文章摘要
毛亚飞,林晓骥,李晶,阮萍,周晓辉,严杰.甲型副伤寒杆菌侵袭蛋白(spaO)基因原核表达及免疫性和保护作用研究[J].中华流行病学杂志,2006,27(4):347-350
甲型副伤寒杆菌侵袭蛋白(spaO)基因原核表达及免疫性和保护作用研究
Construction of prokaryotic expression system of Salmonella paratyphi A spaO gene and immunogenicity and immunoprotection of the expressed product
收稿日期:2005-07-14  出版日期:2014-10-17
DOI:
中文关键词: 甲型副伤寒杆菌  spaO基因  原核表达  免疫原性/免疫保护
英文关键词: Salmonella paratyphi A  spaO gene  Prokaryotic expression  Immunogenicity/immunoprotection
基金项目:
作者单位E-mail
毛亚飞 浙江大学医学院病原生物学教研室  
林晓骥 浙江大学城市学院  
李晶 浙江大学城市学院  
阮萍 浙江绍兴文理学院医学院病原生物学教研室  
周晓辉 浙江大学医学院病原生物学教研室  
严杰 浙江大学医学院病原生物学教研室 Med_bp@zju.edu.cn 
摘要点击次数: 2078
全文下载次数: 0
中文摘要:
      目的研究甲型副伤寒杆菌spaO基因重组表达产物rSpaO免疫性和保护作用,并了解甲型副伤寒杆菌临床菌株spaO基因携带和表达情况。方法扩增并克隆甲型副伤寒杆菌临床株 JH01 spaO基因,构建原核表达系统;采用SDS-PAGE分析rSpaO表达情况,Ni-NTA亲和层析法收集 rSpaO,采用Western blot鉴定其免疫反应性;采用PCR和ELISA检测98株甲型副伤寒杆菌临床菌株 spaO基因携带和表达频率;观察rSpaO对甲型副伤寒杆菌50001株感染小鼠的免疫保护作用。结果与报道的相关序列比较,克隆的spaO基因核苷酸和氨基酸序列同源性分别为99.45%-99.89%和 99.01%-100%;rSpaO表达量为细菌总蛋白的75%左右;甲型副伤寒杆菌全菌抗血清能识别rSpaO, rSpaO免疫家兔可产生抗体;94.9%(93/98)甲型副伤寒杆菌临床菌株含有spaO基因,91.4%(85/93) spaO+菌株表达SpaO。rSpaO灌喂或皮下注射免疫小鼠(500μg)受甲型副伤寒杆菌50001株攻击后的存活率分别为58.3%和50.0%;若加入5μg rLTB,存活率分别上升88.3%和75.0%。结论甲型副伤寒杆菌临床菌株有较高的spaO基因携带率和表达率;rSpaO有良好的免疫原性和一定的免疫保护作用,可作为甲型副伤寒杆菌基因工程疫苗的候选抗原。
英文摘要:
      Objective To study the immunogenicity and immunoprotection of the recombinant expressing product(rSpaO) of S. paratyphi A spaO gene, and to demonstrate the frequencies of spaO gene carrying and expressing in S. paratyphi A isolates. Methods The spaO gene of a clinical S. paratyphi A strain JH01 was amplified and then cloned. After sequencing of the cloned spaO gene, a prokaryotic expression system of the gene was constructed. SDS-PAGE were applied to examine the rSpaO expression. Ni-NTA affinity chromatography was performed to collect rSpaO. Immunogenicity of rSpaO was determined by Western blot assay. A PCR assay and an ELISA were established to respectively detect the carrying and expressing frequencies of the spaO genes in 98 S. paratyphi A isolates. The immunoprotective effects of rSpaO in S. paratyphi A strain 50001 infected mice were observed. Results In comparison with the reported corresponding sequences, the nucleotide and putative amino acid sequence homologies of the cloned spaO gene were 99.45%-99.89% and 99.01%-100%, respectively. The expression output of rSpaO was approximately 75% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rSpaO. rSpaO could efficiently induce rabbits to produce specific antibody. 94.9% (93/98) of the S. paratyphi A isolates had spaO gene and 91.4% (85/93) of the spaO + strains could express SpaO. 58.3% and 50.0 % of the mice that oral-taken or subcutaneous injected with 500 μg of rSpaO for immunization were survival after challenged by lethal dose of S. paratyphi A strain 50001. When co-immunized with 5 μg rLTB, the survival rates of the mice increased to 88. 3% and 75.0%, respectively. Conclusion The S. paratyphi isolates had relatively high carrying and expressing frequencies-of spaO gene. rSpaO showed a fine immunogenicity and a certain immunoprotective effect, which could be used as an antigen candidate for developing genetic engineering vaccine of S. paratyphi.
查看全文   Html全文     查看/发表评论  下载PDF阅读器
关闭