人血清白蛋白的生物制备工艺优化

Optimization of biological preparation of human serum albumin

通过中国仓鼠卵巢细胞(Chinese hamster ovary cell, CHO)表达系统制备人血清白蛋白(Human serum albumin,HSA),为HSA及其融合蛋白质药物的制备及功能研究提供依据。首先构建含有目的基因的重组表达质粒pMH3-HSA,通过有限稀释法和Dot blot检测筛选阳性单克隆,随后扩大培养并悬浮驯化,然后运用5 L一次性生物反应器进行高密度放大培养,以无血清培养基B001和F001分别作为基础和流加培养基,以55 r/min、DO 20%~40%、pH 6.8~7.4、Tm 37℃为基础参数进行发酵控制,通过细胞计数、SDS-PAGE、Elisa、Western Blot等实验方法,检测发酵过程中的细胞生长状态以及目标蛋白HSA的表达情况,同时与毕赤酵母表达系统制备的HSA进行比较。结果表明:1)成功构建并筛选出了含外源HSA基因的高表达单克隆CHO细胞株;2)在5 L一次性生物反应器中进行流加培养,HSA的最高产量达180μg/mL;3)CHO表达系统产物几乎无降解。综合表明HSA蛋白更适合在CHO系统中进行表达,其蛋白完整性优势在该系统中表现得尤为明显。

The human serum albumin was prepared by the expression system of CHO cells, which provided a basis for the preparation and function research of HSA and its protein-fusion drugs. Firstly, the recombinant expression plasmid pMH3-HSA was constructed. Then the positive monoclones were screened by finite dilution and Dot Blot. High density amplification culture by 5 L disposable bioreactor was used after suspension domestication, the serum-free medium B001 and F001 were used as basic and flow media, respectively, the fermentation parameters were 55 r/min, DO 20%-40%, pH 6.8-7.4, and Tm 37 ℃. The cell growth status and expression of target protein HSA during fermentation were detected by cell counts, SDS-PAGE, Elisa and Western Blot, and compared with the HSA protein prepared in the Pichia expression system. The results showed that the highly expressed monoclonal CHO cell line containing exogenous HSA gene was successfully constructed and screened, the maximum yield of HSA reached 180 μg/mL in 5 L fed batch disposable bioreacter, and the product of CHO expression system had almost no degradation. In summary, this study indicated that HSA was more suitable for expression in the CHO system, and its advantage of protein integrity was particularly obvious in this system.