杆状病毒表面展示甲型流感病毒核蛋白的免疫原性及保护效果的初步评价  

Evaluation of Influenza A Virus Nucleoprotein Based on Baculovirus Surface-display Technology

作　　者：张立霞[1] 周剑芳[1] 于在江[1] 舒跃龙[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所国家流感中心,北京102206

出　　处：《病毒学报》2013年第3期265-272,共8页Chinese Journal of Virology

基　　金：国家863课题(2010AA022901);国家科技重大专项(2009ZX10004-016)

摘　　要：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. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with differ- ent subtype influenza viruses. Here, NP of an avian H5N1 （A/Hubei/1/2010, HB） was expressed by bac- ulovirus surface-display technology and its immunogenicity as well as protective mechanism was investiga- ted in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides （NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF）were identified by IFN-r ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4＋ and CD8＋ T cells , and the re- sponse of CD8＋ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 （HIN1） showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.

关 键 词：NP蛋白 H5N1禽流感病毒 杆状病毒表面展示技术 免疫原性和保护性 

分 类 号：R373.9[医药卫生—病原生物学]