为了优化在真核细胞中猪流行性腹泻病毒(PEDV)S蛋白的分泌表达,且构建稳定表达的细胞系,本研究设计合成了 9个信号肽引物,通过同源重组的方式得到表达PEDV变异株AH2012/12株S全长蛋白的9个重组表达质粒,瞬时转染HEK 293T细胞后,通过Western-blot验证蛋白外分泌表达水平,结果显示,筛选出的含跨膜蛋白酶丝氨酸信号肽的S蛋白具有较好的外分泌表达特性;利用G418筛选,获得稳定表达PEDVS蛋白的单克隆细胞,通过传代验证不同代次的蛋白表达水平,成功构建了可稳定外分泌表达PEDV S的CHO细胞系.结果显示,可在100 mL CHO-PEDV/S细胞培养上清中获得约5 mg的PEDV S蛋白,提高了 PEDV S蛋白的表达和分泌,为进一步研制PEDV亚单位疫苗奠定了基础.
Screening exocrine signal peptide and construction of cell lines stably expressing PEDV S protein
The purpose of this study was optimize the expression of PEDV S protein in eukaryotic cells and to construct a cell line stably expressing PEDV S protein.In this study,nine signal peptide primers were designed and synthesized.Additionally,nine recombinant expression plasmids expressing the full-length S protein of the PEDV variant AH2012/12 strain were obtained through homologous recombina-tion.The levels of protein secreted expression were confirmed by.Western-blotting analysis after transient transfection into HEK 293T cells.The spike protein with the signal peptide of transmembrane protease serine demonstrated efficient secretion characteristics and chosen for further study.Subsequently,monoclonal cells stably expressing PEDV S protein consistently were obtained through G418 screening.The protein expression levels of different generations were verified through cell passaging,and CHO cell lines capable of stably exocrine expression of PEDV S protein were successfully engineered.It was determined that approximately 5 mg of PEDV S protein could be obtained from 100 mL of CHO-PEDV/S cell culture supernatant,which could enhance the expression and secretion of PEDV S protein.This research lays the groundwork for the futher development of PEDV subunit vaccines.
国家科技期刊平台
NSTL
万方数据
