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目的克隆、表达并纯化NP-M1-HSP60融合蛋白,免疫Balb/c小鼠,评价其免疫原性及免疫保护性,为候选甲型流感通用疫苗抗原提供新靶标。方法合成NP-M1-HSP60融合基因,构建pET28a-NP-M1-HSP60重组质粒,表达并纯化NP-M1-HSP60融合蛋白,抗原与SP01水包油佐剂混匀,制备候选甲型流感通用疫苗,滴鼻免疫Balb/c小鼠,评价其免疫原性及免疫保护性。结果成功构建pET28a-NP-M1-HSP60表达载体,表达并纯化了NP-M1-HSP60融合蛋白;与SP01水包油乳剂佐剂混合免疫小鼠后,小鼠血清IgG抗体效价较对照组显著提升,鼻、肺灌洗液中sIgA水平显著升高,IgG亚型分析及细胞因子分泌细胞实验表明该候选疫苗可诱导相对均衡的Th1和Th2反应。免疫后小鼠分别对100LD50剂量的A/Beijing/501/2009(H1N1)毒株,100LD50剂量的PR8(H1N1)毒株和50LD50剂量的A/ostrich/Suzhou/097/2003(H5N1)毒株的攻击分别具有100%、100%和67%保护效果。结论 NP-M1-HSP60融合蛋白抗原具有一定的效果,为发展甲型流感通用疫苗提供了实验依据。
Objective To clone, express and purify NP-M1-HSP60 fusion protein and immunize Balb / c mice to evaluate the immunogenicity and immunoprotective properties of NP-M1-HSP60 fusion protein. Methods The NP-M1-HSP60 fusion gene was synthesized and the recombinant plasmid pET28a-NP-M1-HSP60 was constructed. NP-M1-HSP60 fusion protein was expressed and purified. The antigen was mixed with SP01 oil-in-water adjuvant to prepare a candidate universal influenza A , Balb / c mice were intranasally immunized to evaluate their immunogenicity and immunoprotective properties. Results The pET28a-NP-M1-HSP60 expression vector was successfully constructed and NP-M1-HSP60 fusion protein was expressed and purified. After being mixed with the SP01 oil-in-water emulsion adjuvant, the IgG titer of serum was significantly higher than that of the control group SIgA levels in nasopharyngeal and lung lavage fluid were significantly increased. Analysis of IgG subtypes and cytokine secreting cells indicated that the vaccine candidate could induce relatively balanced Th1 and Th2 responses. The immunized mice were challenged with 100LD50 doses of A / Beijing / 501/2009 (H1N1) strain, 100LD50 dose of PR8 (H1N1) strain and 50LD50 dose of A / ostrich / Suzhou / 097/2003 (H5N1) Attacks have 100%, 100% and 67% protection, respectively. Conclusion NP-M1-HSP60 fusion protein antigen has certain effect, which provides an experimental basis for the development of influenza A influenza vaccine.