【作者】 刘文培；

【导师】 董长垣；

【作者基本信息】 武汉大学 ， 病原生物学， 2005， 硕士

【摘要】 快速、有效、经济地从生物材料如动物、植物、微生物和离体培养物中高通量获取高活性、高纯度的目的生物大分子是生物制品生产的中心环节。传统的技术手段很多,国内外病毒纯化和制备多采用经典的离心技术、PEG或过硫酸铵沉淀技术,透析法、层析技术等等。这些传统方法在大批量生产和推广应用上均存在各种弊端。本研究以蓝舌病毒（Bluetongue virus,BTV）为模型,结合有限的离心和琼脂糖蛋白质A沉淀目标生物大分子的背景杂质,达到获取高纯度,高活性生物大分子的目的。该方法与传统的技术不同,沉淀的不是目标物质,而是背景杂质,故命名为琼脂糖蛋白质A反向免疫共沉淀技术（Protein A intermediary reverse co-immunoprecipitation,PARIP）。 反复冻融破碎裂解的未感染病毒的Vero细胞800rpm离心10分钟后,取上清,制备成抗原,以常规方法免疫家兔,制备抗Vero细胞碎片和抗小牛血清的多克隆抗体。在一定的温度、pH值、反应时间和振荡速度等条件下,利用蛋白质A能与IgG抗体非特异性牢固结合的特点,将免疫家兔所得多克隆抗体耦联到琼脂糖Protein A上;再利用抗体抗原特异性结合的特点,以此耦联了抗体的琼脂糖Protein A去吸附细胞培养病毒增殖混合悬液（经12000rpm,15min离心去沉淀）中的Vero细胞碎片及小牛血清抗原。发生上述反应的试管以1000rpm,15min低速离心,可使介质琼脂糖Protein A及其上附着的抗原抗体复合物沉淀下来,上清即为纯化的含单一病毒成分的悬液。琼脂糖双向免疫扩散检测纯化后的病毒悬液,悬液中不含抗Vero细胞碎片及小牛血清抗原的抗体。介质琼脂糖ProteinA上吸附的抗原抗体复合物可用缓冲液B洗去后继续使用。用透射电镜观察病毒纯化效果,纯化后的病毒悬液背景清晰,病毒粒子多且轮廓清楚。高效液相色谱检测纯化前后病毒样品的各成分含量,得到纯化前病毒样品中有多个较高的峰,而纯化后病毒样品中只含有单一的峰。病毒峰面积占所有峰面积的98.9%,小牛血清杂质的含量控制在5ng/mL以下,效果显著,符合中国生物制品质量检测的要求。在Vero细胞上检测病毒悬液的TCID₅₀,纯化前混合有杂质的病毒悬液TCID₅₀为4x10^-5.25/ml,纯化后病毒悬液TCID₅₀为4×10^-4.7/ml,在10⁰,10^-1,10^-2,10^-3这四个稀释度下,所计算纯化得率分别79.81%,77.90%,78.13%,55.42%。此结果表明纯化前后病毒粒子的生物学活性损失不大。以上实验研究结果表明:该实验方法操作简便,效果显著,切实更多还原

【Abstract】 It is an indispensable approach of getting high amount, high infectivity and high purity products with high throughput in the purification and preparation of biological products. The traditional methods of purifying are centrifugation, precipitation and chromatography, etc. These methods had many defections and could not meet the need completely. This article will introduce a novel technology of purifying active bluetongue viruses （BTV） isolated and systematically studied as a model in our laboratory, which is named as the technology of purifying viruses by reverse co-immunoprecipitation and affinity with agarose Protein A for getting the purified BTV particles. The antibodies of the bovine serum and Vero cell fragments were made from immuned rabbits. Then use agarose Protein A to adsorb these antibodies under the optimized temperature, pH value and time. Use the antibodies-Protein A complexes to adsorb the antigens of the bovine serum and Vero cell fragments in BTV preparation proliferated on the culture monolayer of Vero cell under the optimized temperature, pH value and time. The immune complexes from these antibodies to antigens of the bovine serum and Vero cell fragments were adsorbed on the agarose Protein A and can be precipitated by centrifuging the reaction test tube at low speed, and the purified viral particles can be retained in the suspension. The immune complexes from these antibodies to antigens of the bovine serum and Vero cell fragments were adsorbed on the agarose Protein A can be washed down with fit buffer, and agarose Protein A can be used again. Agarose double immunodiffusion shows that there are no bovine serum and Vero cell fragments’antigens in the viral suspension. The electron microscopic detection on the negative staining virus showing that the background of the purified virus suspension and pellet are clean and clear（Fig.4）. HPLC shows that there is a single peak among suspension of the purified virus（Fig.5）. The ratio of virus peak’s square among all peaks is 98.9%,which meets the need of Chinese biological product quality detection. The TCID50 of the viral suspension on the Vero cell line is 4×10^-5.25/ml before purification and 4×10^-4.4/ml after purification. The recovery of virus under the titer of 10°, 10 ^-1, 10^-2,10^-3are 79.81%, 77.90%, 78.13%, 55.42% separately. From TCID50 it can be seen that the viral infectivity is maintained.The conclusion is that this method to purify BTV is significant and feasible. And it is a base for producing high purification, high yield and high infectivity bluetongue virus usingas a nature oncolytic virus and using for other aids. Further more, this method holds the character of high throughput. We can purify all kinds of biological product with this principal.更多还原