灭活口蹄疫病毒抗原的离子交换色谱纯化
发布时间：2018-08-28 19:48
【摘要】：口蹄疫(Foot-and-mouth disease,FMD)是由口蹄疫病毒(Foot-and-mouthdisease virus,FMDV)引起的偶蹄动物的烈性传染性疾病。曾多次在世界范围内爆发,造成严重的经济损失。接种灭活口蹄疫病毒疫苗是预防口蹄疫最有效的手段。随着对疫苗安全性和质量要求的不断提高,对其进行有效的分离纯化日益受到重视。色谱分离具有分辨率高、易于规模放大的优势,在动物疫苗分离纯化中具有潜在的应用前景。但是如何实现结构复杂、尺寸达到28 nm的病毒抗原颗粒在有效分离纯化的同时,保持颗粒结构的稳定,获得高的抗原收率,仍然是一个挑战。本文旨在通过研究灭活FMDV在离子交换色谱分离过程中,影响其分离效率和颗粒稳定性的关键因素,以建立高效的纯化工艺。主要研究内容包括如下几方面:(1)选取DEAE-FF、DEAE-650M、以及DEAE-POROS三种具有相似配基密度和粒径,但孔径显著不同的阴离子交换介质,考察了介质孔径对灭活FMDV动态结合载量(Dynamic binding capacity,DBC)的影响。发现灭活 FMDV 在 DEAE-POROS(孔径214 nm)和DEAE-650M(孔径106 nm)介质上的DBC分别达到11.53和10.03mg/mL,而在小孔径的琼脂糖介质DEAE-FF(孔径32nm)介质上的DBC仅为前两者的1/10。激光共聚焦实验表明灭活FMDV在DEAE-FF介质上的吸附仅限于微球的表面薄层区域,而在大孔DEAE-POROS介质上的吸附能扩散进入微球内部。(2)研究了离子交换介质孔径对灭活FMDV纯化收率和稳定性的影响。经过DEAE-FF、DEAE-650M、以及DEAE-POROS离子交换层析后,灭活FMDV的收率分别为54.46%、66.32%和68.42%。通过高效液相凝胶过滤色谱法(HPSEC)分析发现,灭活FMDV与介质吸附-解吸作用会导致其裂解成无免疫活性的12S,且裂解程度随着介质孔径的增大而减小。利用差示扫描量热仪(Differential Scanning Calorimetry,DSC)分析灭活FDMV吸附在介质上发生解聚的机理。灭活FMDV在溶液中发生裂解生成12S的转变温度Tm1为48.52℃。而吸附在三种介质上之后的Tm1值分别降低为41.73℃,44.04℃和45.37℃,表明在层析介质上的吸附导致灭活FMDV更易于发生裂解,而吸附在大孔径的DEAE-POROS介质上的灭活FMDV的稳定性相对较高。(3)建立了大孔DEAE-POROS介质纯化灭活FMDV的工艺。对缓冲液的电导率、进样蛋白浓度、停留时间等参数进行了优化。优化条件下,灭活FMDV的IEC收率达94%。经过进一步超滤浓缩和凝胶过滤色谱(SEC)精制,最终灭活FMDV收率为79%,纯化倍数为173,宿主DNA的去除率达95%以上。本文研究结果表明大孔介质纯化灭活FMDV相比传统的琼脂糖介质在动态结合载量、活性收率和结构稳定性上具有明显的优势,并探讨了可能的作用机理,对灭活FMDV的色谱纯化具有一定的指导意义。
[Abstract]:Foot and mouth disease (Foot-and-mouth disease,FMD) is a severe infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (Foot-and-mouthdisease virus,FMDV). There have been many outbreaks around the world, causing serious economic losses. Inoculation with inactivated foot-and-mouth disease virus vaccine is the most effective method to prevent foot-and-mouth disease. With the improvement of the safety and quality of vaccine, more and more attention has been paid to the effective separation and purification of vaccine. Chromatographic separation has the advantages of high resolution and easy scale amplification, and has a potential application prospect in the purification of animal vaccine. However, it is still a challenge to achieve the complex structure of virus antigen particles of 28 nm, while maintaining the stability of the particle structure and obtaining high antigen yield. The purpose of this paper is to establish an efficient purification process by studying the key factors affecting the separation efficiency and particle stability of inactivated FMDV in ion exchange chromatography. The main contents are as follows: (1) three anion exchange media, DEAE-FF,DEAE-650M, and DEAE-POROS, which have similar ligands density and particle size, but have different pore sizes, are selected to investigate the effect of pore size on the dynamic binding capacity of FMDV (Dynamic binding capacity,DBC). It was found that the DBC of inactivated FMDV on DEAE-POROS (aperture 214 nm) and DEAE-650M (aperture 106 nm) media was 11.53 and 10.03 mg / mL, respectively, while the DBC on small aperture agarose medium DEAE-FF (pore size 32nm) was only 1 / 10 of the former. Laser confocal experiments show that the adsorption of inactivated FMDV on DEAE-FF media is confined to the thin layer area of the surface of the microspheres. However, the adsorption energy on the macroporous DEAE-POROS medium diffuses into the microspheres. (2) the effect of the pore size of the ion exchange medium on the purification yield and stability of the inactivated FMDV was studied. After DEAE-FF,DEAE-650M, and DEAE-POROS ion exchange chromatography, the yields of