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

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.