重组抗狂犬病病毒抗体在HEK293 EBNA1细胞中的瞬时表达优化  

Optimization of Recombinant Anti-rabies Virus Antibody Transient Expressed in HEK293 EBNA1 Cells

作　　者：陈继军[1] 秦海艳[1] 安晨[1] 乔玉玲[1] 李晓进[1] 毛晓燕[1] 

机构地区：[1]兰州生物制品研究所有限责任公司,甘肃兰州730000

出　　处：《现代生物医学进展》2015年第33期6439-6443,6454,共6页Progress in Modern Biomedicine

基　　金：甘肃省青年科技基金计划(145RJYA290)

摘　　要：目的:优化重组抗体在悬浮无血清培养的HEK293 EBNA1瞬时表达,提高重组抗体表达量。方法:将HEK293 EBNA1细胞适应于无血清悬浮培养,筛选适宜的无血清培养基。使用PEI转染质粒进入细胞,瞬时表达重组抗体;使用Modde软件进行实验设计(DOE),优化转染质粒量、轻重链比例、PEI量等影响瞬时表达的条件。培养上清经亲和纯化,获得目标抗体,用快速免疫荧光灶抑制试验(RFFIT)测定抗体活性,用BCA法测定纯化抗体浓度。结果:293SFMII为适宜的HEK293 EBNA1细胞无血清悬浮培养基。对抗体表达影响最大的因素是轻重链比例(P=0.00000),其次为质粒浓度(P=0.00086),最后为PEI(P=0.00257)。优化的转染条件为:质粒用量0.61μg/106细胞,轻重链比例2:1,PEI 2.67μg/106细胞,优化后抗体表达量有显著提高(P=0.007)。结论:通过DOE优化获得了重组抗狂犬病病毒抗体的高水平表达,抗体表达量提高了至少50倍。Objective： Optimization of the recombinant antibody transient transfect expression of serum-free suspension cultured HEK293 ENBA1 cells to improve the titer of recombined antibody transient expression. Methods： HEK293 EBNA1 cell was adapted by serum-free suspension culture. The suitable medium was selected. Recombinant antibody plasmids were transfected by PEI. Antibodies were expression in serum-free suspension cultured HEK293 EBNA1. Transient expression parameters were optimized by Design of Experiment （DOE） with the Modde software, including plasmid concentration, the proportion ofplasmid encoding the light and heavy chain. Recombinant antibodies were expressed under serum-free conditions by optimized parameters. They were obtained by Mabselect SuRe affinity from culture supematant. Activation of purified antibodies was determined by rapid fluorescent focus inhibition test （RFF1T）. Results： 293SFMII was suitable for HEK293 EBNA1 serum-free suspension cultures. The most effective factor for recombinant antibody transient expression was the proportion of light and heavy chain （P=0.00000）, the second was plasmid concentration （P=0. 00086） and the last was PEI concentration （P=0.00257）. Optimized transient expression parameters： plasmid concentration 0.61 μg/106 cells, light chain： heavy chain 2：1, PEI concentration 2.67 μg/106 cells. Significant improve of recombinant antibody titer was observed in transient expression with optimized parameters （P=0.007）. Conclusion： Recombinant antibody titers had obviously increased by the transient expression parameters been optimized through DOE, it increased at least 50 fold.

关 键 词：HEK293 EBNA1 无血清悬浮培养 抗体瞬时表达 实验设计 

分 类 号：Q812[生物学—生物工程]