct immunoelectron microscopy. It was estimated, using antibodies with a less than a twofold difference in virus-neutralizing activity, that IgG was 500 times more efficient than was IgA for coating transmissible gastroenteritis virions. Guinea pig complement enhanced the antibody coating with IgG, but did not increase virus-neutralizing activity of IgG or IgA. 相似文献