嗜肺军团菌ip基因的克隆及其原核表达_临床医学论文.docVIP

嗜肺军团菌ip基因的克隆及其原核表达_临床医学论文 嗜肺军团菌ip基因的克隆及其原核表达_临床医学论文 【摘要】 　　目的 克隆嗜肺军团菌主要免疫原蛋白ip 基因, 构建重组质粒pET-ip,并在原核系统中表达。方法 采用聚合酶链反应(PCR)， 从嗜肺军团菌基因组DNA 中扩增军团菌主要免疫原蛋白ip 基因 ,并将其定向克隆至原核表达载体pET-32a(+),构建原核表达重组质粒pET- ip,经限制性内切酶鉴定、PCR及测序分析后,转化宿主菌大肠杆菌BL21, IPTG诱导表达,产物进行SDS电泳、免疫印迹分析鉴定。结果 扩增出了792 bp 完整的ip 基因,构建的原核表达重组质粒pET- ip 表达出49 kDa Trx-IP的融合蛋白质，Western证实获得蛋白为所需要的目的蛋白。结论 成功构建了军团菌ip 基因的原核表达载体, 并在大肠杆菌中得到了高效表达。 【关键词】 军团菌;ip基因;克隆;基因表达 　　Abstract: Objective To clone the ip gene of Legionella pneumophila, to construct recombinant plasmid and to detect its expression in prokaryotic cell. Methods The ip gene was amplified from the genomic DNA of Legionella pneumophila with PCR, and then was inserted into the prokaryotic expression vector pET32a(+).The prokaryotic expression recombinant plasmid pETip was constructed.After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, the E. coli BL21 containing the recombinant plasmid pETip was induced with IPTG. The expression of ip was subsequently detected by SDSpolyacrylamine gel electrophoresis and Westernblot analysis. Results The ip gene of 792 bp long was amplified and the recombinant plasmid pETip was constructed. SDSPAGE analysis showed that the Trx IP fusion protein of approximately 49 kDa in size was expressed. The expressed proteins could be confirmed by western blot. Conclusion The prokaryotic expression recombinant plasmid pETip was constructed successfully and its expressed efficiently in E. coli. 　　Key words：legionella pneumophila；ip gene；clone； gene expression 　　嗜肺军团菌是引起军团病的主要病原体，通过空气传播，进入呼吸道的病原体在肺泡上皮细胞和单核巨噬细胞内增殖而发病。随着人们生活水平的提高，其危害性日益受到关注，目前尚无理想的防治措施，研制有效的军团菌疫苗是十分必要。嗜肺军团菌主要免疫原基因ip编码产生的29kDa外膜蛋白（29 kDa immunogenic protein，IP）［1］，为嗜肺军团菌1～10血清型所共有，具有种特异性，是机体免疫应答反应的主要免疫原。生物信息学分析显示其含有丰富的B细胞、T细胞表位，具有高度保守性，故选ip 基因为构建DNA疫苗抗原候选基因。本研究通过PCR扩增获得嗜肺军团菌ip基因，将其定向克隆入原核克隆载体pET-32a(+)，构建重组质粒pET-ip，经鉴定后,并使ip基因在原核系统中得到表达，为进一步研究IP蛋白的结构、功能及其免疫原性和保护性功能等奠定基础。 　　1 材料和方法 　　1.1 材料 嗜肺军团菌L1标准株及相应兔血清抗体由中国疾病预防控制中心传染病控制所惠赠