【作者】 姜骞；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 本实验用IPTG诱导含有猪传染性胃肠炎病毒（TGEV）重组N基因质粒（PHN）的大肠杆菌DH5α，表达重组TGEV N蛋白，经Ni²⁺-NTA金属螯合层析柱纯化后获得纯化的N蛋白。对纯化条件进行优化，得到最佳纯化重组TGEV N蛋白的方法：将大肠杆菌DH5α菌体沉淀物经裂解液（含有1mmol／L PMSF）作用后，依次经过2倍体积的Buffer A（pH8.0）、Buffer B（pH6.3）和Buffer C（pH5.9）洗柱后，收集Buffer C（80mmol／L咪唑）洗脱产物。经SDS-PAGE分析分子量为47ku，Western-Blot检测呈阳性，纯化的N蛋白溶液经Bradford法测定蛋白质含量为154.7μg／ml-479.7μg／ml，推算每克大肠杆菌菌体沉淀物可获得纯化重组TGEV N蛋白1.52mg。 以重组TGEV N蛋白作为诊断抗原建立了两种检测抗体的诊断方法。在Dot-ELISA中其抗原最适包被量为1.25μg／点，抗原预点膜4℃可保存5周以上，特异性实验表明其与猪轮状病毒、猪流行性腹泻病毒等肠道腹泻性病毒均无交叉反应。在间接ELISA中，最佳抗原包被量为0.51μg／孔，封闭液为含5％脱脂奶粉的PBST，待检血清最适稀释倍数为200倍，作用时间为30min，底物液最佳作用时间为10min。 以纯化的重组TGEV N蛋白免疫BALB／c小鼠制备诊断用单克隆抗体。按常规方法进行细胞融合，用间接ELISA方法进行筛选，采用有限稀释法，经过3次克隆筛选，最终获得一株分泌抗重组TGEV N蛋白单克隆抗体的杂交瘤细胞（命名为E10F10D5），其染色体平均计数为87±10对，间接ELISA检测制备的腹水抗体效价达1:12800。以全病毒为抗原对杂交瘤细胞（E10F10D5）产生的腹水进行Western-Blot及间接ELISA检测分析，结果均为阳性。证明制备的单克隆抗体可以识别天然TGEV N蛋白的某一抗原表位。优化了裂解肠内容物中TGEV的方法。为建立诊断TGEV抗原的检测方法奠定了物质基础。 本研究首次在国内建立以重组TGEV N蛋白为抗原检测TGEV抗体的两种检测方法：DOT-ELISA和间接ELISA；制备了一株抗TGEV N蛋白的杂交瘤细胞系。为TGEV的疫情监测、及时而准确的诊断奠定物质基础。更多还原

【Abstract】 E.coli DH5a harbouring recombinant plasmid pProExHTb-N was induced with IPTG to express the recombinant nucleoprotein (rNP) of transmissible gastroenteritis virus (TGEV).The rNP collected after cell extraction was applied onto the nickel nitrilo-tri-acetic acid (Ni-NTA) resin. The optimized procedure of purification was lysing the E.coli by Buffer A (containing Immol/LPMSF) .washing the resin with 2+Buffer A (pH8.0),2+BufferB (pH6.3), 2X BufferC (pH5.9) and collecting the elution of Buffer C (80mmol/L imidazole) .The molecular weight of purified rNP was 47ku analyzed by SDS-PAGE.The result of Western-Blot showed that the rNP can act to sera of anti-TGEV. The concentration of the elution including rNP was identified 154.Vug per ml-4V9.Vug per ml by Bradford’s assay, at the same ratio 1.52mg rNP can be abtained per gram E.coli sedimentDot-ELISA and indirect-ELISA were established by the rNP. The optimal amount of the rNP coated in Dot-ELISA was 1.25g per dot. The pre-dot membrane can be kept 5 weeks long at 4 C. The specific assay showed that rNP have no cross-reaction with porcine epidemic diarrhea coronavirus (PEDV) porcine rotavirus (PRV) .In indirect-ELISA the optimal amount of coated antigen was 0.5lg per well,the optimal blocking solution was 5% dry milk,the ideal dilution of serum was 200 and the fit reaction time was 30mins the adequatable reacrion time of substrate was 10min.The monoclonal antibody (McAb) detecting TGEV was performed by immunizing the BALB/c mice with purified rNP. Cells fusion was carried out by standard method and hybridmas with rNP-antibody-positive supernatants were subcloned third by the limiting dilution. One hybridoma cell line secreting monoclonal antibody specific against rPN was picked out by indirect-ELISA designated E10F10D5. The even number of chromosomes per hybridoma was 8V +10 paires .The antibody liter of ascites v/as 1:12800 determined by indirect-ELISA. TGEV was applicated as antigen in Western-Blot and indirect-ELISA to analyze the McAb. The result showed that the McAb could indetified certain antigenic epitope of TGEV N protein. Method to dispose TGEV in intestinal sample was optimized.Two methods to detect antibodies of TGEV were established firstly by using rNp as antigen coated in Dot-ELISA and indirect-ELISA in domestic. One hybridoma cell line secreting monoclonal antibody recognizing TGEV N protein was produced. All the work founded material base for TGE diagnosis accurately and rapidly.Postgraduate: QianJiang Major: Preventive Veterinary ScienceSupervisor: Professor Li YiJing更多还原