不同CHO工程细胞株表达的VEGFR-Fc唾液酸化修饰的研究  

On the sialylation levels of VEGFR-Fc from different CHO cell strains

作　　者：刘培[1] 宋礼华[1,2] 范清林[2] 刘道琴[2] 张伟[2] 魏利[2] 赵婷[2] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032 [2]安徽安科生物工程(集团)股份有限公司,合肥230000

出　　处：《安徽医科大学学报》2016年第6期778-782,共5页Acta Universitatis Medicinalis Anhui

摘　　要：目的比较中国仓鼠卵巢细胞（CHO）工程细胞株表达的VEGFR-Fc的唾液酸化程度的差异,为CHO工程细胞株的筛选提供参数依据。方法从唾液酸单糖、寡糖链这两个层次分析VEGFR-Fc的唾液酸化程度。唾液酸单糖的定量：酸水解释放出的唾液酸用荧光试剂1,2-diamino-4,5-methylenedioxybenzene（DMB）衍生后,采用荧光-亲水保留液相色谱（HILIC-FLD）检测。寡糖链的结构分析：糖苷酶PNGase F释放出的寡糖链经邻氨基苯甲酰胺（2-AB）衍生后,一部分利用阴离子交换色谱原理分析寡糖链上唾液酸的平均数;一部分经HILIC原理分析VEGFR-Fc寡糖链指纹图谱。结果 6株工程细胞株表达的VEGFR-Fc唾液酸含量在3-12 mol NANA/mol蛋白之间,参考品Aflibercept为10.46 mol NANA/mol蛋白;每个寡糖链的唾液酸平均数在0.28-1.0之间;寡糖链的特征指纹谱图显示VEGFR-Fc有12个共有峰,各样品、参考品均有以上共有峰,但各峰的峰高有差异。结论不同的CHO工程细胞株所表达的糖蛋白的唾液酸化程度存在差异。Objective To describe the sialylation levels of VEGFR-Fc from different CHO cell strains for screening out the better CHO cell strain. Methods The analysis of sialic acid was from two levels： monosaccharide constituent and structural analysis of the glycan chains of glycoconjugates. The sialic acids were liberated by mild acid hydrolysis and then derivatized using 1,2-diamino-4,5-methylenedioxybenzene（ DMB）. The derivatives were separated by hydrophilic interaction liquid chromatography on an amide column. The second level was to remove the oligosaccharides from VEGFR-Fc by Peptide N-glycolsidase F（ PNGase F）,and derivatized with 2-aminobenzamide（ 2-AB）. The labels could then be analyzed by anion exchange and hydrophilic interaction chromatography coupled to fluorescence detector. Results The sialic acid content of 6 VEGFR-Fc isoforms varied between 3 - 12 mol NANA / mol protein,and Aflibercept contained 10. 46 mol NANA / mol protein. The average content of sialic acid in each oligosaccharide chain was 0. 28 - 1. 0. The typical finger chromatogram of VEGFR-Fc oligosaccharides was built and 12 characteristic peaks were pointed out. Conclusion Combined together,these two levels analysis sufficiently demonstrate there is a difference in the degree of salivary acid acidification of VEGFR-Fc from different CHO cell strains.

关 键 词：唾液酸 唾液酸化 糖基化 HILIC-FLD 

分 类 号：R917.796[医药卫生—药物分析学]