【作者】 曹冰玉；

【导师】 王永山； 范红结；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 传染性法氏囊病(Infectious Bursal Disease, IBD)是由传染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)引起的以侵害雏鸡淋巴组织,特别是中枢免疫器官一法氏囊为主要特征的传染病。该病不仅引起患病动物死亡,还会导致机体免疫抑制,使机体的免疫防御能力降低和疫苗免疫接种失败,每年给养禽业带来巨大经济损失。近年来,由于各地出现的IBDV变异株、强毒株的毒力和抗原性存在一定差异,可逃避现行疫苗的免疫,IBD的流行呈现更为复杂的态势,给该病的防控造成较大困难。IBDV VP2蛋白是病毒主要的结构蛋白和宿主保护性抗原,因此针对VP2蛋白及IBDV检测方法的研究是当前重要的研究内容。本文将流行毒株IBDV VP2基因分别在大肠杆菌和杆状病毒表达系统中进行表达,获得了针对VP2蛋白的单克隆抗体。试验内容主要包括以下三个部分：1.传染性法氏囊病病毒VP2基因在大肠杆菌中的表达将从安徽省传染性法氏囊病(IBD)免疫失败的病鸡法氏囊组织中扩增的VP2基因定向克隆入原核表达载体pGEX-4T-1中,构建了原核表达质粒pGEX-VP2,并转化大肠杆菌Rosetta (DE3).经IPTG诱导后,用IBDV双抗体夹心ELISA检测,结果呈阳性反应：SDS-PAGE分析结果显示,重组VP2融合蛋白的分子质量为68 ku,表达量约占菌体总蛋白量的16.0%,表达产物主要以包涵体形式存在于菌体内；Western-blotting结果显示,重组VP2蛋白与IBDV抗体发生反应,形成一条特异性蛋白带,表明获得的重组蛋白具有良好的抗原反应性。2.传染性法氏囊病病毒VP2基因在昆虫细胞中的表达为制备针对传染性法氏囊病病毒(IBDV)流行毒株的重组VP2蛋白,将从安徽省IBD免疫失败的病鸡法氏囊组织中扩增的VP2基因插入到杆状病毒表达系统供体质粒pFastBacHTA中,转化大肠杆菌DH10Bac感受态细胞,并成功获得重组杆状病毒表达质粒rBacHT-VP2,用脂质体法转染Sf9细胞,获得重组杆状病毒vBacHT-VP2.经Western-blotting分析,在蛋白分子量约53 ku处出现特异性蛋白条带；以间接免疫荧光试验(IFA)检测vBacHT-VP2感染的Sf9细胞,具有特异性免疫荧光；电镜观察,重组VP2蛋白能够自组装成病毒样颗粒(VLPs)。3.传染性法氏囊病病毒VP2蛋白单克隆抗体的制备与应用用大肠杆菌表达的传染性法氏囊病病毒(IBDV)重组VP2蛋白、杆状病毒表达的重组VP2蛋白以及纯化的IBDV分别免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术建立了9株分泌抗IBDV VP2蛋白的单克隆抗体(Monoclonal Antibodies, mAbs)杂交瘤细胞系。间接ELISA检测9株mAbs的细胞培养上清液效价为4×10-2～1.6×10-3,腹水效价为10-2～10-5。Western-blotting结果表明,其中4株mAbs在蛋白分子量约53ku处有特异性条带,5株则未显示蛋白带。相加ELISA结果显示,单克隆抗体A12G、B12F、F9C对应相同的抗原表位,其余6株针对不同抗原表位。在夹心ELISA中,9株mAbs与新城疫病毒、传染性支气管炎病毒、减蛋综合征病毒、禽流感病毒H9N2亚型、Sf9细胞培养上清液和CEF裂解物均无交叉反应。将9株杂交瘤细胞系进行传代培养、液氮冻存与复苏实验,杂交瘤细胞仍保持稳定分泌单克隆抗体的能力。用本实验制备的mAbs建立的IBDV夹心ELISA检测方法具有良好的特异性。更多还原

【Abstract】 Infectious bursal disease virus (IBDV), the etiological agent of infectious disease, destructs lymphatic tissue, especially the lymphocytes in the bursa of Fabricius in young chickens. It causes death and immunodepression, and results in immunity decrease and vaccination failure. This disease continues to pose significant losses in the commercial poultry industry. The epidemic situation of IBD is complex, while the vIBDV (virulent IBDV) and vvIBDV (very virulent IBDV) appeare for the past few years which can escape from common vaccinal immounization, because of the virulence and antigencity changing. IBDV VP2 protein is the main structural protein and host protective antigen, therefore, studies on VP2 protein and the detection methods for VP2 are important works at present. In this study, we have expressed the prevalent IBDV VP2 gene in Escherichia coli and baculovirus expression system respectively. Nine monoclonal antibodies (mAbs) against VP2 protein were developed. The thesis includes 3 parts:1. Expression in Escherichia coli of VP2 gene of infectious bursal disease virusA VP2 gene of infectious bursal disease virus (IBDV) was