CAJ | 학술논문

目的了解甲型副伤寒沙门菌临床菌株外膜蛋白(ompX)基因分布、序列保守性及其产物免疫原性和免疫保护性.方法采用PCR扩增甲型副伤寒沙门菌临床菌株ompX基因.利用大肠埃希菌表达系统表达rOmpX,产物采用Ni-NTA亲和层析法提纯.采用免疫扩散法、ELISA和Western blot鉴定rOmpX抗原性和免疫反应性.采用小鼠感染模型了解rOmpX对甲型副伤寒沙门菌感染的免疫保护作用,微量肥达试验检测rOmpX免疫小鼠血清抗体凝集伤寒和副伤寒沙门菌效价.结果所有甲型副伤寒沙门菌临床菌株均有ompX基因,其核苷酸和氨基酸序列相似性分别高达99.2％～ 100.0％和98.4％～100.0％.rOmpX免疫家兔可产生高效价抗体,利用该抗体与甲型副伤寒患者血清标本进行ELISA试验,95.6％(65/68)的标本呈阳性.100 μg和200 μug rOmpX对感染小鼠的免疫保护率分别为93.3％(14/15)和100.0％(15/15).rOmpX免疫小鼠血清对甲型副伤寒和伤寒沙门菌H抗原凝集效价为1∶10～1∶40.结论 ompX基因重组表达产物可作为甲型副伤寒沙门菌基因工程疫苗候选抗原.
목적 료해갑형부상한사문균림상균주외막단백(ompX)기인분포、서렬보수성급기산물면역원성화면역보호성.방법 채용PCR확증갑형부상한사문균림상균주ompX기인.이용대장애희균표체계통표체rOmpX,산물채용Ni-NTA친화층석법제순.채용면역확산법、ELISA화Western blot감정rOmpX항원성화면역반응성.채용소서감염모형료해rOmpX대갑형부상한사문균감염적면역보호작용,미량비체시험검측rOmpX면역소서혈청항체응집상한화부상한사문균효개.결과 소유갑형부상한사문균림상균주균유ompX기인,기핵감산화안기산서렬상사성분별고체99.2％～ 100.0％화98.4％～100.0％.rOmpX면역가토가산생고효개항체,이용해항체여갑형부상한환자혈청표본진행ELISA시험,95.6％(65/68)적표본정양성.100 μg화200 μug rOmpX대감염소서적면역보호솔분별위93.3％(14/15)화100.0％(15/15).rOmpX면역소서혈청대갑형부상한화상한사문균H항원응집효개위1∶10～1∶40.결론 ompX기인중조표체산물가작위갑형부상한사문균기인공정역묘후선항원.
Objective To determine the distribution and sequence conservation of outer membrane protein X (ompX) gene in Salmonella paratyphi A isolates as well as the immunogenicity and irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity of rOmpX were detected by immune-diffusion test,ELISA and Western blot assay.The immuneprotective effect of rOmpX against infection of Salmonella paratyphi in mice was determined and the agglutinative titers of sera from rOmpX-immunized mice was measured by micro-Widal' s test.Results All the tested Salmonella paratyphi A isolates had ompX gene with high nucleotide or amino acid sequence identity (99.2％-100.0％ or 98.4％-100.0％).When rOmpX was induced to rabbits to produce high level antibody and combined with antiserum against whole cell of Salmonella paratyphi A,the results displayed a positive Western hybridization signal.Results from ELISA demonstrated that 95.6％ (65/68) of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100 μg or 200 μg rOmpX displayed an immune-protective rate of 93.3％ (14/15) or 100.0％ (15/15).Sera from those rOmpX-immunized mice provided 1 ∶ 10-1 ∶ 40 agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.Conclusion The recombinant expression product of ompX gene could be used as a candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A infection.