乙肝病毒S抗原和preS1抗原表位融合蛋白(S/preS1)在CHO细胞中的稳定高效表达  

Stable and efficient expression of hepatitis B virus S antigen and preS1 epitope fusion protein(S/preS1) in CHO cells

作　　者：杨振西 李世崇[2] 刘红[2] 张苗 叶玲玲[2] 吴彦卓 徐明波 陈昭烈[2] 

机构地区：[1]安徽大学生命科学学院,安徽合肥230601 [2]军事医学科学院生物工程研究所,北京100071 [3]北京双鹭药业股份有限公司,北京100049

出　　处：《生物工程学报》2013年第12期1808-1816,共9页Chinese Journal of Biotechnology

基　　金："重大新药创制"科技重大专项资助课题(No.20012X09301-001-005)资助~~

摘　　要：含前S蛋白的重组乙型肝炎疫苗是第3代乙肝疫苗研发的重点，有望替代现在广泛使用的基因工程疫苗。构建表达乙肝病毒S抗原和preS1抗原表位（21．47位氨基酸）融合蛋白（S／preS1）的真核表达载体HMRcHEF53u／Neo-S／preSl并转染CHO—S细胞，经ELISA和有限稀释克隆筛选获得了S／preS1表达效率高、体外培养生物学性状好的CHO细胞系10G6。Westernblotting分析证实10G6表达的S／preSI同时保留s和preSl的天然免疫原性。10G6细胞在以活细胞密度和preSI／S浓度为评价指标的连续批次培养过程中保持着稳定的目的产物表达效率和良好的生长特性。采用无血清流加培养工艺，10G6细胞的活细胞密度和preSl／s浓度分另1达至07×10^6～10×10^6cells／mL和17～20mg／L。Hepatitis B surface antigen （HBsAg） carrying preS sequences could be an ideal candidate for a new hepatitis B virus （HBV） vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein （S/preS1） in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS 1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS 1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS 1 production at 17-20 mg/L with viable cell density at 7×106-10×106 cells/mL.

关 键 词：乙肝病毒表面抗原 PreS1抗原表位 疫苗 CHO细胞 表达 

分 类 号：R392[医药卫生—免疫学]