流感病毒为载体重组病毒株rHCV/FLU的构建及初步评价

Construction and efficacy evaluation of a recombinant influenza virus as a vector expressing HCV antigen

目的构建及拯救以流感病毒为载体嵌合HCV抗原表位的重组病毒株r HCV/FLU并对其进行评价。方法利用反向遗传学技术,选择流感病毒A/Puerto Rico/8/34(PR8)为载体,将HCV 1b亚型的E1抗原基因经序列优化插入到PR8流感病毒NS1片段的特定位置,构建重组质粒p NS1-HCV,测序正确的重组质粒p NS1-HCV与PR8的7个重组质粒p HW191-PB2、p HW192-PB1、p HW193-PA、p HW194-HA、p HW195-NP、p HW196-NA、p HW197-M转染共培养的COS-1和MDCK细胞,经拯救、筛选、鉴定获得流感病毒为载体嵌合HCV抗原表位的重组病毒株,命名为r HCV/FLU,并通过血凝试验、RT-PCR、Western blot、电镜观察病毒形态等方法对重组病毒鉴定。接着,重组病毒接种SPF鸡胚扩大培养、超滤浓缩、蔗糖密度梯度离心纯化。纯化获得的重组病毒抗原,滴鼻免疫Balb/c小鼠,0 d、14 d免疫2次,设置104TCID50、105TCID50、106TCID503个剂量组,初步评价其免疫效果。结果成功拯救重组病毒株r HCV/FLU,血凝效价(HA)为29-10,病毒滴度Log10(TCID50/ml)为6.5。滴鼻免疫小鼠二免后14 d取小鼠血清及脾淋巴细胞,3个剂量组针对流感病毒的血抑(HI)效价均值分别为80、640和1 280,初步证明重组病毒株r HCV/FLU在小鼠体内能够产生较强体液免疫和细胞免疫应答反应。结论成功制备的重组病毒株r HCV/FLU在动物体内具有较好的免疫效果,为新型HCV疫苗的研制提供新的研究策略。

HCV is an important infectious disease that endangers human health. So far no vaccine is available.This study designed to evaluate the immune efficacy of the recombinant r HCV/FLU virus, providing theexperimental evidence for the HCV vaccine. By using reverse genetics（RG）, a recombinant PR8 influenza virusvector expressing the E1 antigenic epitopes was rescued and inserted into the influenza nonstructural（NS1） proteingene to construct p NS1-HCV, which was respectively cotransfected with the influenza virus seven plasmids of PB2,PB1, PA, HA, NP, NA and M into COS-1-MDCK cells. Sequence analysis of transfectant viral RNA has verifiedthat the transfectant virus contained the chimeric RNA, and the E1 was detected by Western blotting; therecombinant morphological characteristic was observed with electron microscope. Recombinant r HCV/FLU viruswere amplified by SPF chicken embryos, and purified by 30%-60% sucrose gradient. The purified recombinantvirus was detected and showed 29~10hemagglutination titers and 6.5log10（TCID50/ml） viral titers. Female Balb/c micewere vaccinated intranasally with purified r HCV/FLU. The boost immunization was operated two weeks after theprime immunization, and the sera and spleen cellsuspensions of mice were collected two weeks afterboost immunization. The hemagglutination inhibition（HI） titers of the high, middle and low dose groups were1 280, 640 and 80, respectively. The resultssuggestedthat r HCV/FLU virus can induce humoral and cellular immune responses, which provide a promising candidate for HCV vaccine design.