【作者】 王海春；

【导师】 朱鸿飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2012， 硕士

【摘要】 牛结核病（bovine tuberculosis）是一种人畜共患的慢性消耗性传染病，主要由牛分枝杆菌（Myeobacterium bovis）感染所引起，可以通过病畜传染给人或其他动物。该病不仅严重阻碍了我国奶牛养殖业的发展，也严重危害着人类的健康。国际上普遍采用“检疫-扑杀”的措施进行该病的防控。有关牛结核病的诊断方法有很多种，主要以提纯牛结核菌素（PPD）皮内变态反应试验为主，但该方法在判定结果时存在一定的主观性，易出现假阳性和假阴性，为结核病的防控带来了诸多不利。γ-干扰素（IFN-γ）释放试验是牛结核病体外免疫学检测新方法，该方法操作简单、特异性和灵敏性高，并且为非侵入性检测，可以重复多次检测，检测结果更为客观、可靠，在澳大利亚等多个发达国家推行使用。目前，国内也开始小范围使用牛分枝杆菌IFN-γ ELISA检测试剂盒，但由于所使用的试剂盒主要依赖进口，价格昂贵，严重阻碍了其在临床检测中的应用。鉴于以上现实考虑，本研究从以下四个方面进行了相关探究，为进一步建立牛IFN-γ免疫学检测方法提供基础。1.牛IFN-γ的原核表达及鉴定用RT-PCR方法扩增出牛IFN-γ的基因片段，并成功构建原核重组质粒pET-32a（+）-IFN-γ和pGEX-6p-1-IFN-γ，将重组质粒转化至BL21（DE3）大肠杆菌感受态细胞中，经0.5mM IPTG诱导后成功表达重组牛IFN-γ，分别命名为rIFN-γ-His和rIFN-γ-GST。SDS-PAGE电泳结果显示，两种重组蛋白均为可溶性表达，大小分别为36ku、43ku。Western blotting及ELISA分析结果显示，rIFN-γ-His和rIFN-γ-GST具有良好的免疫原性。MDBK/VSV抗病毒试验结果显示，rIFN-γ-His和rIFN-γ-GST具有一定的抗病毒活性，rIFN-γ-His抗病毒活性为4056IU/mg，rIFN-γ-GST抗病毒活性为5078IU/mg。2.牛IFN-γ的杆状病毒表达及鉴定以pET-32a（+）-IFN-γ为模板扩增出牛IFN-γ的基因片段，定向克隆至pFastBacTMHTA中构建重组质粒pFastBacTMHTA-IFN-γ，转化至DH10Bac感受态细胞中获得重组穿梭质粒Bacmid-IFN-γ，转染sf21昆虫细胞获得重组杆状病毒Bac-IFN-γ。应用sf21昆虫细胞悬浮培养技术大量表达目的蛋白，命名为rIFN-γ-Bac。SDS-PAGE电泳结果显示，rIFN-γ-Bac为可溶性表达。Western blotting分析结果显示，重组牛IFN-γ被进行了有效的糖基化修饰，包括大小分别为16.8ku、20.8ku和22ku的三种重组蛋白。BOVIGAM牛分枝杆菌IFN-γ ELISA检测试剂盒检测结果显示，rIFN-γ-Bac有良好的免疫原性。MDBK/VSV抗病毒试验结果显示，rIFN-γ-Bac具有很高的抗病毒活性，抗病毒活性为1.05×10⁵IU/mg。蛋白质谱鉴定结果显示，所获得的6段多肽序列与牛IFN-γ中的氨基酸序列完全匹配，进一步证实了所表达的rIFN-γ-Bac确为牛IFN-γ。3.抗牛IFN-γ单克隆抗体的制备及鉴定用纯化后的rIFN-γ-Bac免疫6～8周龄的BALB/c小鼠，在PEG4000融合剂的作用下进行小鼠脾脏细胞与SP2/0细胞融合，经筛选后获得1A3、2A7、4A5、3E4、3E5、2G3共6株单克隆抗体细胞株。亚类鉴定结果表明，1A3、4A5、2G3属于IgG2b型，3E5属于IgG1型，2A7、3E4属于IgM型。间接ELISA检测结果显示，6株单克隆抗体均可以特异性的识别rIFN-γ-His、rIFN-γ-GST和rIFN-γ-Bac。间接ELISA检测腹水效价均达到1:10⁵以上，经HiTrap^TM protein G HP亲和层析柱纯化出高纯度的鼠抗牛IFN-γ单克隆抗体。以PPD刺激牛PBMC产生牛IFN-γ，将细胞固定后进行间接免疫荧光试验，结果显示1A3、2A7、3E4、3E5能与刺激后的细胞结合产生特异性荧光，表明1A3、2A7、3E4、3E5可以结合天然牛IFN-γ。4.抗牛IFN-γ多克隆抗体的制备及鉴定用纯化后的rIFN-γ-His、rIFN-γ-GST分别免疫新西兰白兔，收集血清后用HiTrap^TM protein GHP亲和层析柱纯化出高纯度的抗牛IFN-γ多克隆抗体。间接ELISA方法测定多抗的效价可达到1:4×10⁵以上。以PPD刺激牛PBMC产生牛IFN-γ，将细胞固定后进行间接免疫荧光试验，结果显示多抗能特异性结合天然牛IFN-γ。用纯化后的多抗包被ELISA板，联合BOVIGAM试剂盒中的HRP标记鼠抗牛IFN-γ单克隆抗体，采用双抗体夹心法检测PPD刺激的结核病阳性牛血浆，结果显示所制备的多抗能捕获处理血浆中的天然牛IFN-γ，具有良好的亲和力和较高的特异性。通过优化试验确定了多抗的最佳包被浓度为5μg/mL，最佳封闭剂为10%FBS，最佳显色时间为25min。综上所述，我们成功表达并纯化出具有良好反应原性的重组牛IFN-γ， rIFN-γ-Bac抗病毒生物学活性最佳。在此基础上我们进行了牛IFN-γ单克隆抗体和多克隆抗体的初步制备及鉴定，所获得的多克隆抗体对天然牛IFN-γ具有良好的亲和力和较高的特异性。为进一步建立牛IFN-γ免疫学检测方法奠定了基础。更多还原

