目的 选取嗜肺军团菌mip/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。
英文摘要:
Objective To generate and express fusion vector withmip/flaAadvantages epitope genes of Legionella pneumophila by select mip and flaAadvantages epitope genes for future research onLegionella pneumophilaprotein vaccine. Methods Following analysis of secondary structure and surface properties such as: physical and chemical properties, hydropathy, plasticity, antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods, the region which active epitope may exist was selected as advantages epitope region. Then, the recombinant plasmid pET-mip, pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification andT4ligase connection, and induced the expression in E.coli. Results Many potential antigenic epitopes in Mip and FlaA were identified, and the selected advantages epitope regions were cloned and expressed successfully. Moreover, themip/flaAtwo advantages associated epitope fusion proteins were also successfully expressed. Conclusion DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes forLegionella pneumophila Mip and FlaA. And prokaryotic expression vector pET-mip/flaAwith advantages epitope genes has been successfully constructed and efficiently expressed.
