线性化聚乙烯亚胺介导的高效瞬时转染条件的优化  

Optimization of polyethylenimine linear-mediated high-efficiency transient transfection conditions

作　　者：陈玥如 赵忆宁 唐悦 王婉 傅生芳 李雄雄 Chen Yueru;Zhao Yining;Tang Yue;Wang Wan;Fu Shengfang;Li Xiongiong(No.2 Research Laboratory,Lanzhou Institute of Biological Products Co.,Ltd.,Vaccine Engineering Technology Center of Gansu,Lanzhou 730046,China)

机构地区：[1]兰州生物制品研究所有限责任公司第二研究室,甘肃省疫苗工程技术研究中心,兰州730046

出　　处：《国际生物制品学杂志》2023年第3期156-160,共5页International Journal of Biologicals

基　　金：国家科技重大专项(2013ZX09402302)。

摘　　要：目的优化线性化聚乙烯亚胺(polyethylenimine linear,PEI)介导的高效瞬时转染条件,提高重组蛋白在人胚胎肾(human embryonic kidney,HEK)293F悬浮细胞中的瞬时表达效率.方法构建和鉴定重组质粒增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)/pcDNA3.1+,通过瞬时转染的方式将质粒转入HEK293F细胞中进行表达.对转染试剂PEI与重组质粒的比例以及收获时间进行优化,通过倒置显微镜观察、SDS-PAGE和流式细胞仪检测目标蛋白的表达量,确定目标蛋白在HEK293F细胞中的最佳表达条件.采用优化后的条件在T500摇瓶(工作体积120 ml)中进行扩大表达.结果构建的重组质粒EGFP/pcDNA3.1+序列正确,在重组质粒浓度为3.0μg/ml、DNA∶PEI为2∶1、转染后24 h添加终浓度为2 mmol/L的丙戊酸钠、转染后4 d收获条件下,目的蛋白的表达量最高.此条件也适用于放大培养体积至T500的瞬时转染.结论优化了一种快速有效的基于PEI的瞬时转染方法来高水平表达重组蛋白.Objective To optimize polyethylenimine linear(PEI)-mediated high-efficiency transient transfection conditions to improve the transient expression efficiency of recombinant proteins in human embryonic kidney(HEK)293F suspension cells.Methods The recombinant plasmid enhanced green fluorescent protein(EGFP)/pcDNA3.1+was constructed and identified.The recombinant plasmid was transfected into HEK293F cells by transient transfection for expression.The ratio of transfection reagent PEI to recombinant plasmid and the harvesting time were optimized,and the expression of target protein was determined by inverted microscopy,SDS-PAGE and flow cytometry to determine the optimal expression conditions of the target protein in HEK293F cells.The optimized conditions were expanded in T500(working volume 120 ml)shake flasks.Results The recombinant plasmid EGFP/pcDNA3.1+constructed was correct.The highest expression of target protein was obtained 4 d after transfection at 3.Oμg/ml recombinant plasmid concentration,DNA:PEI of 2:1 and a final concentration of 2 mmol/L valproic acid sodium salt added 24 h after transfection.This condition was also suitable for transient transfections with an enlarged culture volume of T500.Conclusion A rapid and effective PEIbased transient transfection method is optimized to prepare recombinant proteins for high-level expression.

关 键 词：线性化聚乙烯亚胺 瞬时转染 人胚胎肾293F细胞 增强型绿色荧光蛋白 

分 类 号：R973.2[医药卫生—药品]