乙肝疫苗评价技术及其应用研究

发布时间：2018-04-23 06:37

本文选题：人/鼠嵌合四聚体 + HBV/HLA-A2.1双转基因小鼠　；参考：《第二军医大学》2007年硕士论文

【摘要】： 通常MHC I类四聚体被用于定量分析表位特异的CD8+T细胞。MHC I类四聚体是由4个MHC I类复合物和一个带有荧光标的亲和素分子组成的。其中每个MHC I类复合物是由分别表达、纯化的MHC分子的重链和β2微球蛋白与合成的表位肽在折叠缓冲液中共折叠所形成的。这种方法势必会形成较多的多聚物和自身折叠产物,而且同时纯化两种单体是很费时的。为了解决这些问题,在本研究中,我们将串联设计的人/鼠嵌合MHC分子的重链(HLA-A2α1, HLA-A2α2,MHC-H2Dα3)与β2微球蛋白克隆到pET17b载体上,并表达为融合蛋白。将重组的融合蛋白与HLA-A2限制性表位肽两者共折叠形成人/鼠嵌合MHC I类复合物。这样就会提高产量,也可以节省大量时间。此外,我们还采用了凝胶迟滞的方法来检测人/鼠嵌合MHC I类复合物生物素化的效率。我们将最终形成的人/鼠嵌合四聚体用于体外定量检测HLA-A2转基因鼠上所产生的HLA-A2限制性细胞免疫水平。由于缺少合适的动物模型,在体内对人乙肝疫苗免疫原性的临床前评价一直受到了制约。在本项研究中,我们构建了能够同时表达HBV和HLA两种基因的HBV/HLA-A2.1双转基因小鼠。此双转基因鼠不仅表现出对HBV各种抗原的耐受,而且能够有效地递呈HLA-A2限制性的抗原表位。我们用HLA-A2限制性HBc或HBs的表位疫苗免疫此双转基因小鼠,借助CTL实验、酶联免疫斑点实验和四聚体染色的手段证明其能够被有效的诱导出针对HLA-A2限制性HBc和HBs特异性的免疫应答。总之,我们构建的有生物活性的人/鼠嵌合四聚体以及HBV/HLA-A2.1双转基因鼠国内外均未见报道。人/鼠嵌合四聚体能恰当的定量反映HLA-A2限制性细胞免疫水平。同时,HBV/HLA-A2.1双转基因小鼠能够在多方面评价人乙肝疫苗的免疫原性:抗体的产生、γ干扰素的分泌水平、免疫小鼠的特异性CTL活性和有效降低体内抗原水平。这些结果也证明了HBV/HLA-A2.1双转基因小鼠作为评价人乙肝病毒疫苗免疫原性动物模型的可用性。
[Abstract]:In general, MHC I tetramer is used to quantitatively analyze epitope specific CD8 T cells. MHCI tetramer is composed of four MHC I complexes and a fluorescent target affinity molecule. Each MHC class I complex is formed by co-folding the heavy chain and 尾 2 microglobulin of the purified MHC molecule with the synthesized epitope peptide in the folding buffer solution. This method is bound to form more polymers and self-folding products, and it takes time to purify the two monomers simultaneously. In order to solve these problems, we cloned HLA-A2 伪 1, HLA-A2 伪 2 MHC-H2D 伪 3 and 尾 2 microglobulin of human / mouse chimeric MHC molecule designed in tandem into pET17b vector and expressed as fusion protein. The recombinant fusion protein was co-folded with HLA-A2 restriction epitope peptide to form a human / mouse chimeric MHC class I complex. This will increase production and save a lot of time. In addition, we also used the gel hysteresis method to detect the biotin efficiency of human / mouse chimeric MHC class I complex. The resulting human / mouse chimeric tetramer was used to quantitatively detect the HLA-A2 restricted cellular immunity in HLA-A2 transgenic mice in vitro. Due to the lack of suitable animal models, preclinical evaluation of the immunogenicity of human hepatitis B vaccine has been restricted in vivo. In this study, we constructed HBV/HLA-A2.1 double transgenic mice which could express both HBV and HLA genes. The two transgenic mice not only showed tolerance to various antigens of HBV, but also presented HLA-A2 restricted epitopes effectively. We immunized the double transgenic mice with epitope vaccines of HLA-A2 restricted HBc or HBs, and we used CTL experiment. Enzyme linked immunoblot assay (Elisa) and tetramer staining showed that it could effectively induce a specific immune response to HLA-A2 restricted HBc and HBs. In a word, the bioactive human / mouse chimeric tetramer and HBV/HLA-A2.1 double transgenic mice have not been reported at home and abroad. Human / mouse chimeric tetramer is an appropriate quantitative reflection of HLA-A2 restricted cellular immunity. At the same time, HBV / HLA-A2.1 transgenic mice could evaluate the immunogenicity of human hepatitis B vaccine in many ways: the production of antibody, the secretion level of interferon 纬, the specific CTL activity of immunized mice and the effective reduction of antigen level in vivo. These results also demonstrate the availability of HBV/HLA-A2.1 double transgenic mice as an animal model for evaluating the immunogenicity of human hepatitis B vaccine.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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