【摘要】：快速、有效、經濟地從生物材料如動物、植物、微生物和離體培養(yǎng)物中高通量獲取高活性、高純度的目的生物大分子是生物制品生產的中心環(huán)節(jié)。傳統的技術手段很多,國內外病毒純化和制備多采用經典的離心技術、PEG或過硫酸銨沉淀技術,透析法、層析技術等等。這些傳統方法在大批量生產和推廣應用上均存在各種弊端。本研究以藍舌病毒(Bluetongue virus,BTV)為模型,結合有限的離心和瓊脂糖蛋白質A沉淀目標生物大分子的背景雜質,達到獲取高純度,高活性生物大分子的目的。該方法與傳統的技術不同,沉淀的不是目標物質,而是背景雜質,故命名為瓊脂糖蛋白質A反向免疫共沉淀技術(Protein A intermediary reverse co-immunoprecipitation,PARIP)。 反復凍融破碎裂解的未感染病毒的Vero細胞800rpm離心10分鐘后,取上清,制備成抗原,以常規(guī)方法免疫家兔,制備抗Vero細胞碎片和抗小牛血清的多克隆抗體。在一定的溫度、pH值、反應時間和振蕩速度等條件下,利用蛋白質A能與IgG抗體非特異性牢固結合的特點,將免疫家兔所得多克隆抗體耦聯到瓊脂糖Protein A上;再利用抗體抗原特異性結合的特點,以此耦聯了抗體的瓊脂糖Protein A去吸附細胞培養(yǎng)病毒增殖混合懸液(經12000rpm,15min離心去沉淀)中的Vero細胞碎片及小牛血清抗原。發(fā)生上述反應的試管以1000rpm,15min低速離心,可使介質瓊脂糖Protein A及其上附著的抗原抗體復合物沉淀下來,上清即為純化的含單一病毒成分的懸液。瓊脂糖雙向免疫擴散檢測純化后的病毒懸液,懸液中不含抗Vero細胞碎片及小牛血清抗原的抗體。介質瓊脂糖ProteinA上吸附的抗原抗體復合物可用緩沖液B洗去后繼續(xù)使用。用透射電鏡觀察病毒純化效果,純化后的病毒懸液背景清晰,病毒粒子多且輪廓清楚。高效液相色譜檢測純化前后病毒樣品的各成分含量,得到純化前病毒樣品中有多個較高的峰,而純化后病毒樣品中只含有單一的峰。病毒峰面積占所有峰面積的98.9%,小牛血清雜質的含量控制在5ng/mL以下,效果顯著,符合中國生物制品質量檢測的要求。在Vero細胞上檢測病毒懸液的TCID_(50),純化前混合有雜質的病毒懸液TCID_(50)為4x10~(-5.25)/ml,純化后病毒懸液TCID_(50)為4×10~(-4.7)/ml,在10~0,10~(-1),10~(-2),10~(-3)這四個稀釋度下,所計算純化得率分別79.81%,77.90%,78.13%,55.42%。此結果表明純化前后病毒粒子的生物學活性損失不大。以上實驗研究結果表明:該實驗方法操作簡便,效果顯著,切實
[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.
【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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