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流感病毒的核蛋白(Nucleoprotein,NP)高度保守,具有型特异性,能够诱导产生细胞介导的免疫应答,从而起到保护作用,抵抗不同亚型流感病毒的攻击。本研究利用杆状病毒表面展示技术,将杆状病毒GP64蛋白的信号肽(Signal peptide,SP)和胞质尾(Cytoplasmic tail,CT)与甲型流感病毒(A/Hubei/1/2010(H5H1))的NP蛋白融合表达,从而将NP蛋白展示在杆状病毒的表面,并对该病毒NP蛋白的免疫原性和保护效果进行了研究。Westernblot实验证明NP蛋白可以在Sf9细胞中有效表达。免疫电镜试验显示NP蛋白已成功展示在杆状病毒表面。通过小鼠感染实验,ELISA结果证明NP疫苗可以诱导产生高水平的IgG血清抗体。ELISPOT结果表明NP蛋白的2个细胞免疫抗原表位(NP57-66IQNSITIERM和NP441-450RTEIIKMMES)可以诱导小鼠脾淋巴细胞产生IFN-γ,利用NP57-66、NP441-450和NP蛋白均可刺激CD4+和CD8+T细胞产生IFN-γ,但主要以CD8+诱导为主,表明重组病毒可以产生有效的细胞免疫反应。利用小鼠鼠肺适应株A/PR/8/34(H1N1)进行攻毒实验,结果表明该重组病毒感染小鼠后可以降低小鼠肺病毒载量,减轻肺组织损伤,减少体重下降,并可以保护50%小鼠存活。
Nucleoprotein (NP) of influenza virus is highly conserved and type-specific, which can induce cell-mediated immune response and thus protect against the attack of different subtypes of influenza virus. In this study, the signal peptide (SP) and cytoplasmic tail (CT) of the baculovirus GP64 and the influenza A virus (A / Hubei / 1/2010 (H5H1 )) NP protein fusion expression, so that the NP protein is displayed on the surface of the baculovirus, and the virus NP protein immunogenicity and protective effect were studied. Western blotting experiments showed that NP protein can be efficiently expressed in Sf9 cells. Immunoelectron microscopy showed that the NP protein has been successfully displayed on the baculovirus surface. Through mouse infection experiments, ELISA results demonstrate that the NP vaccine can induce high levels of IgG serum antibodies. ELISPOT results showed that two cellular immunological epitopes of NP protein (NP57-66IQNSITIERM and NP441-450RTEIIKMMES) could induce IFN-γ production in mouse splenic lymphocytes, and both NP57-66, NP441-450 and NP proteins could stimulate CD4 + and CD8 + T cells produce IFN-γ, but predominately induced by CD8 +, indicating that the recombinant virus can produce an effective cellular immune response. The challenge experiment was carried out by using mouse lung-adapted strain A / PR / 8/34 (H1N1). The results showed that the recombinant virus could reduce the lung virus load, reduce lung tissue damage and reduce body weight loss in mice Can protect 50% of mice survive.