[Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice].

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 pVAXlhsp65 caused higher expression level of Hsp65 in 293T cells,and enhanced Thl 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.Conclusion 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. Key words: Introns;  Vaccines,DNA;  Immunity;  Heat-shock proteins