重组流感病毒H1N1活菌疫苗的构建及其免疫效果的初步研究

目的获得两种流感病毒重组沙门氏菌X4550(asd-pVAX1-HA)和X4550(asd-pVAX1-NA)活菌疫苗,并对其免疫效果进行初步的研究。方法将本室已构建好的重组质粒pVAX1-HA及pVAX1-NA双酶切后得到HA、NA基因,将其克隆入asd-pVAX1构建重组的表达质粒。将重组质粒转染COS-7细胞,用免疫细胞化学方法鉴定重组质粒体外的表达。进一步将重组质粒转化减毒鼠伤寒沙门氏菌X4550,以2×109CFU/只的剂量三次口服免疫Balb/c小鼠。结果asd-pVAX1-HA和asd-pVAX1-NA均能在COS-7细胞中表达;免疫小鼠不仅可以检测到HA、NA特异性的血清IgG及IgA抗体;刺激黏膜免疫反应,产生sIgA抗体,而且能抵抗40LD50同型病毒滴鼻的攻击。结论H1N1流感病毒DNA疫苗的减毒沙门氏菌运送系统在体内能够成功释放所携带的质粒,并且能够刺激机体产生保护性免疫应答。

Construction of bacterial carrier vaccines against H1N1 subtype of influenza virus and its immunogenicity in Balb/c mice

Objective To construct attenuated S. typhimurium carrier vaccine harbouring influenza HA and NA genes and to study its immunogenicity. Methods HA and NA genes were provided by double restriction enzyme digestion of constructed recombinant plasmid pVAX1-HA and pVAX1-NA, and then inserted into asd-pVAX1. The expressions of these two recombinant plasmids in COS-7 cells were detected by immunocytochemistry. Furthermore, the recombinants asd-pVAX1-HA and asd-pVAX1-NA were transformed into an attenuated S. typhimurium strain X4550, named as X4550 (asd-pVAX1-HA) and X4550 (asd-pVAX1-NA) respectively, and administered to Balb/c mice orally. Results X4550 (asd-pVAX1-HA) and X4550 (asd-pVAX1-NA) could induced HA-specific and NA-specific serum IgG, IgA, and sIgA antibodies. These immunization were capable of inducing the effective immunoprotection against subcutaneous challenge of 40LD50 influenza virus. Conclusion The results demonstrate that two Salmonella-based delivering systems can release harboured DNA vaccines in vivo, and elicit protective immune responses.