| 陈继军 秦海艳 安晨 乔玉玲 李晓进 毛晓燕.重组抗狂犬病病毒抗体在HEK293 EBNA1 细胞中的瞬时表达优化[J].,2015,15(33):6439-6443 |
| 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 TransientExpressed in HEK293 EBNA1 Cells |
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| DOI： |
| 中文关键词: HEK293 EBNA1 无血清悬浮培养 抗体瞬时表达 实验设计 |
| 英文关键词: HEK293 EBNA1 Serum-free suspension culture Antibody transient expression Design of experiment（DOE） |
| 基金项目:甘肃省青年科技基金计划（145RJYA290） |
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| 中文摘要: |
| 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0.61 ug/106细胞，轻重链比例2:1，PEI 2.67 ug/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。 |
| 英文摘要: |
| 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 of plasmid 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 supernatant. Activation of purified antibodies was determined by rapid fluorescent focus inhibition test
(RFFIT).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 ug/106
cells, light chain: heavy chain 2:1, PEI concentration 2.67 ug/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. |
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