摘要： 为建立重组Ⅱ型人源化胶原蛋白Ⅱ-23k的分离与纯化工艺，先通过对不同离子交换柱纯化效果的比较，确定层析柱类型；再考察发酵液的澄清过滤、浓缩换液、离子交换层析的纯化效果，建立Ⅱ-23k的分离与纯化工艺。结果表明，与Q强阴离子、SP强阳离子和CM弱阳离子交换柱相比，DEAE弱阴离子柱层析效果最佳，在缓冲液pH为7.0时，经DEAE柱流穿模式层析，得到的Ⅱ-23k纯度大于95%，回收率大于90%。采用切向流过滤（tangential flow filtration，TFF）的中空纤维微滤进行发酵液的澄清过滤、TFF平板膜包超滤进行浓缩换液、离子交换层析进行纯化，建立澄清过滤—浓缩换液—DEAE柱流穿—冻干的分离与纯化工艺，得到的Ⅱ-23k纯度约95%，无色素残留，浓缩换液的平均回收率约75%，DEAE柱流穿的平均回收率约78%，总回收率最高接近60%。研究建立了基于TFF和一步柱流穿模式层析的重组Ⅱ型人源化胶原蛋白分离与纯化工艺，为其放大生产及工业化制备提供参考。

关键词： 重组Ⅱ型人源化胶原蛋白,  分离与纯化,  离子交换层析,  流穿模式,  切向流过滤 

Abstract: To establish a separation and purification process of recombinant humanized type Ⅱ collagen Ⅱ-23k, the type of chromatography column was first determined by comparing the perification performance of different ion-exchange columns. Then, the purification effects of clarification and filtration, concentration and buffer exchange, and ion exchange chromatography of the fermentation broth were investigated to establish the separation and purification process of Ⅱ-23k. The experimental results showed that compared to the Q strong anion, SP strong cation and CM weak cation exchange columns, the DEAE weak anion column performed the best. Using the DEAE column in flow-through mode, Ⅱ-23k yielded with a purity over 95% and a recovery rate greater than 90% at the buffer pH of 7.0. The clarification and filtration of the fermentation broth was carried out by tangential flow filtration (TFF) hollow fiber microfiltration, the concentration and buffer exchange were carried out by TFF flat membrane ultrafiltration, and the purification was carried out by ion exchange chromatography. Consequently, a separation and purification process was established, comprising the following steps: clarification and filtration, concentration and buffer exchange, DEAE flow-through chromatography, and freeze-drying.The purity of Ⅱ-23k obtained was about 95%, with no pigment residue. The average recovery rate of concentration and buffer exchange was about 75%, and the average recovery rate of DEAE column flow-through was about 78%. The overall process recovery reached nearly 60%. This study established a separation and purification process of recombinant humanized type Ⅱ collagen based on TFF and one-step column flow-through mode chromatography, which provides a reference for its large-scale production and industrial preparation.

Key words: recombinant humanized type Ⅱ collagen,  separation and purification,  ion exchange chromatography,  flow-through mode,  TFF 

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作者简介: 孟子璇，硕士研究生，研究方向为重组蛋白制备与检测，E-mail：mengzixuan0222@163.com