title: [Construction of prokaryotic expression system of Salmonella paratyphi A spaO gene and immunogenicity and immunoprotection of the expressed product].
abstract: OBJECTIVE
  To study the immunogenicity and immunoprotection of the recombinant expressing product (rSpaO) of S. paratyphi A spaO gene, and to demonstrate the frequencies of spaO gene carrying and expressing in S. paratyphi A isolates.
  
  
  METHODS
  The spaO gene of a clinical S. paratyphi A strain JH01 was amplified and then cloned. After sequencing of the cloned spaO gene, a prokaryotic expression system of the gene was constructed. SDS-PAGE were applied to examine the rSpaO expression. Ni-NTA affinity chromatography was performed to collect rSpaO. Immunogenicity of rSpaO was determined by Western blot assay. A PCR assay and an ELISA were established to respectively detect the carrying and expressing frequencies of the spaO genes in 98 S. paratyphi A isolates. The immunoprotective effects of rSpaO in S. paratyphi A strain 50001 infected mice were observed.
  
  
  RESULTS
  In comparison with the reported corresponding sequences, the nucleotide and putative amino acid sequence homologies of the cloned spaO gene were 99.45%-99.89% and 99.01%-100%, respectively. The expression output of rSpaO was approximately 75% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rSpaO. rSpaO could efficiently induce rabbits to produce specific antibody. 94.9% (93/98) of the S. paratyphi A isolates had spaO gene and 91.4% (85/93) of the spaO+ strains could express SpaO. 58.3% and 50.0% of the mice that oral-taken or subcutaneous injected with 500 microg of rSpaO for immunization were survival after challenged by lethal dose of S. paratyphi A strain 50001. When co-immunized with 5 microg rLTB, the survival rates of the mice increased to 88.3% and 75.0%, respectively.
  
  
  CONCLUSIONS
  The S. paratyphi isolates had relatively high carrying and expressing frequencies of spaO gene. rSpaO showed a fine immunogenicity and a certain immunoprotective effect, which could be used as an antigen candidate for developing genetic engineering vaccine of S. paratyphi.
authors.affiliation: Department of Medical Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 310031, China.
authors.forename: Ya-fei
authors.initials: YF
authors.lastname: Mao
authors.forename: Xiao-ji
authors.initials: XJ
authors.lastname: Lin
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Ruan
authors.forename: Xiao-hui
authors.initials: XH
authors.lastname: Zhou
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
mesh_terms: D000818:Animals; D000917:Antibody Formation; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D022562:Salmonella Vaccines / Q000276:immunology; D012482:Salmonella paratyphi A / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D022562:Salmonella Vaccines; C101106:SpaO protein, Salmonella sp.