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-protective effect related to its recombinant expressed products

作　　者：李明雷[1] 方海俊[1] 范兴丽[2] 张佳[2,3] 严杰[4] 孙爱华[2,3] 

机构地区：[1]浙江省义乌市中心医院,322000 [2]浙江医学高等专科学校 [3]温州医学院检验医学院和生命科学学院 [4]浙江大学医学院病原生物学系

出　　处：《中华流行病学杂志》2013年第12期1219-1222,共4页Chinese Journal of Epidemiology

基　　金：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）

摘　　要：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Westemblot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。100μg和200ggrOmpX对感染小鼠的免疫保护率分别为93．3％（14／15）和100．O％（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。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 immono-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. eoli 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 immune- protective 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 IX g 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.

关 键 词：甲型副伤寒杆菌 外膜蛋白X基因 免疫原性 

分 类 号：R378.22[医药卫生—病原生物学]