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题名	层析技术结合冷乙醇沉淀纯化人血清白蛋白
作者	杨海荣
学位类别	硕士
答辩日期	2004
授予单位	中国科学院过程研究所
授予地点	中国科学院过程研究所
导师	苏志国
关键词	层析 纯化 人血清白蛋白 冷乙醇沉淀
其他题名	Purification of Human Serum Albumin with Comination of Chromatography and Cold Ethanol Precipitation
中文摘要	人血清白蛋白（HSA）的传统生产方法—冷乙醇沉淀法由于所得产品纯度不高，生产周期长，劳动强度大，已逐渐不能满足越来越高的产品质量要求，但其灭菌消毒 作用则是一大优点。现代层析技术的广泛使用为HSA的纯化方法开辟了新思路。而 将层析技术结合冷乙醇沉淀用于HSA的纯化则可以有效利用二者各自的优点，并能 很好的避免二者的不足之处。本文采用将层析技术与冷乙醇沉淀方法相结合的工艺思 路用于壬始A的纯化，得到一条纯度高、收率理想、周期短的HSA纯化新路线。首先将冰冻血浆冻融离心后进行了一步冷乙醇沉淀，采用的方法为Kistler和 N讹chxnalln改良法，并将该法的前两步乙醇沉淀合并为一步，以缩短处理时间，提高 工艺集成化程度，冷乙醇沉锭后离心所得上清用于下一步制备HSA的原料，沉淀可 用于制备lgG。该步骤HSA收率为95.5％，纯度由冻融离心后的59％提高到76.5％。对冷乙醇沉淀后所得上清先进行了一步脱盐脱乙醇处理，以除去其中的乙醇和盐 分对后续层析的影响，采用的介质为SePhadeX G25。脱盐脱乙醇后的含HSA组分进 行了第一步层析处理，通过比较选用了CM Sepharose FF作为纯化的介质，选用缓冲 系统为20mmol／L NaZHPO4＋10mmol／L柠檬酸（pH6.2），在该pH下介质只吸附杂质，而HSA直接穿透，这样可以加快物流速度，缩短处理时间。经该步骤处理后，HSA 纯度提高到90.5％，单步HSA收率为94.5％。在此后的第二步层析处理过程中，考察了阴离子交换层析和疏水层析对HSA的 纯化效果，并最终采用疏水层析方法。疏水层析过程中通过综合比较Butvl SePharose FF，0cty1SePharoseFEPheny1SePharOSeFF这三种疏水介质，最终选用了Butyl Sepharose FF，该介质疏水性适中，可以保证温和条件下洗脱HSA，选用的缓冲系统 为20mmol／L Na2HPO4／NaH2PO4（pH6.2）。该过程采用的纯化策略是先以高盐缓冲液平衡层析柱使HSA吸附，然后通过逐级降低缓冲液盐浓度的方式先洗脱杂蛋白，最后使HSA脱附。经该步层析处理后，最终得到电泳银染呈单带的HSA纯品，分析其纯度大于99％，明显高于现有冷乙醇沉淀方法96％左右的产品纯度，单步过程HSA收率为90％，从而使得整个工艺过程总收率为81.2％，与传统冷乙醇沉淀法制备HSA80％左右的总收率相当。本工艺如能进行条件的进一步优化，特别是疏水层析步骤洗 脱条件的优化，则整个工艺的总收率还有进一步提高的空间。与传统冷乙醇工艺相比 较，该工艺最终产品纯度更高，收率也有相当保证，生产周期相对较短，且层析过程 可以在常温下操作，易实现自动化控制，从而降低了劳动强度。
英文摘要	Human serum albumin (HSA) has been used to treat a number of diseases with high dosage, so extremely high purity is required in large scale production. HSA has long been produced by cold ethanol precipitation with purity about 95%, and final recovery 80%. With the development of modern chromatography, its application in purification of HSA has been increasingly studied in the last few years. In view of the advantage of virus elimination in cold ethanol precipitation, people began to study out a new art for the purification of HSA with the combination of chromatography and cold ethanol precipitation. In this paper, a new process was developed for purification of human serum albumin from plasma. The process features a combination of modern chromatography and cold ethanol precipitation, hoping to get the product with higher purity, higher final recovery and shorter production cycle. The plasma was first treated with cold ethanol using the method of Kistler&Nitschmann to eliminate possible virus contamination, integrating the first two steps of this method into one step. With the treatment, the purity of HSA increased from 59% to 76.5%, and the recovery of HSA in this procedure was 95.5%. After cold ethanol precipitation, the supernatant was put onto Sephadex G25 to remove salt and ethanol, buffer exchange was accomplished at the same time, then the supernatant after desalting and de-ethanol was put through a cation exchange chromatographic column containing CM Sepharose FF. The operation was in a flow-through mode in which non-albumin protein fractions were retained by the column and albumin fraction passed through. In this way, a fast separation was achieved. After this procedure, the purity of HSA reached 90.5%, and the recovery of HSA in this procedure was94.5%. Purification of albumin from the flow-through fraction was performed with hydrophobic interaction chromatography on Butyl Sepharose FF which binds albumin tightly on high salt concentration. Trace impurities were flushed away from the column and finally HSA was eluted with stepwise reduction of salt concentration. High purity of 99% was obtained as one band in SDS-PAGE with silver staining. Recovery of HSA in this procedure was 90%, thus the final recovery of HSA in the whole process came to 81.2%. Compared with the traditional process of cold ethanol precipitation, the chromatographic purification can be operated under room temperature, and is faster and more efficient.
语种	中文
公开日期	2013-09-16
页码	78
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1424]
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	杨海荣. 层析技术结合冷乙醇沉淀纯化人血清白蛋白[D]. 中国科学院过程研究所. 中国科学院过程研究所. 2004.

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