【作者】 汪招雄；

【导师】 何启盖；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本论文包含2个部分。 1.检测猪伪狂犬病毒单抗夹心ELISA方法的建立和应用 伪狂犬病（Pseudorabies, PR）是由伪狂犬病病毒（Pseudorabies Virus, PrV）引起的包括家畜和多种野生动物的一种以发热、奇痒及脑脊髓炎为症状的重要传染病。该病给养猪业造成了巨大的经济损失,猪是本病毒的天然宿主和贮存者。准确、及时地检测出病原是防止疾病扩散的重要措施之一。单克隆抗体的各种诊断方法在其它疾病的检测中得到广泛的应用,显示出灵敏性高、特异性强等优点。应用单克隆抗体来检测PrV在我国报道较少,为了能从感染PrV的动物体内和肉食品中检测出病原,本课题开展了以下研究。 1.1 将猪伪狂犬病毒Ea株在IBRS-2细胞上增殖培养后,纯化后免疫Balb/C小鼠,取其脾细胞与SP2/0的骨髓瘤细胞融合。经ELISA筛选,获得两株分泌抗PrV单克隆抗体（MAb）的杂交瘤细胞,分别命名为4A6F5和3G7E3。免疫荧光试验和交叉试验证明两种单抗能特异性地与PrV反应,小鼠腹水中MAb的ELISA效价均为1:100×2¹²。这两株单克隆抗体为病毒抗原的诊断研究提供良好的材料。 1.2 用纯化的病毒免疫健康猪,制备了抗PrV血清,提纯了IgG;将效价较高的一株单克隆抗体和抗血清IgG分别作为捕获抗体和检测抗体,用于建立检测PrV的双抗体夹心间接ELISA方法,并优化了反应条件,检测结果显示,该方法对PrV的检测灵敏度达31.2ng（0.312μg/ml）,与乙型脑炎病毒（JEV）、猪繁殖与呼吸障碍综合症（PRRSV）、细小病毒（PPV）和猪流感病毒（SIV）等病毒无交叉反应。批间和批内变异系数较小,介于为4.87%和1.07%之间。应用本方法对人工感染PrV的40日龄仔猪组织进行检测,肺和脑PrV抗原检出率最高,其次是脾、心、肝和肌肉。应用该方法和PCR对159份临床病料进行平行检测,二者的符合率可达到77.4%。实验结果表明:双抗体夹心间接ELISA方法具有敏感性高、特异性强和重复性好的特点,可应用于发病动物组织中PrV检测。 1.3 从武汉市及其周边地区市场收集97头份猪肉样本、57头份羊肉样本、50头份牛肉样本,应用双抗体夹心ELISA和聚合酶链式反应（PCR）方法平行检测样本中的伪狂犬病毒。检测结果显示:97头份猪肉样中,双抗体夹心ELISA方法PrV检出19份（19.6%）,gD-PCR检出25份（25.6%）;牛样本中ELISA阳性数为0,PCR阳性2份（4%）;羊样本中两种方法均为阴性。这2种方法均为阳性和阴性的总份数为12和170份,总符合率达到了89.2%,对部分ELISA阳性的样本,病毒分离也为阳性。 2 猪传染性胸膜肺炎毒素Ⅲ在巴斯德毕赤酵母中的表达和应用 胸膜肺炎放线更多还原

【Abstract】 This thesis consisted of two parts.1 Development of monoclonal antibody-based capture ELISA for detection of pseudorabies virus （PrV）Pseudorabies virus （PrV）, a member of Alpha Herpesvirus, is the causative agent of Pseudorabies （Aujeszky’s disease）, one of the most severe and economically important infectious diseases of domestic and wild animals. Swine was the natural host and reservoir of PrV.1.1 Balb/C mice were immunized with purified PrV. After fusion of SP2/0 myeloma cells with the stimulated splenocytes, two hybridoma secreting monoclonal antibody against PrV, designated as 3G7E3 and 4A6F5, were identified by ELISA. The ELISA titers of 3G7E3 and 4A6F5 in ascites were both 1:100×2¹². No cross reactions were found when Japanese encephalitis virus （JEV）, Porcine respiratory and reproduce syndrome virus （PRRSV）, porcine parvavirus （PPV）, hog cholera virus （HCV） and swine influenza virus （SIV） were tested. The specific reactivity of Mabs with PrV was confirmed by IFA and Mab-based sandwich ELISA. These results indicated that specific hybridomas secreting Mab against PrV were developed and possessed the potential of usage in development for detection of PrV antigen .1.2 The Pig anti-PrV serum was produced through immunization of animals with purified PrV, followed by isoaltion of IgG. Using 3G7E3 as capture antibody and purified IgG as detector antibody, a sandwich ELISA was developed to detect pseudorabies virus （PrV）. After optimization, the microplates were coated with 100μl monoclonal antibody at concentration of 12.2 μg/ml and the working concentration of IgG was determined as 16.0 μg/ml. No cross reaction was found when JEV, PRRSV, PPV, HCV and SIV were tested by this technique. The detection limit of the test was 0.312μg/ml of PrV. The varition indexes between or within batches were 