| 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].Chinese journal of Epidemiology,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 |
| Received:July 14, 2005 Revised:July 14, 2005 |
| DOI： |
| KeyWord: 甲型副伤寒杆菌 spaO基因 原核表达 免疫原性/免疫保护 |
| English Key Word: Salmonella paratyphi A spaO gene Prokaryotic expression Immunogenicity/immunoprotection |
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| Abstract: |
| 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。 |
| English Abstract: |
| 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. |
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