细胞穿透肽递送的流感病毒M2胞外区通用DNA疫苗在小鼠体内的免疫效力  

Immune efficacy of 3M2e-based universal influenza DNA vaccine delivered by cell penetrating peptides in mice

作　　者：刘洪港 赵美霞 任维岗 李军伟 LIU Honggang;ZHAO Meixia;REN Weigang;LI Junwei(Medical Innovation Team for Infectious Diseases,Nanjing Second Hospital,Nanjing 210003,Jiangsu Province,China)

机构地区：[1]南京市第二医院传染病医学创新团队,江苏南京210003 [2]青岛农业大学动物医学院,山东青岛266109 [3]南京市感染病临床医学中心,江苏南京210003

出　　处：《中国生物制品学杂志》2024年第9期1030-1036,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(32072837);江苏省社会发展社科类项目(BE2021603)。

摘　　要：目的设计通用甲型流感病毒DNA疫苗,并检测其在小鼠体内的免疫效力。方法合成含H1N1、H3N2、H9N2亚型甲型流感病毒M2(3M2e)胞外结构域的DNA序列,克隆至载体pVAX1中,构建真核表达质粒pVAX1-3M2e。将质粒pVAX1-3M2e分别与细胞穿透肽(cell penetrating peptides,CPPs)RVG9dR、Protamine、RVG9dR+Protamine多肽复合物(质量分数1∶1)按不同质量分数混合(1∶0.5、1∶1、1∶2、1∶4、1∶8),进行凝胶阻滞试验,以确定DNA与CPPs的正确结合;并通过细胞荧光试验在细胞水平(293T细胞)检测CPPs的递送效率。选择RVG9dR+Protamine递送质粒pVAX1-3M2e经肌内免疫雄性BALB/c小鼠(多肽复合物两种剂量:25μg pVAX1-3M2e+500μg多肽复合物、50μg pVAX1-3M2e+1000μg多肽复合物,每组8只小鼠),检测通用甲型流感病毒DNA疫苗对3种亚型流感病毒(H1N1、H3N2、H9N2)的交叉保护作用。结果DNA与RVG9dR、Protamine、RVG9dR+Protamine的最佳结合比分别为1∶2、1∶2、1∶1;RVG9dR+Protamine在1∶20的质量分数下对质粒pVAX1-3M2e具有超强的递送效率;50μg pVAX1-3M2e+1000μg多肽复合物对3种亚型流感病毒攻毒组小鼠的保护率均为100%。结论由RVG9dR+Protamine多肽复合物递送的质粒pVAX1-3M2e在小鼠中提供了针对3种亚型甲型流感病毒的交叉保护。Objective To design an effective universal DNA vaccine for influenza A virus and detect its immune efficacy in mice.Methods The DNA sequence containing the M2(3M2e)extracellular domain of H1N1,H3N2 and H9N2 subtypes of influenza A virus was synthesized and cloned into vector pVAX1 to construct eukaryotic expression plasmid pVAX1-3M2e.The plasmid pVAX1-3M2e was mixed with cell penetrating peptides(CPPs)RVG9dR,Protamine,and RVG9dR+Protamine peptide complex(1∶1)at different mass fractions(1∶0.5,1∶1,1∶2,1∶4,and 1∶8),separately,and gel retardation was performed to determine the proper binding of DNA and CPPs.The delivery efficiency of the CPPs was detected by cell fluorescence assay at cellular level(293T cells).Male BALB/c mice were immunized intramuscularly with plasmid pVAX1-3M2e delivered by RVG9dR+Protamine(two doses:25μg pVAX1-3M2e+500μg polypeptide complex,50μg pVAX1-3M2e+1000μg polypeptide complex,8 mice in each group)to detect the cross-protection effect of universal influenza A virus DNA vaccine against three subtypes of influenza viruses(H1N1,H3N2 and H9N2).Results The optimum binding ratios of DNA to RVG9dR,Protamine,and RVG9dR+Protamine were 1∶2,1∶2,and 1∶1,respectively.RVG9dR+Protamine had superior delivery efficiency to plasmid pVAX1-3M2e at the mass fraction of 1∶20.The protective rates of 50μg pVAX1-3M2e+1000μg polypeptide complex against three subtypes of influenza virus in mice were all 100%.Conclusion The plasmid pVAX1-3M2e delivered by RVG9dR+Protamine polypeptide complex provides cross protection against three subtypes of influenza A virus in mice.

关 键 词：甲型流感病毒 M2胞外结构域 通用DNA疫苗 细胞穿透肽 交叉保护 

分 类 号：S851[农业科学—预防兽医学]