【摘要】 本文研究了以鸡恒定链基因(invariant chain,Ii)为载体的新城疫病毒(Newcastle disease virus,NDV)F基因片段的基因疫苗及其小鼠的免疫应答。首先,用重叠延伸法经3轮PCR获得以NDV-F343表位基因(327-359AA)取代鸡恒定链中Ⅱ类分子相关肽段(class-Ⅱassociated invariant chain peptide,CLIP)片段序列,构建了基于恒定链的NDV内源性靶向基因疫苗(C1-△CLIP-Ii-F343)。其次,在观察C1-△CLIP-Ii-F343和无恒定链载体的C1-F343转染真核细胞中,发现它们均能在COS-7细胞中高效表达。最后,将25只6~8周龄的雌性Balb/c小鼠(Mus musculus)随机分为5组,用C1-△CLIP-Ii-F343和非靶向性疫苗(C1-F343)进行免疫,以C1-△CLIP-Ii、pEGFP-C1和生理盐水作为对照。经3次免疫后,取小鼠尾部血对小鼠的体液免疫进行检测。结果表明,在C1-△CLIP-Ii-F343和C1-F343免疫组小鼠血清中均可检测到针对NDV的特异性抗体,其滴度分别达到1/6400和1/1600。进一步Westernblot研究结果表明,抗体与接种NDV的鸡胚尿囊液和原核表达的PET-NDV-F306蛋白均产生特异性结合。研究结果提示,鸡恒定链作为载体的NDV基因疫苗能够刺激小鼠产生特异性的体液免疫应答,为新城疫疫苗开发提供了一个新的思路。更多还原

【Abstract】 In this paper a gene vaccine C1-△CLIP-Ii-F343 containing Newcastle disease virus(NDV) F epitope based on chicken invariant chain(Ii) as carrier and its immune response in mice are studied. First the NDV-F epitope gene vaccine, an endogenous targeting fragment, was constructed by overlap extension method via three cycles of PCR, in which class-Ⅱ associated invariant chain peptide (CLIP) of chicken Ii was substituted with NDV F epitope, F343 (327-359 AA). Secondly the transfection of COS7 cells with C1-△CLIP-Ii-F343 and C1-F343 (no Ii carrier) was observed. And both could efficiently express in the cost cells. Finally twenty-five female Balb/c mice(Mus musculus) of 6~8 weeks old were randomly divided into five groups and injected with C1-△CLIP-Ii-F343 and C1-F343, as experimental controls with C1-△CLIP-Ii, pEGFP-C1 and saline, respectively. After immunizations three times, the humoral immune response was estimated. The C1-△CLIP-Ii-F343 and C1-F343 could induce the mice to produce specific antibody to NDV-F and the titers could reach 1/6400 and 1/1600, respectively. Western blot results showed that the antibodies could bind specifically with NDV allantoic fluid of chicken embryo injected with NDV and PET-NDV-F306 protein of prokaryotic expression. The results suggest that the C1-△CLIP-Ii-F343 epitope vaccine based on Ii as carrier can stimulate specific humoral immune response in mice and it may be a potential candidate gene for NDV vaccine development.更多还原