amplified from bursa of Fabricius of chickens with immunoprophylaxis defeat in Anhui province and cloned into the prokaryotic expression vector pGEX-4T-1. The expression plasmid pGEX-VP2 was constructed and transformed into competent Escherichia coli Rosetta (DE3), the target protein was expressed under the induction with IPTG and was positive in the detection of IBDV sandwich ELISA. SDS-PAGE analysis showed that the VP2 fusion protein was approximately 68 ku in molecular weight, and it made up 16.0% of the total bacterial proteins as inclusion bodies in E. coli. Western-blotting showed that the VP2 protein could react with IBDV antibody, indicating that the VP2 protein possessed good antigenicity. 2. Expression of VP2 gene of infectious bursal disease virus in insect cellsTo develop the recombinant VP2 protein of the current infectious bursal disease virus (IBDV), the VP2 gene of IBDV isolated from chicken with immunoprophylaxis defeat in Anhui province was cloned into pFastBacHTA donor plasmid. The recombinant donor plasmid pFastBacHT-VP2 containing VP2 was constructed and transformed into competent E. coli DH10Bac cells. After screening, the recombinant expression Bacmid rBacHT-VP2 was obtained and used to transfect insect Sf9 cells with Lipofectamine reagent to produce recombinant baculovirus vBacHT-VP2. The protein band of approximate 53 ku was detected in western blotting, the Sf9 cells infected with vBac-VP2 could generate specific fluorescent light in the indirect immunofluorescence assay (IFA), and the self-assembly virus-like particles (VLPs) could be observed by electron microscopy.3. Preparation and application of monoclonal antibodies against VP2 protein of infectious bursal disease virusRecombinant VP2 protein of infectious bursal disease virus (IBDV) of AH1 strain expressed by Escherichia coli and recombinant baculovirus and purified IBDV antigen were used to immunize BALB/c mice, nine hybridomas secreting anti-VP2 monoclonal antibodies(mAbs) were established by hybridoma technique. The antibodies titers were 4×10-2～1.6×10-3 in culture supernatants and 10-2～10-5 in ascetic fluids by indirect-ELISA. The recombinant VP2 protein could react with four mAbs and the specific protein bands were 53 ku in western-blotting. Additivity ELISA indicated that A12G、B12F and F9C were corresponding to the same antigenic epitope, and the other six mAbs were different. The nine mAbs were specific to IBDV and didn’t react with NDV, IBV, EDAV, AIV H9N2, culture supernatant of Sf9 cells and lysate of CEF in sandwich ELISA. The stability experiment indicated that the nine mAbs posed the ability of secreting antibodies after subculturing, freezing and thawing test. The sandwich ELISA established by the mAbs for detecting IBDV showed highly specificity.更多还原