Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of andrographolide on the expression of eosinophil granulocytes and possible mechanisms / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Andrographolide, the active component in andrographis paniculata, has potent anti-inflammatory actions. This study aimed to evaluate the effects of andrographolide on eosinophil granulocytes (EOS) and the expression of eotaxin and IL-5 in mice with asthma.</p><p><b>METHODS</b>BALB/c mice were randomly assigned into normal control, asthma, budesonide treatment and andrographolide treatment groups (n=8 each). Mice in the latter three groups were sensitized and challenged with ovalbumin (OVA) to induce asthma. ELISA was used to detect the concentrations of eotaxin and IL-5 in bronchoalveolar lavage fluid (BALF) and peripheral blood. The expression of eotaxin mRNA and IL-5 mRNA in lung tissues was detected by real-time quantitative PCR.</p><p><b>RESULTS</b>Andrographolide treatment significantly decreased EOS count in BALF (P<0.05) and the effect of andrographolide was better than the effect of budesonide. Andrographolide treatment significantly down-regulated the expression of eotaxin and IL-5 in BALF, lung eotaxin mRNA expression and blood IL-5 expression (P<0.05), but the effects of andrographolide were poorer than the effects of budesonide. Andrographolide treatment resulted in a decrease in blood eotaxin expression and lung IL-5 mRNA expression and the effects of andrographolide were similar to budesonide.</p><p><b>CONCLUSIONS</b>Andrographolide can down-regulate the expression of IL-5 and eotaxin and thus suppress the inflitration of EOS in a mouse model of asthma.</p>

Improvement of immunogenicity of Streptococcus pneumoniae psaA DNA vaccine by prime and boost strategy / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study on the preparation of Streptococcus pneumoniae psaA DNA vaccine and to analyse the immunogenicity by the prime-boost strategy.</p><p><b>METHODS</b>The psaA gene was amplified from the genome of Streptococcus pneumoniae by PCR, and then was inserted into plasmid pVAX1 and pET28a to construct recombinant expression vectors respectively. 293T cells were transiently transfected with pVAX1-psaA, and RT-PCR analysis of total cell RNA extracts showed successful expression of psaA. BALB/c mices (n = 5) were intramuscularly injected with 100 microg psaA DNA vaccine for three times, and then boosted with 50 microg recombinant PsaA protein. The antibody response against PsaA was measured by ELISA.</p><p><b>RESULTS</b>The psaA gene was amplified and subcloned successfully. The constructed psaA DNA vaccine was confirmed by DNA sequencing, and the recombinant PsaA protein was purified by the one-step Ni(2+) affinity chromatography. Expression of the PsaA was observed in cells transfected with pVAX1-psaA. The animal experiment results showed that the anti-PsaA level of the DNA prime-protein boosting mice was higher significantly than the other groups (t = 87.518, P < 0.05).</p><p><b>CONCLUSION</b>The psaA DNA vaccine was prepared successfully, and the immunogenicity of Streptococcus pneumoniae psaA DNA vaccine could be improved significantly by the DNA prime and protein boost strategy.</p>