新型PCV2衣壳融合蛋白在HEK293F细胞瞬时表达研究  

Research on the Transient Expression of a Novel PCV2 Capsid Fusion Protein in HEK293F Cells

作　　者：罗清平[1] 彭妍 ALI Mohsin 庄英萍[1,2] 郭美锦 LUO Qingping;PENG Yan;ALI Mohsin;ZHUANG Yingping;GUO Meijin(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;Shanghai Guojia Biochemical Engineering Research Centre Co.LTD,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]上海国佳生化工程技术研究中心有限公司,上海200237

出　　处：《华东理工大学学报（自然科学版）》2024年第1期62-70,共9页Journal of East China University of Science and Technology

摘　　要：猪圆环病毒2型(Porcine Circovirus type 2,PCV2)衣壳(Capsid)蛋白是制备抗猪圆环2型病毒亚单位疫苗的有效免疫抗原,能在大肠杆菌及杆状病毒/昆虫细胞表达系统中获得表达,然而在哺乳细胞中的表达研究仍然缺乏。瞬时表达条件考察结果显示,采用宿主HEK293F细胞及PEI-40kDa转染试剂能获得35%病毒PCV2 Capsid基因细胞转染效率。转染试剂PEI(Polyethylenimine)与DNA比例差异会影响复合物形成大小及形态,复合物形成15~80 nm颗粒有利于转染效率的提高。实验以免疫逃逸型猪圆环病毒2b型NDSU41513病毒株,设计了新型PCV2 Capsid (△1-41aa)-Fc(pig) protein(PCFP)分子。在3 L反应器中成功实现了HEK293F细胞瞬时表达PCFP,表达水平达到3.8 mg/L。PCFP蛋白能够自主装形成约为41 nm类病毒颗粒,诱导小鼠机体内产生强烈的体液免疫反应,具有很高的应用潜力。Porcine circovirus type 2(PCV2)capsid protein is the only immunodominant structural protein of PCV2 for preparing vaccines against PCV2 infection.The protein can be expressed in Escherichia coli and baculovirus/insect cell expression system.However,studies on the expression of the protein in mammalian cells remain elusive.Here,we investigated several factors that determine the transient expression of the PCV2 capsid protein in mammalian cells.The results showed that a 35%efficiency of the PCV2 capsid gene transfection could be obtained by using HEK293F as the host cell and PEI-40kDa as the transfection reagent.The size and morphology of the PEI/DNA complex was determined by the ratio of the two components,and a particle size range of 15—80 nm contributed to a high cell transfection efficiency.A novel PCV2 Capsid(△1-41aa)-Fc(pig)protein(PCFP)was designed from the immune escape virus strain of PCV2b 41513.PCFP was transiently expressed in HEK293F cells cultivated at a 3 L bioreactor,and the expression level reached 3.8 mg/L.PCFP self-assembled into 41 nm virus-like nanoparticles,which effectively stimulated the humoral immune response in mice.

关 键 词：猪圆环病毒2型 瞬时表达 HEK293F 类病毒颗粒 疫苗 

分 类 号：Q81[生物学—生物工程]