重组人血清白蛋白在CHO细胞中的高效表达及其培养工艺优化  

High level expression of recombinant human serum albumin in CHO cells and optimization of its culture technology

作　　者：牛宇辉 李洪珊 李殿玉 吴贝 李向茸[1] 冯若飞[1] NIU Yuhui;LI Hongshan;LI Dianyu;WU Bei;LI Xiangrong;FENG Ruofei(Key Laboratory of Biotechnology&Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,Gansu Province,China)

机构地区：[1]西北民族大学生物医学研究中心生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《中国生物制品学杂志》2023年第9期1054-1061,1071,共9页Chinese Journal of Biologicals

基　　金：国家民委中青年英才计划资助项目[批准号(2018)98];兰州市人才创新创业项目(2019-RC-20)。

摘　　要：目的在中国仓鼠卵巢(CHO)细胞中高效表达人血清白蛋白(human serum albumin,HSA),并优化其培养工艺,为HSA的规模化生产奠定基础。方法利用基因重组技术构建HSA的真核表达载体,并将其电转染至全悬浮CHO细胞中,利用G418及有限稀释法筛选获得可稳定高表达HSA的单克隆细胞株,通过在基础培养基中添加葡萄糖、丁酸钠及补料培养基等方式进行细胞培养工艺优化,以提高HSA表达量;最终在5 L生物反应器上进行放大培养验证。结果成功构建了重组表达质粒pcDNA3.1-HSA,并在全悬浮CHO细胞中实现分泌表达后,又经过2次单克隆筛选,获得高表达HSA的2次单克隆细胞株CHO-rHSA-7H2A9和CHO-rHSA-7H2D12,表达量分别达到29.37和25.26 mg/L;通过培养工艺优化使HSA表达量达到100.00 mg/L左右;最终使5 L生物反应器上HSA表达量提升至166.16 mg/L,与第1次单克隆细胞上清中HSA表达量相比,提高了30倍左右。结论实现了HSA在CHO细胞中的高效表达,为进一步在生物制品领域规模化生产HSA及解决市场供应问题奠定了基础。Objective To achieve efficient expression of human serum albumin(HSA)in Chinese hamster ovary(CHO)cells and optimize its culture technology,so as to lay a foundation of the large-scale production of HSA.MethodsThe eukaryotic expression vector of HSA was constructed by gene recombination technology,and then electrotransfected into fully suspended CHO cells.The monoclonal cell lines with stable and high expression of HSA were screened by G418 and limited dilution method.By adding glucose,sodium butyrate and supplementalmedium to the basal medium,the cell culture process was optimized to improve the expression of HSA.Finally,the scale-up culture verification was carried out in a 5 L bioreactor.ResultsThe recombinant expression vector pcDNA3.1-HSA was successfully constructed and expressed in fully suspended CHO cells.After two monoclonal screening,the secondary monoclonal cell lines CHO-rHSA-7H2A9 and CHOrHSA-7H2D12 were obtained with high HSA expression of 29.37 mg/L and 25.26 mg/L respectively.The HSA expression level reached about 100.00 mg/L by optimizing the culture process and wasfinally increased to 166.16 mg/L in the 5 L bioreactor,which was about 30 times higher than that in the supernatant of the first monoclonal cells.Conclusion The high level expression of HSA in CHO cells was achieved,which laid a foundation of the further large-scale production of HSA in the field of biological products and solving the market supply problems.

关 键 词：重组人血清白蛋白 CHO细胞 高效表达 

分 类 号：Q819[生物学—生物工程]