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目的选取嗜肺军团菌mip和flaA优势抗原表位基因,构建mip/flaA二联优势抗原表位基因融合表达载体,并在原核系统中表达,为后续制备嗜肺军团菌蛋白疫苗提供初步的实验基础。方法运用生物信息学方法对Mip和FlaA蛋白的二级结构和表面特性如理化性质、亲水性、可塑性、抗原指数以及胞外区等方面进行分析,选择其活性表位可能存在的区域为优势抗原表位区。通过PCR扩增和T4连接酶构建pET-mip、pET-flaA和pET-mip/flaA优势抗原表位基因融合表达载体,并诱导其在大肠杆菌中表达。结果 Mip和FlaA都存在多个潜在的抗原表位位点,选取其优势抗原表位区域进行克隆和表达获得成功,并成功表达了mip/flaA二联优势抗原表位融合蛋白。结论 DNA Star软件和Expasy在线蛋白分析系统能够成功预测嗜肺军团菌Mip和FlaA抗原的表位;选取其优势抗原表位成功构建了pET-mip/flaA二联原核表达载体,并高效表达。
Objective To select the genes of mip and flaA epitopes of Legionella pneumophila and construct the mip / flaA binary dominant epitope fusion gene expression vector and express in prokaryotic system to provide a preliminary experiment for producing Legionella pneumophila protein vaccine basis. Methods Bioinformatics methods were used to analyze the secondary structure and surface properties of Mip and FlaA proteins such as physicochemical properties, hydrophilicity, plasticity, antigenic index and extracellular region. It is suggested that the regions where the active epitopes may exist are dominant Epitope region. The pET-mip, pET-flaA and pET-mip / flaA dominance gene fusion expression vectors were constructed by PCR amplification and T4 ligase, and induced to express in E. coli. Results There were many potential epitopes in Mip and FlaA. The dominant epitopes of Mip and FlaA were successfully cloned and expressed, and the mip / flaA dual dominant epitope fusion protein was successfully expressed. Conclusion DNA Star software and Expasy online protein analysis system can successfully predict the epitopes of MpI and FlaA antigens of Legionella pneumophila. The pET-mip / flaA binary prokaryotic expression vector was successfully constructed and expressed efficiently.