溶液环境对乙肝表面抗原（HBsAg）在疏水层析中的影响
文献类型：学位论文
| 作者 | 原航 |
| 学位类别 | 硕士 |
| 答辩日期 | 2010-06-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 苏志国 |
| 关键词 | 乙肝表面抗原 构象变化 稳定性 分离纯化 介质污染 |
| 其他题名 | The effects of solution environment on Hepatitis B surface antigen (HBsAg) during hydrophobic interaction chromatography |
| 学位专业 | 生物化工 |
| 中文摘要 | 乙肝表面抗原（HBsAg）是乙肝疫苗的主要成分。它是一种病毒样颗粒，由蛋白质和脂类通过非共价键作用而形成。因为HBsAg亚基数目多，结构复杂，稳定性差，所以在保存和层析过程中容易发生变构、聚集或者解聚，对其溶解性以及免疫原性等性质会产生较大影响。因此，如何保持蛋白质在层析过程中的构象稳定性已成为多聚亚基蛋白质分离纯化的核心问题和关键技术之一。本论文以HBsAg作为多聚亚基蛋白质的代表，考察不同溶液环境（温度、酸度、聚乙二醇（PEG）、离子类型、盐浓度）对HBsAg构象的影响；然后根据不同盐对HBsAg的影响对疏水层析进行了优化，最后对汉逊酵母细胞表达的HBsAg的纯化工艺进行了整合。论文结论如下： 1.溶液环境条件下，HBsAg在常温下比较稳定，在温度超过60ºC时稳定性明显下降；pH值小于4.0时引起HBsAg不可逆聚集，但在pH 5.0时HBsAg的聚集是部分可逆的；不同分子量的PEG对于溶液中HBsAg的构象影响较小，但是对酶联免疫检测（ELISA）的结果影响较大；不同离子对HBsAg的影响基本符合Hofmeister序列，不同之处是SO42-比F-更易引起HBsAg的聚集。 2.在溶液环境中，(NH4)2SO4对HBsAg有不利影响。但通过考察不同盐条件下，疏水层析介质对HBsAg的静态吸附性能，并结合疏水层析的结果发现，(NH4)2SO4是最适合疏水层析的盐，有着最高的活性回收率和纯化倍数。 3.工艺优化时发现，汉逊酵母细胞破碎液会对疏水层析介质和离子交换层析介质造成不可逆的污染，并且疏水层析介质的抗污染能力强于离子交换层析介质。通过牛血清白蛋白（BSA）对疏水层析介质的预处理，以及层析后介质的碱洗等操作，使疏水层析介质耐受破碎液污染的能力进一步提高。同时也确定了HBsAg纯化工艺路线是先疏水层析，后离子交换层析。 4.综合上述实验结果，进行工艺整合。通过疏水层析—离子交换层析这两步层析方法，HBsAg的总活性回收率为61.5%，纯化倍数为44.5。经过进一步的超滤浓缩和凝胶过滤层析之后，整条纯化工艺的总活性回收率由实验室前期工艺的24.4%提高到37.9%，纯化倍数也增加到46.7。 通过本研究，初步得到了溶液环境对HBsAg构象影响的规律，并将实验结果成功运用于层析优化和工艺整合，最终开发出一条高收率、低成本，且工艺简单的HBsAg纯化工艺。这是降低疫苗生产成本，减轻我国政府经济负担的有效办法，具有非常现实的意义。 |
| 英文摘要 | Hepatitis B surface antigen (HBsAg), a virus-like particle whose structure is maintained by the non-covalent interaction of proteins and lipids, is the primary component of the hepatitis B vaccine. Because of its complex structure and unstability, HBsAg undergoes conformational change, aggregation or disassociation in store or purification, which reduces the bioactivity and solubility of protein, or alters its immunogenicity. Therefore, how to keep the conformation steady in chromatography becomes the key problem in the separation and purification of multi-subunit protein. In this paper we studied the effects of temperature, acidity, polyethylene glycol (PEG), ionic type and salt concentration on HBsAg conformation, and optimized the purification procedure of HBsAg expressed by Hansenula polymorpha. The following results were obtained: 1. In the solution environment, HBsAg was stable at normal temperature, but began to denature from 60ºC. The aggregation of HBsAg at pH 3.0 and 4.0 was irreversible, but partly reversible at pH 5.0. PEGs of different molecular weight had little influence on HBsAg conformation, but much influence on the ELISA results. The effect of ionic type on HBsAg was generally in accordance with Hofmeister sequence except that SO42- caused more aggregation than F-. 2. (NH4)2SO4 had negative effect on the stability of HBsAg in the solution environment. But after the investigation of the adsorbing ability of hydrophobic chromatographic adsorbent to HBsAg and hydrophobic chromatography of HBsAg in different salt solution, we found (NH4)2SO4 was the most appropriate salt for hydrophobic chromatography because of the highest activity recovery and purification-fold. 3. The cell disruption of Hansenula polymorpha fouled chromatographic adsorbent irreversibly. It was found that hydrophobic chromatographic adsorbent was much more difficult to be polluted than ion exchange chromatographic adsorbent. Through the pretreatment by BSA and NaOH washing after chromatography, the ability of anti-fouling of hydrophobic chromatographic adsorbent improved obviously. Therefore hydrophobic interaction chromatography became the first step in purification precedure, followed by ion exchange chromatography. 4. Sum up the above results and then optimize the purification procedure. By combining hydrophobic interaction chromatography and ion exchange chromatography, the activity recovery and purification-fold of the two steps were 61.5% and 44.5 respectively. After adding the ultrafiltration and gel filtration chromatography, the whole activity recovery and purification-fold of the purification procedure increased to 37.9% and 46.7. In this paper, we gained some rationales about the effects of solution environment on HBsAg conformation. And the conclusions were successfully put into the practice of the optimization and combination of procedure. Finally we opened out a simple purification procedure of HBsAg with high recovery and low cost, which is an available way to reduce the productive cost of vaccine and lighten the financial burden of our government. |
| 公开日期 | 2013-09-17 |
| 页码 | 65 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1508]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 原航. 溶液环境对乙肝表面抗原（HBsAg）在疏水层析中的影响[D]. 北京. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：过程工程研究所
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