人源性抗体在HEK293T细胞中瞬时表达条件的优化  

Optimization of Human Antibody Gene Transient Expression in HEK293T Cell

作　　者：陶俊[1] 向军俭[1] 王宏[1] 龚义平[1] 邓宁[1] 

机构地区：[1]暨南大学生命科学技术学院抗体工程中心,广州510632

出　　处：《生物技术通报》2010年第11期137-141,共5页Biotechnology Bulletin

基　　金：国家科技计划"863"专题课题(2009AA02Z112)

摘　　要：以HEK293T细胞为宿主对抗体基因的转染和表达条件进行优化。以GFP(green fluorescence protein)和人源性抗b FGF抗体1A2为报告基因,考察了3种转染试剂磷酸钙,PEI,FuGene HD的转染效率,确定FuGene HD的转染效率最高后,对DNA∶FuGene HD的比例,加入氯喹的浓度,转染后的孵育时间,最佳的低温培养温度以及加入组蛋白抑制剂的浓度进行了优化。结果显示,FuGene HD的转染效率最高,达到56.7%。当转染的DNA=2μg/4×106细胞时,DNA∶FuGene HD的最佳比例为1∶4(W/V);加入氯喹能够增强FuGene HD的转染效率,最佳浓度为100μmol/L,转染后的最佳孵育时间为6h;低温诱导能够提高293T细胞表达抗体的能力,最佳的温度为33℃;加入组蛋白抑制剂丁酸钠或丙戊酸能够明显提高抗体的表达量,丙戊酸的效果优于丁酸钠。通过对转染试剂,转染方法,培养温度,组蛋白抑制剂的加入等条件进行优化使人源性抗bFGF抗体的表达量从1.5mg/L提高到了15.1mg/L。The study was to optimize the condition of human antibody gene transient expression in HEK 293T cell.The transfection reagent and conditions,ratio of DNA∶FuGene were optimized by using GFP（green fluorescence protein）as report gene.The Influences of chloroquine on transfection efficiency were investigated.The effects of low temperature and histone deacetylase inhibitors on antibody gene expression were investigated using human pIgG-1A2 which containing human anti-bFGF antibody gene as report gene.The best transfection reagen is FuGene HD and the optimal transfection efficiency was obtained when the ratio of foreign DNA∶FuGene HD= 1∶4（W/V）,DNA= 2 μg /4×10^6 cells.Chloroquine enhanced the transfection efficiency of FuGene HD,and it was found that low temperature can enhance antibody expression.Histone deacetylase inhibitors,such as sodium butyrate and valproic acid can improve antibody expression,and the effect of valproic acid was better than sodium butyrate.Through optimizing transfection reagen,transfection methodes,culture temperature and added histone deacetylase inhibitors,the expression of human anti-bFGF antibody in 293T cells was increased ten-fold from 1.5 mg/L to 15.1 mg/L.

关 键 词：人源性抗体 瞬时表达 优化 HEK293T 

分 类 号：Q789[生物学—分子生物学] R392[医药卫生—免疫学]