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由禽白血病病毒和鸡白痢沙门氏菌感染引起的禽白血病和鸡白痢是两种严重的禽类传染病，不仅造成禽类大量死亡，产生巨大的经济损失，还可能感染人体，影响健康。由于缺乏有效的疫苗，目前主要通过诊断淘汰病鸡，净化鸡群来达到防控的目的。快速、准确的诊断检测对禽病的有效防控至关重要, 但是由于缺乏标准物质，诊断试剂质量良莠不齐，导致检测结果的准确性往往难以判断。将诊断标志物研制成标准物质，对现有的诊断技术或诊断试剂盒等进行标定，使检测结果具有可溯源性、可比性和准确性，对禽病的防控具有重要意义。禽白血病病毒抗原蛋白P27和鸡白痢沙门氏菌感抗体是这两种疾病诊断检测的重要标志物。为此，本论文建立了针对该两种禽病诊断用标准物质的纯化及稳定工艺。主要研究结果如下：（1）针对用于禽白血检测的抗原蛋白P27，利用组氨酸标签建立了两步Ni亲和层析纯化工艺：首先通过吸附式Ni亲和层析纯化大肠杆菌重组表达带有His-tag的P27蛋白，经酶切去除标签后再次进行穿透式Ni亲和层析进行精制。最终P27纯度达到99%，ELISA活性回收率为30%。利用Tricine电泳及十二烷基硫酸钠毛细管电泳（CE-SDS）对纯化过程中P27的稳定性进行研究，发现-80℃保存能有效抑制原料液中P27末端His-tag的脱落，加入酶抑制剂和咪唑能有效抑制酶切过程中的蛋白裂解。最终精制得到的P27蛋白分子质量与理论分子量完全相符，可溯源性和纯度均能满足标准物质的制备要求。（2）针对用于鸡白痢沙门氏菌检测的抗体，构建了Q-XL阴离子交换层析一步纯化鸡白痢沙门氏菌兔抗血清获得高纯度多抗IgG的方法。采用高效液相体积排阻色谱建立了IgG的快速分析和定量方法，并利用多抗IgG与杂蛋白等电点的差异性指导阴、阳离子交换层析介质种类和pH条件的筛选。结果表明阴离子交换介质Q-XL在pH 5.5条件下，能够去除所有杂蛋白，纯度达99%，回收率为67.5%。进一步采用差示扫描荧光进行稳定剂快速筛选，其中20%（w/v）山梨醇稳定效果最佳，使裂解温度Tm1和Tm2分别提高5.52和8.84℃，在70℃加速稳定性中效果显著。最终制备的兔抗血清多抗IgG具有高纯度、高收率及高稳定性，且制备工艺简单，可作为标准物质，促进鸡白痢的防控。 （3）考察了利用提取的鸡白痢沙门氏菌提取O-抗原多糖，制备超大孔亲和介质用于纯化特异性IgG，以及用于间接酶联免疫吸附诊断鸡白痢的可行性。经等温滴定量热仪检测，通过酸解法提取出的O-抗原与多抗IgG具有高度特异性的亲和作用。采用激光共聚焦显微镜观察O-抗原成功接枝到环氧活化的超大孔微球上，静态吸附结果表明所制备的亲和介质能特异性吸附IgG蛋白，载量为0.46 mg/g-介质。将O-抗原包被用于鸡白痢的间接酶联免疫吸附法诊断，结果表明检测具有特异性，最佳抗原包被浓度为40 μg/mL，IgG最低检测限为46.92 ng/mL，从而为鸡白痢的准确诊断提供新的方法。;Avian leukemia and pullorum disease caused by avian leukemia virus and salmonella pullorum are two of most serious poultry epidemics that not only cause huge economic losses through the massive death of poultry, but also may infect human bodies and affect health. Therefore, effective prevention and control is vital to achieve complete elimination, but so far there is no effective vaccine. Diagnosis and cleaning out diseased chickens are the major strategy for prevention and control purposes. Rapid and accurate diagnosis of the desease is therefore extremanly important, but largely limited by lack of effective standard substances to ensure a reliable diagnosis results for poultry diseases. Developing diagnostic markers into reference materials can be used to calibrate existing detection techniques or detection kits, so that the results are traceable, comparable and accurate, and of great significance for the prevention and control of poultry diseases. Avian leukosis virus (ALV) antigen protein P27 and anti-Salmonella pullorum polyclonal immunoglobulin G (IgG) is generally used as a diagnostic marker for the coresponding desease. In this paper, we aimed at establishing purification and stabilization processes for these two diagnostic markers to be used as reference materials. The main results and findings are as follows:(1) A method for the purification of avian leukosis virus antigen protein P27 by two-step Ni affinity chromatography using his-tag was established. The his-taged P27 protein expreesed by recombinant E. coli was firstly purified by Ni affinity chromatography through bound-elution mode. After removing the his-tags on P27 by enzymatic digestion, the P27 was further purified by Ni affinity chromatography through a break-thrgough mode. After purification, the