title: [Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice].
abstract: OBJECTIVE
  To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.
  
  
  METHODS
  As model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining.
  
  
  RESULTS
  Compared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance.
  
  
  CONCLUSIONS
  Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.
authors.affiliation: Shanghai Public Health Clinical Center Affiliated to Fudan University, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.forename: Xiao-yong
authors.initials: XY
authors.lastname: Fan
authors.forename: Qing
authors.initials: Q
authors.lastname: Qu
authors.forename: Yu-ping
authors.initials: YP
authors.lastname: Luo
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Lowrie
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D018834:Chaperonin 60 / Q000235:genetics / Q000276:immunology*; D005260:Female; D005822:Genetic Vectors; D007438:Introns / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D019444:Vaccines, DNA; C074119:heat-shock protein 65, Mycobacterium