呼吸道合胞病毒融合前F蛋白分泌表达条件的优化和建立  

Optimization and establishment of secretory expression conditions of RSV prefusion F protein

作　　者：陈玥如 赵忆宁 唐悦 王剑 王宇 李雄雄 CHEN Yue-ru;ZHAO Yi-ning;TANG Yue;WANG Jian;WANG Yu;LI Xiong-xiong(The Second Research Laboratory,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,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,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第4期5-9,共5页Progress In Microbiology and Immunology

摘　　要：目的通过表达条件的优化,提高融合前呼吸道合胞病毒(respiratory syncytial virus,RSV)F蛋白在HEK 293F细胞中的表达。方法将优化后的融合前RSV F基因片段插入到pLexm载体中,构建重组表达质粒,通过瞬时转染的方式将重组质粒转入HEK293F细胞中进行表达。对转染试剂线性化聚乙烯亚胺(polyethylenimine linear,PEI)与重组质粒的比例、添加丙戊酸钠(valproic acid sodium salt,VPA)的浓度以及收获时间进行摸索和优化,通过Western blot和ELISA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。结果成功构建了含有融合前RSV F基因的重组表达质粒,并确定在重组质粒质量浓度为3.0μg/mL,DNA∶PEI为1∶0.5,在转染后24 h添加终浓度为4 mmol/L的VPA,转染后第6天收获目的蛋白的表达量最多。结论成功构建了含有融合前RSV F基因的高效分泌表达载体,确定了目标蛋白在HEK293F细胞中的最佳表达条件,为研究融合前RSV F蛋白的特性奠定了基础。Objective To improve the expression level of prefusion RSV F protein in HEK293 F cells by optimization of expression conditions.Methods The optimized RSV prefusion F gene was inserted into pLexm vector,and then the recombinant plasmid was transfected into HEK293 F cells.The optimal ratio of transfection reagent PEI to DNA,the concentration of VPA and the time of cell harvesting were explored and investigated.The optimal conditions of target gene expression in HEK293 F cells were determined using Western blot and ELISA.Results The recombinant plasmid containing RSV prefusion F gene was successfully constructed.The optimal conditions for prefusion RSV F protein expression were determined as follows:the concentration of the plasmid was 3.0μg/mL,the ratio of DNA to PEI was 1∶0.5,4 mmol/L VPA was added at 24 h post-transfection and the cells were harvested after day 6.Conclusion High-efficiency expression vector containing RSV prefusion F gene was successfully constructed,also it was determined that the target protein optimally expressed in HEK293 F cells.It lays a foundation for studying the characteristics of RSV prefusion F protein.

关 键 词：呼吸道合胞病毒 融合前RSV F蛋白 瞬时转染 条件优化 

分 类 号：R373.1[医药卫生—病原生物学]