4.87% and 1.07%, respectively. The tissue samples collected from experimentally and clinically infected pigs were used to validate the sandwich ELISA. The highest positive rates were obtained from detection of tonsils, brains and lungs. Up to 77.4% agreement between sandwich ELISA and PCR was obtained from detection of 159 clinical samples and new ELISA was more sensitive. These data indicated that a sensitive and specific sandwich ELISA for detection of PrV was developed, providing a useful tool for diagnosis of PrV infection.1.3 By using sandwich ELISA and PCR, the presence of PrV in the pork, mutton and beef samples, which were collected from markets in Wuhan, were investigated. Theresults showed that 19 positive samples were detected in 97 pork samples by DAS-ELISA while 25 positive samples were picked up by PCR. Only two positive samples were detected from 50 beef samples by PCR. All of 50 mutton samples were proved to be PrV free by both PCR and ELISA. Up to 892 % agreement between PCR and DAS-ELJSA was achieved. PrV could be isolated from the ELJSA-positive samples. These data indicated that the latent infection of PrV in pigs might play an important role in present prevalence of Aujeszky’s disease.2. Expresion of actinobacillus pleuropneumoniae ApxIII in yeast system and development of recombiant ApxEQ-ELISAActinobacillus pleuropneumoniae is the etiological agent of porcine contagious pleuropneumonia, a respiratory disease that continues to have a worldwide economic significance. RTXs determine the bacterial pathogenicity, so, detection of antibodies against RTXs is helpful in diagnosis of APP and evaluation of vaccine efficacy. Due to the difficulty in purification of native toxins, the easily-purified recombinant proteins have been used in development of diagnostic methods with high specificty. Comapred to other protein expression system, pichia pastoris system has its unique advantages. This paper describes the expression of ApxIII in the novel system and development of recombinant ApxlII-based ELISA.The full length of gene encoding ApxIIIA of Actinabacillus Pleuropneumoniae was amplified by polymerse chain reaction （PCR） and ligated to pMD-18T for purpose of sequence analysis. The amplicon was further cloned to pPIC9K expressing vector and transformed to GS115 competent cells. The protein with the size of approximate 110 kd was expressed by addition of methanol. The products were used to coat microtiter plates for develoment of antibody-detection ELISA that was optimized through checkerboard assay. The recombinant apxIHA-based ELISA possessd specificity, no false positive results occurred when Hps, PrV and PRRSV were tested. The variation indexes ranged from 8.24% to 2.46%. 82.4 % of agreement between the novel ELISA and IHA was observed after detection of 284 clinical samples, while novel ELISA was more sensitive. These results revealed that our new ELISA was of sensitivity, specificity and reproducibility, suitable for clinical diagnosis of infection caused by ApxIII-producing actinobacillus pleuropneumoniae.更多还原