inactivated FMDV were 54.46% and 66.32%, respectively. By (HPSEC) analysis of high performance liquid phase gel filtration chromatography, it was found that the inactivated FMDV and the adsorption-desorption of the medium would lead to the dissociation of 12s with no immune activity, and the degree of decomposition would decrease with the increase of the pore size of the medium. The mechanism of depolymerization of inactivated FDMV adsorbed on medium was analyzed by differential scanning calorimeter (Differential Scanning Calorimetry,DSC). The transition temperature (Tm1) of inactivated FMDV to form 12s in solution is 48.52 鈩，
本文编号：2210431
[Abstract]:Foot and mouth disease (Foot-and-mouth disease,FMD) is a severe infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (Foot-and-mouthdisease virus,FMDV). There have been many outbreaks around the world, causing serious economic losses. Inoculation with inactivated foot-and-mouth disease virus vaccine is the most effective method to prevent foot-and-mouth disease. With the improvement of the safety and quality of vaccine, more and more attention has been paid to the effective separation and purification of vaccine. Chromatographic separation has the advantages of high resolution and easy scale amplification, and has a potential application prospect in the purification of animal vaccine. However, it is still a challenge to achieve the complex structure of virus antigen particles of 28 nm, while maintaining the stability of the particle structure and obtaining high antigen yield. The purpose of this paper is to establish an efficient purification process by studying the key factors affecting the separation efficiency and particle stability of inactivated FMDV in ion exchange chromatography. The main contents are as follows: (1) three anion exchange media, DEAE-FF,DEAE-650M, and DEAE-POROS, which have similar ligands density and particle size, but have different pore sizes, are selected to investigate the effect of pore size on the dynamic binding capacity of FMDV (Dynamic binding capacity,DBC). It was found that the DBC of inactivated FMDV on DEAE-POROS (aperture 214 nm) and DEAE-650M (aperture 106 nm) media was 11.53 and 10.03 mg / mL, respectively, while the DBC on small aperture agarose medium DEAE-FF (pore size 32nm) was only 1 / 10 of the former. Laser confocal experiments show that the adsorption of inactivated FMDV on DEAE-FF media is confined to the thin layer area of the surface of the microspheres. However, the adsorption energy on the macroporous DEAE-POROS medium diffuses into the microspheres. (2) the effect of the pore size of the ion exchange medium on the purification yield and stability of the inactivated FMDV was studied. After DEAE-FF,DEAE-650M, and DEAE-POROS ion exchange chromatography, the yields of inactivated FMDV were 54.46% and 66.32%, respectively. By (HPSEC) analysis of high performance liquid phase gel filtration chromatography, it was found that the inactivated FMDV and the adsorption-desorption of the medium would lead to the dissociation of 12s with no immune activity, and the degree of decomposition would decrease with the increase of the pore size of the medium. The mechanism of depolymerization of inactivated FDMV adsorbed on medium was analyzed by differential scanning calorimeter (Differential Scanning Calorimetry,DSC). The transition temperature (Tm1) of inactivated FMDV to form 12s in solution is 48.52 鈩，
本文编号：2210431
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