purity analyzed by SDS-PAGE was 99% and activity recovery measured by ELISA was 30%, respectively. Protein P27 degradation occurs in the preservation of the fermentation raw material and enzymatic digestion process was observed by tricine-PAGE and capillary electrophoresis with sodium dodecylsulfate (CE-SDS). Storing the raw material at -80°C and adding protease inhibitor and imidazole in the enzyme digestion process was found effectively inhibit the degradation. The moledular weight of the final product determined by MALDI-TOF is consistent with the theoretical value, therefore both the traceability and purity satisfy the reference material preparation requirements. (2) A single step purification of polyclonal immunoglobulins G (IgG) against Salmonella pullorum from rabbit serum by anion exchange chromatography was developed. According to the difference of isoelectric point (pI) between IgG and the major impurities, the types of ion exchange media were systematically screened by comparison of their purification results. The results showed that pI of the IgG was 6.04~7.08 determined by capillary iso-electric focusing. In contrast, the purity of IgG was 99.3% and recovery rate was 67.5% after anion exchange chromatography with packing materials of Q-XL. After anion exchange chromatography with packing materials of Q-XL, a purity of 99% analyzed by SDS-PAGE and a recovery of 67.5% measured by high performance size-exclusion chromatography was achived under pH 5.5. In order to prevent the IgG from denaturation, different stabilizers were screened by differential scanning fluorimetry (DSF). 200 g/L sorbitol was found to possess the best protection effect. The two thermal denaturation temperatures of IgG were increased by 5.52 and 8.84℃, respectively, and the stability at 70℃ was significantly improved. The finally prepared polyclonal IgG has high purity, high recovery and high stability, and the preparation process is simple. It can be used for developing reference material to promote the prevention and control of pullorum disease. (3) O-antigen located at the outermost side of the Salmonella pullorum cell was extracted and its suitability as ligand of affinity adsorbent for IgG purification and antigen for indirect enzyme-linked immunosorbent assay (ELISA) of specific IgG anginst the bateria were explored. The O-antigen showed a specific affinity interaction with the anti-serum polyclonal IgG according to the isothermal titration calorimetry (ITC) analysis. By grafting the O-antigen onto the epoxy-activated giga-porous media, affinity adsorbent was successfully preapred, which showed a static adsorption amount of 0.46 mg-IgG/mL-media. When was coated on 96-well as antigen for serological testing, the O-antigen showed specificity in indirect ELISA. Under the optimal coating concentration of 40 μg/mL, the lowest detection limit of IgG was 46.92 ng/mL, thus provided a powerful tool for accurate diagnosis of pullorum disease.
|尤星力. 禽白血病及鸡白痢检测用标准物质的纯化及稳定[D]. 中国科学院大学,2019.|
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