| 李明雷,方海俊,范兴丽,张佳,严杰,孙爱华.甲型副伤寒杆菌外膜蛋白X基因分布及其重组表达产物免疫保护作用的研究[J].Chinese journal of Epidemiology,2013,34(12):1219-1222 |
| 甲型副伤寒杆菌外膜蛋白X基因分布及其重组表达产物免疫保护作用的研究 |
| Distribution of Salmonella paratyphi A outer membrane protein X gene and immune-protectiveeffect related to iIs recombinant expressed products |
| Received:July 06, 2013 |
| DOI：10.3760/cmaj.issn.0254-6450.2013.012.015 |
| KeyWord: 甲型副伤寒杆菌 外膜蛋白x基因 免疫原性 |
| English Key Word: Salmonella paratyphi A Outer membrane protein X gene Immunogenicity |
| FundProject:浙江省卫生高层次创新人才培养工程项目([2012]241);医药卫生科学研究基金项目(2011KYB006) |
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| Abstract: |
| 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Westem 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。御x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.o%。rOmP>X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嵋和200雌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l：10～l：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。 |
| English Abstract: |
| 0bjecfive To determine the distribution and sequence conservation of outermembrane protein X(ompX)gene in Salmonella paratyphi A isolates as well as the immunogenicityand irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi Aisolates was detected by PCR and the amplification products were sequenced after the T-A cloningprocess.OmpX gene product was expressed with E eoli expression system and the expressed rOrnpXwas extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer weTeapplied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity ofrOmpX were detected by immune.diffusion test.ELISA and Westem blot assay.The immuneprotectiveeffect of rOmpX against infection of Salmonella paratyphi in mice was determined and theagglutinative 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 aminoacid sequence identity(99.2%-100.0%or 98.4%-100.0%).Ⅵmell rOmpX was induced to rabbits toproduce high leveI antibody and combined with antiserum against whole eell of Salmonella paraophiA，the results displayed a positive Western hybridization signal.Results from ELlSA demonstrated that95.6%(65/68)of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100¨g or 200 lag rOmpX displayed an immune-protective rate of 93.3%(14/15)or 100.O%(15/15).Sera from thoserOmpX-immunized mice provided 1：10-1：40agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.ConclusionThe recombinant expression product of ompX gene could be used as a candidate antigen for developinggenetic engineering vaccines against Salmonella paratyphi A infection， |
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