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应用反向遗传学技术,选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60 ca(H2N2)型流感病毒的6个内部基因为骨架,与A/California/07/2009株流感病毒2个抗原基因HA、NA分别克隆到polⅠ-polⅡ转录表达载体pAD3000中,构建8个转录表达载体重组质粒,共转染Vero细胞,获得重配A/California/07/2009ca株流感病毒.重配病毒的TCID50为7.5,病毒传4代后其血凝素(HA)滴度稳定在1∶256,半数感染剂量EID50为8,鸡胚传20代,经RT-PCR鉴定未发现重组病毒基因突变,电镜观察重配病毒符合流感病毒的主要特征;蔗糖纯化的病毒经肌肉注射(灭活)及滴鼻(减毒活病毒)两种途径免疫BALB/c小鼠,结果显示:滴鼻免疫和肌肉注射都可以产生较高效价的血凝抑制(HI)抗体,肌肉注射组产生的HI抗体略高(P=0.044),但肌肉注射组检测不到高效价IgA抗体;滴鼻免疫组鼻冲洗液中可以检测到高效价的IgA抗体,同型病毒感染后,IL-1β、TNFα、IFN-α等前炎因子分泌较早,且高于肌肉注射组(P<0.05),可见,喷鼻减毒疫苗比灭活全病毒疫苗能更好地激发黏膜免疫反应.通过对小鼠各个器官病毒载量的检测发现,4天后鼻腔、气管、脑、肺、脾脏没有病毒存在,证明减毒活疫苗株在小鼠上是安全的.以上数据可以初步断定,重组病毒有作疫苗候选株的可能,而且喷鼻疫苗具有降低免疫剂量、同时激活体内体液免疫和细胞免疫的功能.
The six internal genes of influenza A / Ann Arbor / 6/60 ca (H2N2) influenza virus, which are cold-adapted, temperature-sensitive and attenuated, were selected as backbones by back- The HA and NA genes of the two viruses were cloned into the pol Ⅰ-pol Ⅱ transcriptional expression vector pAD3000, respectively, and eight recombinant plasmids were constructed and co-transfected into Vero cells to obtain a reassociated A / California / 07 / 2009ca influenza virus. The TCID50 of the virus was 7.5. After the passage of the virus for 4 generations, the hemagglutinin (HA) titer was stable at 1: 256, the median infection dose EID50 was 8 and the embryo was passaged for 20 passages. No recombinant virus gene was identified by RT-PCR Mutation and electron microscopy were used to identify the main characteristics of the reassortant virus. The sucrose-purified virus was immunized by both intramuscular (inactivated) and intranasal (attenuated live virus) immunization of BALB / c mice. The results showed that intranasal immunization And intramuscular injection could produce high titer of HI antibody. The HI antibody in the intramuscular injection group was slightly higher (P = 0.044), but the intramuscular injection group could not detect the high titer IgA antibody. The intranasal immunization group High titer anti-IgA antibodies can be detected in the wash solution. After being infected with the same virus, IL-1β, TNFα, IF N-α and other pro-inflammatory cytokines secreted earlier, and higher than the intramuscular injection group (P <0.05), we can see that the nasal attenuated vaccine can inactivate the mucosal immune response better than the inactivated whole virus vaccine.After mice The detection of organ viral load found that there was no virus in the nasal cavity, trachea, brain, lung and spleen after 4 days, which proved that the attenuated live vaccine strain was safe in mice.The above data can be preliminary concluded that the recombinant virus has vaccine candidate And that nasal vaccines have the potential to reduce the immunization dose while activating humoral and cellular immunity in vivo.