【作者】 王开艳；

【导师】 李太元； 金宁一；

【作者基本信息】 延边大学 ， 预防兽医学， 2010， 硕士

【摘要】 流行性感冒是由流感病毒引起的急性呼吸道传染病,对人群健康,社会稳定均产生重大影响。降低流感流行的最有效手段仍是接种流感疫苗,然而,由于流感病毒血清亚型众多,变异性强,因此设计开发安全的且可提供长久保护的,对多亚型流感共保护的新型流感疫苗势在必行。根据当前流感的流行现状和高致病性禽流感病毒跨种间传播的现状,本研究在本实验室已构建好的表位盒基础上,加以改进并合成了两个表位盒(H5亚型Th／B,CTL表位盒)。另外,采用基因串联的方式,构建了两种核酸疫苗质粒(pVAX1-NP-ECH5、PVAX1-H3-ETH5-H1HA1)。为了验证其能否用于进一步的体内试验,本研究以脂质体法转染BHK细胞,分别以RT-PCR和IFA对外源基因转录和蛋白表达及表达产物生物学活性进行了鉴定。结果显示RT-PCR均检测到外源基因的mRNA转录情况；IFA检测到了特异性荧光,说明表达产物具有相应的生物学活性。这说明各抗原组分可以在真核细胞中有效表达,为进一步动物实验免疫研究奠定了基础。本研究以小鼠模型作为哺乳动物模型,对之前构建的多表位核酸疫苗进行了免疫研究,对疫苗的免疫原性进行了评估。血清ELISA抗体检测结果显示：添加表位疫苗组(ThB、CTL表位)激发了与抗原单表达组相当的抗体水平,且并在H5亚型流感病毒特异性ELISA抗体水平显示了优势。细胞因子IL-4、IFN-γ检测结果显示：表位组分的加入显著上调了小鼠Th2细胞免疫功能,且CTL表位与NP串联免疫,可有效刺激了T淋巴细胞IFN-γ分泌。淋巴细胞转化试验结果表明：添加表位的DNA疫苗可刺激小鼠产生较强的特异性和非特异性增殖能力,免疫组小鼠的脾脏T淋巴细胞无论是特异性刺激指数还是非特异性刺激指数均明显高于对照组,且优于未添加表位DNA疫苗组。以上实验结果显示,本研究构建的以H3HA和H1HA1融合表达基因为骨架嵌合加入多表位基因的核酸疫苗,小鼠免疫实验显示,其不仅产生针对H3、H1 HA产生了特异性免疫应答(抗体和特异性细胞免疫),还增加了其抗H5亚型流感感染的可能性,为最终获得通用型流感病毒多表位基因工程疫苗进行了有意义的尝试。更多还原

【Abstract】 Influenza is caused by the influenza virus, acute respiratory infections, on human health, have a major impact on social stability. Reduce influenza pandemic remains the most effective means of influenza vaccination, however, due to a number of influenza virus subtypes, variability, and therefore design and development of safe and provide long-term protection against multiple subtypes of influenza of a new influenza vaccine to protect imperative.Based on the current status and the prevalence of influenza highly pathogenic avian influenza virus transmission across species status, this study has been constructed in the laboratory on the basis of a good table-bit box, to improve and synthesized two epitope Box (H5 Asia Model Th/B, CTL epitopes box). In addition, the manner by gene tandem construct two plasmid DNA vaccine (pVAX1-NP-ECH5, pVAX1-H3-ETH5-H1HA1). To test its ability to be used for further in vivo tests, this study liposome transfected BHK cells, RT-PCR and IFA exogenous gene transcription and protein expression and biological activity of expression products were identified respectively. The results showed that both RT-PCR detected mRNA transcription of exogenous genes situation; IFA to detect the specific fluorescence, indicating expression products with the corresponding biological activity. This indicates that the antigen components can be efficiently expressed in eukaryotic cells, in order to further animal experiments laid the foundation for Immunization Research.In this study, mice as a mammalian model, prior to construction of multi-epitope DNA vaccine immunization study, the immunogenicity of the vaccine was evaluated. ELISA serum antibody test results showed that:Add-epitope vaccine group (ThB, CTL epitopes) to stimulate expression of group with a single antigen-antibody level considerably, and in the H5 subtype of influenza A virus-specific ELISA antibody levels showed an advantage. Cytokines IL-4, IFN-γtest results showed:Table-bit components by adding significantly up-regulated Th2 cellular immune function in mice, and the CTL epitope immunization with the NP series, which can effectively stimulate T-lymphocyte IFN-γsecretion. Lymphocyte transformation test results showed that:Add the epitope DNA vaccine can stimulate the mice produced strong specific and non-specific proliferation and immune groups of mice spleen T lymphocytes, whether specific or non-specific stimulation index stimulation index significantly higher than, and superior to add-epitope DNA vaccine group.These results show that this study was constructed in order to H3HA and H1HA1 fusion gene expression for the skeleton, adding multi-epitope chimeric gene DNA vaccine, immunization experiments in mice showed that they not only produce for the H3, H1 HA produced a specific immune response (antibodies and specific cell-mediated immunity) and also increased its anti-H5 subtype of influenza infection in the possibility of ultimately universal influenza virus multi-epitope genetically engineered vaccines meaningful attempt.更多还原