Prokaryotic expression of Salmonella paratyphi A nmpC gene and immunoprotective effect of the expressed product
10.3760/cma.j.issn.0254-5101.2010.12.012
- VernacularTitle:甲型副伤寒杆菌nmpC基因原核表达及表达产物免疫保护作用
- Author:
Ying WU
;
Yanfang WANG
;
Jie YAN
;
Ping RUAN
- Publication Type:Journal Article
- Keywords:
Salmonella paratyphi A;
nmpC gene;
Recombinant expression;
Immunogenicity;
Immunoprotection
- From:
Chinese Journal of Microbiology and Immunology
2010;30(12):1118-1123
- CountryChina
- Language:Chinese
-
Abstract:
Objective To generate a prokaryotic expression system of Salmonella paratyphi A nmpC gene that encoding an outer membrane protein(OMP),and to determine immunogenicity and immonuprotection of the recombinant expressed product rNmpC and carrying and expression frequencies of the nmpC genes in isolates of S.paratyphi A.Methods A nmpC gene clone was obtained from a clinical S.paratyphi A strain JH01 by PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Bio-Rad Agarose Image Pattern Analysis System were applied to examine the expression and yield of rNmpC.Antigenicity and immunoreactivity of rNmpC were determined by immunodiffusion test,Western blot assay and micro-Widal's test.The carrying and expression rates of nmpC genes in 98 S.paratyphi A isolates were detected by PCR and ELISA.By a mouse infection model,the immunoprotective effect of rNmpC against the lethal challenge of S.paratyphi A was determined.Results All the cloned nmpC genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The expression yield of rNmpC was approximately 30% of the total bacterial proteins.rNmpC could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains have nmpC gene as well as express NmpC protein,but no nmpC gene could be detectable in S.typhi,S paratyphi B and S paratyphi C.Immunization with 100 μg and 200 μg rNmpC contributed 41.7%(5/12) and 66.7%(8/12) immunoprotective rates in mice,respectively.The sera from rNmpC immunized mice and survival mice in the NmpC is an unique OMP antigen of S.paratyphi A with conserved sequence,extensive distribution and natural expression.This OMP can be used as one the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A based on its fine immunogenicity and certain immunoprotection.
