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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。
Objective To study the immunogenicity and protective effect of SPAO gene recombinant SPAO gene and to understand the carrying and expression of spaO gene of clinical strains of Salmonella paratyphi A. Methods The SPAO gene of JH01 strain was amplified and cloned, and the prokaryotic expression system was constructed. The expression of rSpaO was analyzed by SDS-PAGE and rSpaO by Ni-NTA affinity chromatography. The immunoreactivity was identified by Western blot. The PCR-ELISA and ELISA were used to detect the frequency of 98 strains of spa Parasiticid paratyphoid bacilli, respectively. The immunoprotective effect of rSpaO on Paramyxobacteriophage 50001 infected mice was observed. Results The nucleotide and amino acid sequence homologies of cloned spaO genes were 99.45% -99.89% and 99.01% -100%, respectively, compared with the reported sequences. The expression level of rSpaO was 75 (P <0.01) .Antibacillus paratyphi A bacterial antisera could recognize rSpaO; rSpaO immunized rabbits could produce antibodies; 94.9% (93/98) clinical isolates of Salmonella paratyphi A contained spaO gene, 91.4% / 93) spaO + strain expresses SpaO. Survival rates of rSpaO-fed or subcutaneously injected mice (500μg) were 58.3% and 50.0%, respectively, when challenged with Salmonella paratyphi A strain 50001; the viability increased by 88.3% And 75.0%. Conclusion The clinical strains of paratyphoid A paratycheides have high spaO gene carrier rate and expression rate; rSpaO has good immunogenicity and certain immunoprotective effect, and can be used as a candidate antigen of Paragonimica paratyphi genetic engineering vaccine.