用层析技术高效纯化多种血浆蛋白质的工艺探索
文献类型：学位论文
| 作者 | 孔英俊 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-12-02 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 层析技术 血浆蛋白 乳腺生物反应器 特异性免疫球蛋白 多维组合液相层析 |
| 其他题名 | The process exploration of the efficient production of multiple plasma proteins by chromatography |
| 学位专业 | 生物化工 |
| 中文摘要 | 现有的低温乙醇法生产血浆蛋白质未能充分利用血浆资源。本论文探索建立一个利用层析技术辅助低温乙醇法从血浆中生产多种蛋白质产物的工艺平台。首先，针对血浆利用率低的问题，本论文开发了从低效价特异性免疫球蛋白中回收有效成分的快速免疫亲和层析方法。本论文以破伤风、乙肝、狂犬病免疫球蛋白为目标产物，制备了相应的三种特异性免疫球蛋白亲和介质，优化三种蛋白的纯化条件，发现制备的介质能够有效纯化相应的目标蛋白。在实验过程中，发现PEG的存在可能通过屏蔽亲和配基上某些非特异性吸附位点，从而提高了蛋白的活性回收率。通过分析亲和配基的大小与固相载体孔径之间的关系，建立了特异性免疫球蛋白亲和层析纯化方法。为了验证该方法的有效性，本论文用其对手足口病、流感免疫球蛋白两种蛋白进行了纯化，结果表明，手足口病免疫球蛋白的收率高于35%；流感免疫球蛋白的收率高于45%。其次，开发了一条包含疏水相互作用层析、离子交换层析、亲和层析、凝胶过滤层析的多维层析工艺路线，充分利用低温乙醇法中被废弃的组分IV沉淀中的微量血浆蛋白。使用该工艺路线，通过收集各层析柱组分、人工换柱、重新进料等手动操作，从组分IV沉淀中，同时纯化运铁蛋白(Tf)、白蛋白(HSA)、抗凝血酶III(AT-III)、α1-抗胰蛋白酶(α1-AT)、结合珠蛋白(Hp)五种目标蛋白，得到了纯度90%以上的五种产品，其中HSA收率为87%；ATIII收率为65%；Tf收率为74%；α1-AT收率为62%；Hp收率为40%。然后，利用本实验室自主研发的自动化多维层析系统，实现了上述多维层析过程的自动化控制。最终得到了纯度90%以上的五种目标蛋白，收率均高于手动操作，其中HSA收率为95%；AT-III收率为70%；Tf收率为80%；α1-AT收率为65%；Hp收率为50%。该系统通过计算机工作站控制设备，让多个层析柱同时工作。整个纯化过程在3小时内完成，与手动操作的3个工作日相比，大大缩短工艺时间，增加设备的处理量和速度。而且，整个流程实现全管道化操作，不接触外界环境，可以避免污染和损失。最后，为了开拓新的血浆蛋白来源，本论文对乳腺生物反应器生产的血浆蛋白进行了分离纯化的研究。本论文以含重组人抗凝血酶(rhAT-III)的转基因羊乳为原料，开发了一条快速纯化rhAT-III的工艺路线。该工艺以等电点沉淀酪蛋白和肝素亲和层析纯化rhAT-III作为主要分离步骤，能够快速有效地将rhAT-III从转基因羊奶中纯化出来，得到的rhAT-III纯度大于99%，收率大于90%。重组蛋白产品有极高的纯度，能避免外源蛋白对人体的损害。工艺重复性好，满足大规模生产需要。产物的结构分析表明rhAT-III的氨基酸序列、二硫键位置、糖基化位点、二级、三级结构与血浆来源的AT-III一致。 |
| 英文摘要 | Traditionally, the production of plasma protein is based on the cold ethanol precipitation. However, this method could not make full use of plasma components. This thesis provides a technology platform for efficient production of multiple plasma proteins by the combination of conventional cold ethanol precipitation and chromatographic purification methods. Firstly, an immunoaffinity chromatography method was developed to solve the low utilization ratio in the purification of the specific immunoglobulin from plasma. Three kinds of immunoaffinity chromatography media were prepared for purification of the tetanus, hepatitis B and rabies immune globulin, respectively. After optimization of the chromatographic conditions, the three kinds of immune globulin were all effectively purified. It was found that the presence of PEG in mobile phase increased the protein activity recovery probably by shielding the non-specific adsorption sites. Moreover, an affinity chromatography method for the purification of specific immunoglobulin was established by correlating ligand size with pore size of the supporting matrix. By this method, the hand-foot-mouth disease and influenza immunoglobulin were successfully purified with a yield of 35% and 45%, respectively. Secondly, a multidimensional chromatography process, including gel filtration, ion exchange, hydrophobic interaction, and heparin affinity chromatography, was developed to make full use of plasma fraction IV, which was discarded in the cold ethanol precipitation process. After the multidimensional purification, five proteins involving human serum albumin (HSA), transferrin (Tf), antithrombin-III (ATIII), alpha 1-antitrypsin (α1-AT) and haptoglobin (Hp) were successfully purified with purities over 90% from fraction IV by manual operation. The protein recovery was as follow: 87% for HSA, 74% for Tf, 65% for ATIII, 62% for α1-AT and 40% for Hp. Thirdly, the automatic control of the above multidimensional chromatography process was achieved using an automatic system designed by our laboratory for the integration of multidimensional protein chromatography. The same five proteins were obtained with purities over 90%, and their recoveries were higher than the former manual operation process: 95% for HSA, 80% for Tf, 70% for ATIII, 65% for α1-AT and 50% for Hp. This computer-controlled system integrated several chromatographic columns that work simultaneously to achieve efficient high throughput protein purification. The whole multidimensional chromatography process was finished in 3 hours, much shorter compared with 3 work days by manual operation. Moreover, this enclosed system ensured that the contamination and protein loss of the purification process were significantly reduced. Finally, to develop new source of plasma protein, transgenic milk was used as the raw material for the investigation. A facile process was introduced for the purification of recombinant human antithrombin III (rhAT-III) from transgenic goat milk. The rhAT-III was rapidly purified with the purity of 99% by isoelectric precipitation and heparin affinity chromatography. The overall protein yield of this method was higher than 90%. This method had a good reproducibility and was easily scaled up. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT-III were measured and found to be the same as those of the human AT-III. |
| 语种 | 中文 |
| 公开日期 | 2013-09-22 |
| 页码 | 132 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1632]  |
| 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 | 孔英俊. 用层析技术高效纯化多种血浆蛋白质的工艺探索[D]. 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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