中国兽医科学2024，Vol.54Issue(7)：873-880,8.DOI:10.16656/j.issn.1673-4696.2024.0120
猪流行性腹泻病毒S蛋白外分泌信号肽筛选及稳定表达细胞系的构建
Screening exocrine signal peptide and construction of cell lines stably expressing PEDV S protein
摘要
Abstract
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.关键词
猪流行性腹泻病毒/S蛋白/信号肽/稳定表达/亚单位疫苗Key words
porcine epidemic diarrhea virus/spike protein/signal peptide/stable expression/subunit vaccine分类
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王传红,胡朝阳,李彬,宋旭,刘诗雨,张格格,徐红,王祎,赵永祥,郭荣利,范宝超..猪流行性腹泻病毒S蛋白外分泌信号肽筛选及稳定表达细胞系的构建[J].中国兽医科学,2024,54(7):873-880,8.基金项目
国家重点研发计划项目(2022YFD1800801) （2022YFD1800801）
江苏省农业科技自主创新资金项目(CX(23)3076) （CX(23）
