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灭活病毒及病毒颗粒的结构表征和稳定性研究
杨延丽
学位类型博士
导师苏志国 ; 张松平
2015-10
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词灭活病毒 病毒样颗粒 口蹄疫病毒 乙肝病毒表面抗原 稳定
其他摘要

灭活病毒及病毒样颗粒(Virus like particles,VLPs)是两类重要的疫苗抗原,都具有多聚亚基的大颗粒组装体结构,在生产和储存过程中易于发生结构变化,影响疫苗的安全性和有效性。如何稳定这种大颗粒组装体结构是疫苗工程研究领域的一个重要课题。本论文以灭活口蹄疫病毒(Foot and mouth disease virus,FMDV)及乙肝病毒表面抗原病毒样颗粒(Hepatitis B virus surface antigen VLPs,HBsAg-VLPs)为对象,研究了其溶液中和固/液界面上颗粒结构变化的检测方法,分析了层析纯化中颗粒结构变化的机理,探索了影响结构稳定性的因素,主要内容如下:1)建立了高效液相凝胶过滤色谱法(HPSEC)快速检测和分析抗原颗粒组装体结构的技术,应用于FMDV、HBcAg-VLPs、HBsAg-VLPs在不同生产阶段以及储存溶液中颗粒完整性的检测,与传统的超高速离心分析法相比,具有简单、快速、可靠等优点。该方法经中国兽医药品监察所推荐,已被疫苗生产企业使用。2)分析了溶液中FMDV和HBsAg-VLPs的颗粒性质,采用场流分级、圆二色光谱、荧光光谱、动态光散射技术对两种抗原进行了表征,进一步证实了HPSEC方法的可靠性,同时提供了更为全面的结构变化的信息。 3)探索了双偏极化干涉(DPI)分析抗原颗粒在液固相界面的变化,发现HBsAg-VLPs在DEAE离子交换表面发生吸附,其厚度随pH不同而变化。在pH 5.0下,平均吸附层厚度高于HBsAg-VLPs的颗粒直径;在pH值7.0或9.0下, 平均吸附层厚度则小于HBsAg-VLPs的颗粒直径,说明病毒样颗粒在离子交换中可能出现复杂的结构变化。通过对比DPI分析和离子交换层析纯化的实验结果,证明了HBsAg-VLPs在离子交换层析中的活性损失与界面失活密切相关。4)建立了提高颗粒型抗原在生产过程中稳定性的策略。对于FMDV,最适pH范围在7.5-8.0,最适的盐类为铵盐。通过HPSEC及差示扫描量热法(DSC)证实了添加多羟基化合物对于疫苗抗原的颗粒结构具有稳定作用。当溶液中存在多羟基化合物时,超滤过程中的FMDV收率提高了10.4%,离子交换层析的收率从17.1%提高到了84.0%。整个工艺获得的FMDV完整颗粒(146S)的纯度达到98%以上,整体收率达到56.7%。

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Inactivated virus and virus-like particles (VLPs) represent two classes of important vaccine antigens with a common feature of multimeric subunit structure. The complex particulate assembly is susceptible to the environments of production, transportation and storage, resulting in conformational change which may bring about safety issues and decrease the efficiency of the vaccines. It is therefore an important subject to study the stability and stabilization of these particulate antigens. For these purposes, the inactivated foot and mouth disease virus (FMDV) and hepatitis B virus surface antigen VLPs (HBsAg-VLPs) were studied as models. Analytical methods of conformational changes of particulate antigens in solution and at solid-liquid interface, the mechanisms of conformational changes during purification, and factors affecting stabilization were systematiclally studied. The detailed contents and results are as follows.1) We developed a size-exclusion high-performance liquid chromatography (SE-HPLC) method for rapid characterization of the assembly structure of vaccine antigens. The HPSEC method was successfully used to analyze inactivated FMDV, hepatitis B core antigen (HBcAg) VLPs, and HBsAg-VLPs in different processing steps such as fermentation, purification and storage. Compared with ultracentrifugation, HPSEC is simpler, rapid, and reliable. The method has been recommended to companies by China Institute of Veterinary Drugs Control, and applied in industrial practice. 2) FMDV and HBsAg-VLPs were characterized by field flow fraction (FFF) and various spectroscopic techniques including circular dichroism, flurescence spectroscopy and dynamic light scattering. The results further verified the HPSEC method, and provided more detail information of conformational changes. 3) Dual polarization interferometry (DPI) was developed for real-time monitoring the adsorption and conformational changes of HBsAg-VLPs at solid-liquid interface. The thickness of HBsAg-VLPs at DEAE ion exchange surface varied with pH. At low pH like pH 5.0, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HBsAg-VLPs. At pH 7.0 or 9.0, the HBsAg-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness. By comparing the DPI results with chromatography experiments, the significant loss of antigen recovery was proven to be highly related to conformational changes at solid-liquid surface. 4) Stabilization of vaccine antigen particles during processing was investigated. For FMDV, the most stable pH was found between 7.5-8.0, and the most suitable salts were ammonium salts. Addition of multi-hydroxy compounds significantly increased the stability of FMDV as indicated by HPSEC and DSC. By adding multi-hydroxy compounds as purification companion, the FMDV recovery increased for 10.1% during ultrafiltration, and increased from 17.1% to 84.0% during chromatography. The entire process was completed with 56.7% final FMDV recovery, and the purity above 98%. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/21363
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杨延丽. 灭活病毒及病毒颗粒的结构表征和稳定性研究[D]. 北京. 中国科学院研究生院,2015.
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