【作者】 张帆；

【导师】 董悦生；

【作者基本信息】 大连理工大学 ， 生物化工， 2011， 硕士

【摘要】 人血清白蛋白为人体血浆中含量最丰富的蛋白,具有很多重要的生理功能。临床上可作为很多疾病的药品治疗剂,还可作为生物产品的保护剂和赋形剂及细胞培养的添加成分,得到了广泛应用。近年来大规模生产重组人血清白蛋白作为血源白蛋白替代品。针对从高密度发酵液中分离纯化重组人血清白蛋白中存在的固液分离困难、工艺复杂、杂质难去除等问题,本论文从以下方面做了探索性研究。首先,考察了不同乙醇/无机盐双水相体系对重组人血清白蛋白的萃取能力,其中乙醇/磷酸氢二钾双水相体系的回收率最高,且在4-30℃的温度和1-30g/L的重组人血清白蛋白浓度均能保持较高萃取收率。该双水相体系能够直接从发酵液中萃取重组人血清白蛋白,当操作条件为乙醇20%(w/w)/磷酸氢二钾18%(w/w)时,重组人血清白蛋白回收率达到108.13%。综合考虑回收率和相比因素,选择乙醇18%(w/w)/磷酸氢二钾20%(w/w)体系放大实验,0.01-73L规模的平均回收率为100.4%,菌体的平均去除率达到99%。同时该双水相体系能够有效去除发酵液中的部分杂蛋白,还可以去除87.2%的多糖,并使上相的蛋白酶A活性降低了24.4%,部分抑制了蛋白酶B的活性,提高了重组人血清白蛋白的稳定性。双水相萃取将固液分离、萃取、浓缩、初级纯化整合为一步进行。通过研究成相物质的回收,上相中的乙醇和下相中的磷酸氢二钾回收率均在80%以上。其次,根据双水相上相的特点,建立了与之相适应的纯化方法：包括减压蒸馏去除乙醇、Phenyl SepharoseTM疏水层析纯化、DEAE阴离子交换层析去除多聚体等步骤。其中向平衡液中添加含1.2mol/L的磷酸氢二钾,为疏水层析的最优条件,此时收率为74.94%,还原SDS-PAGE电泳结果表明纯化的重组人血清白蛋白达到了电泳纯。而且纯化的重组人血清白蛋白能够长期保存,其中蛋白酶A活性降至上清液的9.18%,蛋白酶B可能被去除。最后,对上述方法分离纯化得到的重组人血清白蛋白纯度、杂质含量、二级结构及分子量进行了分析。高效液相色谱检测其纯度达到了99.77%,色素比值A350/A280为0.028。同时,其多糖含量为2.98μg/mg,蛋白酶A活性降为上清液的2.93%。而且,纯化的重组人血清白蛋白分子量及二级结构与血源白蛋白具有一致性。双水相萃取耦合柱层析技术提供了一种简便的、分离纯化得到高纯度重组人血清白蛋白的方法,具有分离时间短、收率高、杂质去除效果好的优点,具有产业化的前景。更多还原

【Abstract】 Human serum albumin (HSA) is the most abundant protein component contained in plasma and has many functions. HSA has wide applications, such as pharmaceuticals under various clinical conditions, an excipient or stabilizer in other biotechnology based products, a component in cell culture media, and so on. Large scale production of recombination human serum albumin (rHSA) which is as a substitute for the HSA originating in plasma (pHSA) has developed. For the problem in separation and purification of rHSA from high density fermentation broths such as complex process, difficulties in solid-liquid separation and removal of impurities,some technical problems were explored from the following aspects.Firstly, the extraction ability of different ethanol/salt aqueous two-phase systems (ATPS) was investigated. The ethanol/dipotassium hydrogen phosphate system showed the highest recovery for rHSA. When the concentration of rHSA changed from lg/L to 30g/L, and temperature was from 4℃to 30℃, aqueous two-phase extraction (ATPE) of rHSA exhibited high extraction efficiency. And ATPE could recover rHSA directly from a crude culture medium. The recovery (Y) of rHSA reached up to 108.13%when the system was composed of 20%(w/w) ethanol and18%(w/w) dipotassium hydrogen phosphate. Take suitable volume ratio of the top to bottom phase (R) into consideration, extrations which ATPS was composed of 18%(w/w) ethanol and 20%(w/w) dipotassium hydrogen phosphate were scaled up from 0.01 to 73L successfully.The average recovery of rHSA at different scales was 100.4%,and average removal ratios of cells was up to 99%. Simultaneously, partial protein impurities and 87.2%polysaccharides were eliminated, proteinase A (PrA) activity was decreased by 24.4%, proteinase B(PrB) activity was partially inhibited, and thus the stability of rHSA was improved. ATPE has integrated solid-liquid separation, extraction, concentration and primary purification into a single step. Moreover, recycle of phase components were reaserched. More than 80% of ethanol in the top phase and 80% dipotassium hydrogen phosphate in the bottom phase can be recovered.Secondly, according to the characteristics of the top phase, the downstream purification process of rHSA has been established. It included vacuum distillation for removing ethanol, purification by Phenyl SepharoseTM hydrophobic interaction chromatography(HIC), DEAE anion exchange chromatography for removal of polymers,. When the equilibrium solution contained 1.2mol/L K2HPO4, HIC condition was optimum.The recovery of HIC attained 74.9%, and reduced SDS-PAGE analysis showed that rHSA was electrophoretic pure. Moreover, purified rHSA remained stable for a long time because PrA activity was decreased to 9.18% compared with the supernatant, and PrB might be removed.Finally, purity, impurities content, secondary structure and molecular characteristics of purified rHSA has been analyzed. The HPLC purity and A350/A280 of rHSA attained 99.77% and 0.028 respectively. Meanwhile, the content of polysaccharides was reduced to 2.98ug/mg, and PrA activities was 2.93% compared with the supernatant. Furthermore, purified rHSA was essentially identical with pHSA in molecular weight and secondary structure.Integration process of hydrophilic organic solvents/inorganic salts ATPE and chromatography technology has provided a simple process for the separation and purification of rHSA to a high purity level, which has advantages of short time, high yield and removal rate of impurity, and produces large-scale industrial prospect.更多还原