瓊脂糖Protein A反向免疫共沉淀技術(shù)純化高活性BTV方法的建立
發(fā)布時(shí)間：2018-12-23 18:31
【摘要】：快速、有效、經(jīng)濟(jì)地從生物材料如動(dòng)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(chǎn)的中心環(huán)節(jié)。傳統(tǒng)的技術(shù)手段很多,國(guó)內(nèi)外病毒純化和制備多采用經(jīng)典的離心技術(shù)、PEG或過硫酸銨沉淀技術(shù),透析法、層析技術(shù)等等。這些傳統(tǒng)方法在大批量生產(chǎn)和推廣應(yīng)用上均存在各種弊端。本研究以藍(lán)舌病毒(Bluetongue virus,BTV)為模型,結(jié)合有限的離心和瓊脂糖蛋白質(zhì)A沉淀目標(biāo)生物大分子的背景雜質(zhì),達(dá)到獲取高純度,高活性生物大分子的目的。該方法與傳統(tǒng)的技術(shù)不同,沉淀的不是目標(biāo)物質(zhì),而是背景雜質(zhì),故命名為瓊脂糖蛋白質(zhì)A反向免疫共沉淀技術(shù)(Protein A intermediary reverse co-immunoprecipitation,PARIP)。 反復(fù)凍融破碎裂解的未感染病毒的Vero細(xì)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(xì)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(yīng)時(shí)間和振蕩速度等條件下,利用蛋白質(zhì)A能與IgG抗體非特異性牢固結(jié)合的特點(diǎn),將免疫家兔所得多克隆抗體耦聯(lián)到瓊脂糖Protein A上;再利用抗體抗原特異性結(jié)合的特點(diǎn),以此耦聯(lián)了抗體的瓊脂糖Protein A去吸附細(xì)胞培養(yǎng)病毒增殖混合懸液(經(jīng)12000rpm,15min離心去沉淀)中的Vero細(xì)胞碎片及小牛血清抗原。發(fā)生上述反應(yīng)的試管以1000rpm,15min低速離心,可使介質(zhì)瓊脂糖Protein A及其上附著的抗原抗體復(fù)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(kuò)散檢測(cè)純化后的病毒懸液,懸液中不含抗Vero細(xì)胞碎片及小牛血清抗原的抗體。介質(zhì)瓊脂糖ProteinA上吸附的抗原抗體復(fù)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(cè)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(gè)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(zhì)的含量控制在5ng/mL以下,效果顯著,符合中國(guó)生物制品質(zhì)量檢測(cè)的要求。在Vero細(xì)胞上檢測(cè)病毒懸液的TCID_(50),純化前混合有雜質(zhì)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(gè)稀釋度下,所計(jì)算純化得率分別79.81%,77.90%,78.13%,55.42%。此結(jié)果表明純化前后病毒粒子的生物學(xué)活性損失不大。以上實(shí)驗(yàn)研究結(jié)果表明:該實(shí)驗(yàn)方法操作簡(jiǎn)便,效果顯著,切實(shí)
[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(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
本文編號(hào)：2390090
[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(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
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