【Abstract】 Bovine tuberculosis （TB） is a serious chronic and zoonotic disease，which is mainly caused byMycobacterium bovis （M. bovis）. TB infection not only seriously hinders the development of thebreeding industry of cows, but also causes great health issues of human in China. Until now, the―Quarantine-Slaughter‖strategy is adopted in most countries to control the disease. Among thevarious methods currently used to detect bovine TB, the PPD-related skin test is still regarded as the goldstandard for TB diagnosis, though the result of this method is prone to be compromised due to certainfactors. The IFN-γ release assay is another method for TB diagnosis with high specificity and sensitivity.It is easy to carry out and the result is objective and reliable. The imported Mycobacterium bovis IFN-γtest kit is very expensive, limiting its widespread use in the clinic diagnosis. In consideration of thecurrent situation, we did the research about bovine IFN-γ from the following aspects, which provided thebasis for the establishment of IFN-γ immunology test method.1. Cloning, expression and identification of the bovine IFN-γ gene in theprokaryotic expression systemIn the study, bovine IFN-γ gene was amplified by RT-PCR, sequenced and inserted into twoexpression vectors: pET-32a（+） and pGEX-6p-1. The two recombinant-plasmids were designated aspET-32a（+）-IFN-γ and pGEX-6p-1-IFN-γ, respectively. The target proteins named rIFN-γ-His andrIFN-γ-GST were successfully expressed in soluble forms by adding0.5mM IPTG during the inductionperiod. SDS-PAGE analysis showed that the molecular weight of the target proteins was36ku and43ku,respectively. Both western blotting and ELISA analysis conformed that rIFN-γ-His and rIFN-γ-GSThave good immunogenicity. MDBK/VSV antiviral test results showed that both proteins exhibitedantiviral activities, with rIFN-γ-His at4056IU/mg and rIFN-γ-GST at5078IU/mg.2. Expression and identification of bovine IFN-γ via the baculovirusexpression systemIn this study, the bovine IFN-γ gene was PCR-amplified from pET-32a（+）-IFN-γ, and inserted intothe pFast BacTMHTA to get the recombinant-plasmid, named as pFast Bac^TM HTA-IFN-γ. Aftertransformation and transfection, the recombinant baculovirus was obtained. The target protein namedrIFN-γ-Bac was successfully expressed in sf21insect cells during suspension culture. SDS-PAGEanalysis showed that the recombinant protein was expressed in soluble forms. Western blot analysisshowed that the recombinant protein was modified with glycosylation in three different forms:16.8ku,20.8ku and22ku, respectively. Myeobacterium bovis IFN-γ test kit analysis showed that rIFN-γ-Bachad good immunogenicity. MDBK/VSV antiviral test results showed that the rIFN-γ-Bac had highantiviral activity at1.05×10⁵IU/mg. The Protein mass spectrum identification results confirmed therecombinant bovine IFN-γ expression via the Baculovirus Expression System. 3. Preparation and identification of monoclonal antibodies againstbovine IFN-γBALB/c mice were immunized with the purified rIFN-γ-Bac three times. Then the spleen cells werecollected to fuse with SP2/0cells. After screening with indirect ELISA,6strains of monoclonalantibody cells were obtained:1A3、2A7、4A5、3E4、3E5、2G3, respectively. Among the many strains,1A3、4A5、2G3were identified as IgG2b subtype, and and3E5were identified as IgG1subtype, and2A7、3E4were identified as IgM subtype. Indirect ELISA test showed that all these strains can reactwith rIFN-γ-His、rIFN-γ-GST and rIFN-γ-Bac, and the titer of ascites was as high as1:105. Afterpurification with the G affinity chromatography and desalination, high-purity monoclonal antibodieswere obtained with good specificity and sensitivity. The indirect immunofluorescence assay with PBMCinduced With PPD showed that1A3、2A7、3E5、3E4can all react with the wild bovine IFN-γ.4. Preparation and identification of polyclonal antibodies against bovineIFN-γTwo groups of rabbits were immunized with rIFN-γ-His and rIFN-γ-GST for three times,respectively. Then serum was collected and PcAbs were purified. Indirect ELISA test showed that theantibody titer was as high as1:4×10⁵. The indirect immunofluorescence assay with PBMC induced WithPPD showed that the PcAb can react with the wild bovine IFN-γ. The Double-Antibody SandwichELISA assay showed that the PcAb had good specificity and sensitivity to wild bovine IFN-γ in TBpositive plasma induced with PPD. By optimizing this method, the optimal concentration of PcAb was5μg/mL, and the optimal confining liquid was10%FBS, and the optimal color time was25min.To sum up, we successfully expressed and purified recombinant IFN-γ of bovine with goodimmunogenicity, of which rIFN-Bac had high antiviral biological activities. And we also did theresearch about preparation of monoclonal antibodies and polyclonal antibodies against IFN-γ of bovine,of which PcAb had good affinity and high specificity with wild bovine IFN-γ.更多还原