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|关键词||灭活口蹄疫病毒 离子交换色谱 差示扫描量热仪 稳定性 解聚|
口蹄疫（Foot-and-mouth disease，FMD）是由口蹄疫病毒（Foot-and-mouth disease 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10.03 mg/mL，而在小孔径的琼脂糖介质DEAE-FF（孔径32nm）介质上的DBC仅有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，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48.52oC。而吸附在三种介质上之后的Tm1值分别降低为41.73oC, 44.04oC和45.37oC，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，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FMDV的色谱纯化具有一定的指导意义。
Inactivated foot-and-mouth disease virus (FMDV) vaccine is one of the most important vaccines for preventing foot-and-mouth disease, which is a highly infectious disease of ruminants and would result in large economic loss once breakout. Purification is a key step to ensure sufficient immunogenic effect and safety for inactivated FMDV vaccine. Chromatographic techniques possess advantages of scalability and high selectivity, thus providing an alternative strategy for FMDV vaccine purification. It is challenging to obtain high recovery and stable native structure for inactivated FMDV whose size is about 28 nm during chromatography. In this paper, we have investigated the critical factor on purification efficiency and structural stability for purification of inactivated FMDV by ion exchange chromatography(IEC) , and tried to establish a novel purification process for industrial production, including: (1) Three anion-exchange media with similar particle size and ligand density except pore size, including DEAE-FF (32 nm), DEAE-650M (106 nm) and DEAE-POROS (214 nm), were applied and compared for purification of inactivated FMDV. We have investigated the effect of pore size of media on the dynamic binding capacity (DBC) for inactivated FMDV, and results show that the DBC for DEAE-POROS and DEAE-650M were 11.53 and 10.03 mg/mL, while that for DEAE-FF was less than 1/10 of the previous two. And confocal laser scanning microscopy (CLSM) also confirmed that inactivated FMDV mostly confined to a thin shell on the outer surface of DEAE-FF, but permeated into intraparticle region of DEAE-POROS. (2) The effects of pore size of media on the recovery and stability of inactivated FMDV were investigated during IEC process. The recovery of inactivated FMDV after chromatographic process was 68.42%, 66.32% and 54.46% on DEAE-POROS, DEAE-650M and DEAE-FF in sequence. High performance size exclusion chromatography (HPSEC) analysis results indicated that interaction between inactivated FMDV and media was the main reason for FMDV dissociation into smaller subunits 12S, and severer dissociation occured on media with smaller pore size. Possible denaturation of the FMDV on the surfaces of anion exchange media was analyzed by differential scanning calorimetry (DSC). The transition temperature (Tm1), dissociating of inactivated FMDV into 12S, is at about 48.52oC in solution. When inactivated FMDV were absorbed on DEAE-FF, the Tm1 became 41.73oC, indicating increased the possibility of dissociation. The Tm1 for DEAE-650M and DEAE-POROS was 44.04oC and 45.37oC, showing less dissociation and higher thermostability was obtained on DEAE-POROS. (3) We developed a purification process for inactivated FMDV by DEAE-POROS. Inactivated FMDV crude was loaded onto DEAE-POROS IEC at the optimum condition, including conductivity of buffer, the protein concentration of crude and retention time. 94% inactivated FMDV recovery was reliably recovered from crude solution by IEC. After polished by ultrafiltration and size exclusion chromatography (SEC), intact inactivated FMDV with high-purity was obtained. The average total recovery was 79%, with 173-fold increase in purity, and above 95% host DNA was removed. In summary, this work demonstrated that media with large pore size was superior to the conventional agarose-gel based chromatographic media for the purification of inactivated FMDV in several aspects, including static and dynamic binding capacity, thermostability and recovery during chromatographic process. And the mechanism of dissociation of inactivated FMDV on DEAE media has also been investigated. All these results will provide a useful guidance to establish an efficient purification process for FMDV.
|梁山琴. 灭活口蹄疫病毒抗原的离子交换色谱纯化[D]. 北京. 中国科学院研究生院,2017.|
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