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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (2): 171-176    DOI: 10.3785/j.issn.1008-9292.2013.02.007
    
Distribution of Salmonella paratyphi A pagC gene and immunoprotective effect of its recombinant expressed products
ZHANG Jia1,2, FAN Xin-Li2, GE Yu-Mei3, YAN Jie3, SUN Ai-Hua1,2
1.School of Laboratory Medical Science and Life Science,Wenzhou Medical College,Wenzhou 325035,China; 2.Zhejiang Medical College,Hangzhou 310053,China; 3.Department of Medical Microbiology and Parasitology,Zhejiang University School of Medicine,Hangzhou 310058,China
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Abstract  
Objective: To determine the distribution and sequence conservation of pagC gene in Salmonella paratyphi A isolates,and the immunogenicity and immonuprotection of its recombinant expression products(rPagC).
Methods: The distribution of pagC gene in Salmonella paratyphi A isolates and its sequence conservation were examined by PCR and sequencing.A prokaryotic expression system of pagC gene was constructed and the expressed rPagC was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rPagC.The antigenicity and immunoreactivity of rPagC were detected by immunodiffusion test,ELISA and Western Blot assay.The immunopretective effect of rPagC against infection of Salmonella paratyphi A in mice was determined,while the agglutinative effect of sera from rPagC-immunized mice was measured by micro-Widal′s test.
Results: All the Salmonella paratyphi A isolates tested had the pagC gene,the similarity of nucleotide and amino acid sequences was 99.1%-100% and 98.4%-100%,respectively.The constructed prokaryotic expression system expressed rPagC with high efficiency.The rPagC immunized rabbit produced a high level antibody and it also combined with antiserum against whole cell of S.paratyphi A to generate a positive Western hybridization signal.ELISA results indicated that 97.1%(66/68) paratyphoid patients infected with Salmonella paratyphi A were positive for rPagC antibody in their serum specimens.When mice were immunized with 100 μg or 200 μg rPagC,the immunoprotective rates were 73.3%(11/15) or 86.7%(13/15),respectively.The sera from rPagC-immunized mice offered 1∶10-1∶40 agglutination titers with the H antigens of Salmonella paratyphi A and Salmonella typhi.
Conclusion: PagC gene has an extensive distribution in Salmonella paratyphi A isolates.rPagC can be used as the candidate antigen in genetic engineering vaccine due to its fine immunogenicity and powerful immunoprotective effect.


Key wordsSalmonella paratyphi A/immunology      Salmonella paratyphi A/genetics      Gene expression regulation,bacterial      Recombination,genetic      Gene expression      pagC gene      Recombinant expression      Immunogenicity      Immunoprotection     
Received: 28 December 2012      Published: 25 March 2013
CLC:  R 378.24  
Corresponding Authors: 孙爱华(1968-),女,教授,主要从事病原菌致病与耐药机制及实验室诊断方法研究     E-mail: sunah123@126.com
Cite this article:

ZHANG Jia, FAN Xin-Li, GE Yu-Mei, YAN Jie, SUN Ai-Hua. Distribution of Salmonella paratyphi A pagC gene and immunoprotective effect of its recombinant expressed products. Journal of ZheJiang University(Medical Science), 2013, 42(2): 171-176.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.02.007     OR     http://www.zjujournals.com/med/Y2013/V42/I2/171


甲型副伤寒杆菌pagC基因分布及其重组表达产物免疫保护作用

目的:了解甲型副伤寒沙门菌临床菌株pagC基因携带率及序列保守性,确定甲型副伤寒沙门菌pagC基因原核重组表达产物rPagC免疫原性和保护作用。
方法:采用PCR及其产物测序了解甲型副伤寒沙门菌临床菌株pagC基因携带率及序列保守性。构建甲型副伤寒沙门菌pagC基因原核表达系统,Ni-NTA亲和层析法提纯表达的rPagC。采用SDS-PAGE和BioRad凝胶图象分析系统检测rPagC表达情况及其产量。采用免疫扩散法、ELISA和Western Blot鉴定其抗原性和免疫反应性。采用小鼠感染模型了解rPagC对甲型副伤寒杆菌感染的免疫保护效果,微量肥达试验检测rPagC免疫小鼠血清凝集伤寒和副伤寒沙门菌的效果。
结果:所有被检测的甲型副伤寒沙门菌临床菌株均携带pagC基因,其核苷酸和氨基酸序列相似性分别高达99.1%~100%和98.4%~100%。所构建的原核表达系统能高效表达rPagC。rPagC免疫家兔可产生高效价抗体并能与甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号。ELISA结果显示,97.1%(66/68)甲型副伤寒患者血清标本rPagC抗体阳性。100 μg和200 μg rPagC对感染小鼠的免疫保护率分别为73.3%(11/15)和86.7%(13/15)。rPagC免疫小鼠血清对甲型副伤寒沙门菌和伤寒沙门菌H抗原凝集效价高达1∶10~1∶40。
结论:pagC基因在甲型副伤寒沙门菌株中分布广泛。rPagC有良好的免疫原性及较强的免疫保护作用,可作为甲型副伤寒杆菌基因工程疫苗候选抗原。

关键词: 沙门菌,  甲型副伤寒/免疫学,  沙门菌,  甲型副伤寒/遗传学,  基因表达调控,  细菌,  重组,  遗传,  基因表达,  pagC基因,  重组表达,  免疫原性,  免疫保护性 

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