Studies on intermembrane protein transfer and establishment of preparation method of artificial membrane vaccine

人工膜疫苗膜间蛋白转移研究及制备方法的建立

• 批准号: 15590039
• 负责人: UENO Masaharu
• 金额: $ 2.3万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590039/
• 关键词: membrane protein; liposomes; artificial membrane vaccine; CV-1 cell; influenza virus; immunoprecipitation; immunoactivity; adjuvant; 膜たんぱく質; 膜間移行; 免疫能; 中和抗体; HA; NA; 抗体産生能; MDP誘導体

Recentlyy it has been reported that the various membrane proteins are transferred spontaneously from living cell membranes to liposomal membranes. We adopted the phenomenon in developing liposome vaccine against influenza virus. First, we studied viral protein transfer from influenza virus-infected cells to liposomes. Next, biological activity of the liposome vaccine was examined.The membrane proteins were recovered on the liposomes in both cases : One is direct transfer of protein in the condition of coexisting of donor cells and acceptor liposomes ; the other is protein transfer from the sorting buffer to the liposomes, in which buffer virus-infected cells were incubated for 1 hour without liposomes followed by adding liposomes after removing cells. Immunoprecipitation and immunoblotting experiments showed the transfer of viral protein from influenza virus-infected CV-1 celles to liposomes.The antigenic protein-incorporated liposomes were inoculated to BALB/c mice as an artificial membrane vaccine or liposome vaccine. Immunoactivity of the liposome vaccine was almost equivalent to that of inactivated virus. B-30 MDP (6-o-(2-tetradecyl hexadecanoyl)-N-acetylmuramyl-L-aranyl-isoglutamine) as an adjuvant enhanced strongly immunoactivity.

最近有报道称，各种膜蛋白可以自发地从活细胞膜转移到脂质体膜上。我们在研制流感病毒脂质体疫苗时采用了这一现象。首先，我们研究了病毒蛋白从流感病毒感染细胞到脂质体的转移。接下来，对脂质体疫苗的生物活性进行了检测，在两种情况下都在脂质体上回收了膜蛋白：一种是在供体细胞和受体脂质体共存的情况下直接转移蛋白质；另一种是将蛋白质从分选缓冲液转移到脂质体中，在这种情况下，缓冲病毒感染的细胞在没有脂质体的情况下孵育1h，然后在去除细胞后加入脂质体。免疫沉淀和免疫印迹实验表明，病毒蛋白从流感病毒感染的CV-1细胞转移到脂质体中，以人工膜疫苗或脂质体疫苗的形式接种BALB/c小鼠。脂质体疫苗的免疫活性与灭活病毒基本相当。B-30MDP(6-o-(2-十四烷基)hexadecanoyl)-N-acetylmuramyl-L-aranyl-isoglutamine)作为佐剂可显著增强免疫活性。