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To design and construct an HIV-1 subunit vaccine containing the N-terminal heptad repeat (NHR) domain N51 in gp41 of the HIV-1 CRF01_AE recombinant subtype in China and study its immunogenicity.Two pairs of primers were designed to amplify DNA fragment encoding N51Fd gene, which was then subcloned into pFUSE-hIgG1-Fc2 vector. The recombinant plasmid pFUSE/N51Fd was confirmed by DNA sequencing. Western blotting was used to measure the correct expression of the recombinant protein N51FdFc-AE. The N51FdFc-AE protein was used to immunize BALB/c mice, and the specific antibody response was measured by ELISA.A recombinant plasmid pFUSE/N51Fd was successfully constructed and the N51FdFc-AE recombinant protein was expressed effectively in 293T cells. The purified N51FdFc-AE recombinant protein (35 kD) was detected by Western blotting with rabbit anti-gp41 N/C peptide antibodies. The mouse anti-sera could specifically recognize the antigens derived from gp41 NHR. The titers of the specific antibodies were up to 1:102 400 with an average titer of 1:51 200.The recombinant N51FdFc-AE protein can effectively induce the gp41 NHR-specific immune responses in BALB/c mice and could be used as an immunogen for design of HIV-1 subunit vaccine.

To investigate whether the HIV-1 gp41 fusion peptide (FP) could affect the regulatory T cell (Treg) function activated by CD3 antibody.Murine CD4(+);CD25(+); Treg and CD4(+);CD25(-); effector T cells (Teff) were isolated from mice spleens by the immune magnetic beads. The splenocytes were treated with mitomycin C to obtain antigen presenting cells (APCs). CCK-8 assay and CFSE loading were employed to evaluate the effects of FP on the Treg inhibitory function via measuring Teff proliferation activated by CD3 antibody. In addition, IL-10 secretion of activated Treg was detected by ELISA. The distribution of FP and TCR on Treg surface was observed by laser scanning confocal microscope.Through the CCK-8 assay and flow cytometry, we found that Teff cells have significant proliferation stimulated by the CD3 antibodies. When Treg and Teff were co-cultured, the proliferation of Teff was significantly inhibited by Treg. When added with 25 μg/mL FP, the proliferation of Treg+Teff group and Teff group was not significantly affected, but when 5 μg/mL FP was added, the proliferation rate of Treg+Teff group was significantly lower than that of Teff group. IL-10 secretion was low when Treg were not activated, but it significantly increased by CD3 antibodies stimulation. When the concentration of FP was 25 μg/mL, IL-10 level significantly decreased, but 5 μg/mL FP did not significantly influence IL-10 secretion. Through the laser scanning confocal microscope, we found that T cell receptors (TCR) of non-activated Treg showed uniform distribution on the cell surface, and that FP and TCR had no common distribution. When Treg were stimulated by CD3 antibodies, the activated TCR formed half crescent, and FP and the activated TCR had common distribution on cell surface.Treg inhibitory function is significantly inhibited by 25 μg/mL FP in vitro, but 5 μg/mL FP does not affect it. This may be due to the suppression of IL-10 secretion and the influence of the signaling cross-talk between APC and TCR.

To detect the serum anti-nuclear antibody (ANA) and interleukin-21 (IL-21) levels in patients infected with hepatitis C virus (HCV), and study their impact on the liver function of HCV patients.Enzyme-linked immunosorbent(ELISA) assays were used to detect the serum ANA and IL-21 levels in 96 cases of HCV-IgG positive sera, as well as 30 cases of HCV-IgG negative sera. Hitachi 7600 biochemical analyzer was used to test the ALT, AST and ALB of all sera samples.HCV patients have abnormal liver function significantly and their average IL-21 level was much lower than the control group. While the liver function of most ANA positive HCV patients was well-balanced. The IL-21 level of HCV patients with ALTor = 40 U/L or ALT40 U/L were markedly reduced.The production of ANA and IL-21 do help to control the progress of hepatitis and reduce the damage to liver cells in HCV patients.

To study the effects of ConA on the early activation and the immunosuppressive function of CD4+ CD25+ regulatory T cells.CD4+ CD25+ regulatory T cells and CD4+ CD25- effector T cells of BALB/c mice were separated by MACS immunomagnetic beads. After twelve hours of stimulation with different concentrations of ConA, the cells were collected and the percentage of CD69 expression was analyzed by flow cytometry. The CD4+ CD25- effector T cells were stained by CFSE and co-cultured with the CD4+ CD25+ regulatory T cells in a ratio of 2:1. After stimulation with ConA for 3 days, the proliferation of CD4+ CD25- effector T cells suppressed by CD4+ CD25+ regulatory T cells was analyzed by flow cytometry.ConA dose-dependently increased the percentage of CD69 expression of both CD4+ CD25+ regulatory T cells and CD4+ CD25- effector T cells, and enhanced the expression of CD69 on these two groups of cells similarly. ConA also dose-dependently activated the suppressive function of CD4+ CD25+ regulatory T cells to inhibit the proliferation of CD4+ CD25- effector T cells.ConA enhances the activation and the function of CD4+ CD25+ regulatory T cells in vitro. Considering that ConA can mimic the antigen to some extent, this system can be used to evaluate the drug influence on the activation and the function of the regulatory T cells.