琼脂糖Protein A反向免疫共沉淀技术纯化高活性BTV方法的建立

发布时间：2018-12-23 18:31

【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TCID_(50),纯化前混合有杂质的病毒悬液TCID_(50)为4x10~(-5.25)/ml,纯化后病毒悬液TCID_(50)为4×10~(-4.7)/ml,在10~0,10~(-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
[Abstract]:Rapid, efficient and economical extraction of high-throughput biological macromolecules from biomaterials such as animals, plants, microorganisms and in vitro cultures is the central link in the production of biological products. There are many traditional methods for virus purification and preparation, such as classical centrifugation, PEG or ammonium persulfate precipitation, dialysis, chromatography and so on. These traditional methods have various disadvantages in mass production and application. In this study, blue tongue virus (Bluetongue virus,BTV) was used as a model, combined with limited centrifugation and agarose protein A to precipitate background impurities of target biomolecules, in order to obtain high purity and high activity biomolecules. This method is different from the traditional technology, the precipitation is not the target substance, but background impurity, so it is named as agarose protein A reverse immunoprecipitation (Protein A intermediary reverse co-immunoprecipitation,PARIP). The 800rpm of uninfected Vero cells was centrifuged for 10 minutes after repeated freezing and thawing, then the supernatant was extracted and the antigens were prepared. The rabbits were immunized with routine methods to prepare anti-Vero cell fragments and polyclonal antibodies against calf serum. Under the conditions of certain temperature, pH value, reaction time and oscillating speed, the polyclonal antibodies obtained from immunized rabbits were coupled to agarose Protein A by using the characteristics that protein A could bind to IgG antibody firmly and nonspecifically. Using the specific binding characteristics of antibody antigens, the fragments of Vero cells and calf serum antigens from the antibody-agarose Protein A desorbed cell culture mixed suspension of virus proliferation (centrifugation and precipitation after 15 min centrifugation of 12000rpmm-1) were coupled. The medium agarose Protein A and the antigen-antibody complex attached to it could be precipitated by centrifugation at a low speed of 1 000 rpm for 15 min. The supernatant was a purified suspension containing a single viral component. The purified virus suspension was detected by double immunodiffusion with agarose. The suspension did not contain antibodies against Vero cell fragments and calf serum antigen. The antigen-antibody complexes adsorbed on the medium agarose ProteinA can be washed out by buffer B and continue to be used. The purification effect of the virus was observed by transmission electron microscope. The purified virus suspension had a clear background, many virus particles and a clear outline. High performance liquid chromatography (HPLC) was used to detect the contents of each component of the purified virus samples, and the results showed that there were many higher peaks in the purified previrus samples, but only a single peak was found in the purified samples. The virus peak area accounted for 98.9% of all peak areas, and the content of serum impurities in calf serum was controlled below 5ng/mL, and the effect was remarkable, which met the requirement of quality detection of biological products in China. TCID_ (50) of virus suspension was detected on Vero cells. The TCID_ (50) of virus suspension mixed with impurity before purification was 4x10- (-5.25) / ml,. After purification, TCID_ (50) of virus suspension was 4 脳 10 ~ (-4.7) / ml, was 10 脳 10 ~ (-1). Under the dilution of 10 ~ (-2) and 10 ~ (-3), the calculated purification rates were 79.81 and 77.900.78.13 and 55.42, respectively. The results showed that the biological activity loss of virus particles before and after purification was not significant. The experimental results show that the method is simple, effective and practical.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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