ABSTRACT
Objective: To discuss the effect of Gandou decoction (GDD) on the immune index of spleen in TX mice of Wilson's disease model. Method: The mice were divided into normal group, model group and GDD or tetrathiomolybdate(TM)treatment group, with 20 mice in each group. Each group was fed in various ways for 30 successive days. Normal group:10 normal DL mice were randomly selected and feed normally. Model group:20 TX mice were randomly selected and feed with 2 mL·kg-1·d-1ig saline by gavage twice per day. GDD or TM treatment group:80 TX mice were randomly selected and feed with 2 mL·kg-1·d-1 ig Gandou decoction 22,44,66 g·kg-1 or tetrathiomolybdate by gavage twice per day. ICP-MS was used to compare the expressions of trace elements inside the mice's spleens, flow cytometry was applied to detect the mice T lymphocyte subsets of splenic tissue CD4+, CD8+, CD4+/CD8+, and Western blot was used to detect the expressions of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-17 (IL-17) and interleukin-18 (IL-18). Result: Flow ICP-MS results showed that GDD can reduce Cu of mice's spleen, flow cytometry results showed that CD4+and CD8+in model group were increased than those in normal group (P+/CD8+was decreased (P+and CD8+in middle and high-dose GDD groups were decreased (P+/CD8+was increased. According to Western blot detection, compared with normal group, the expressions of IL-2, IL-8, IL-17, IL-18, TNF-α and IFN-γ in the model group were increased (Pα, IFN-γ, IL-2, IL-8, IL-17 and IL-18 in the GDD middle and high or TM group were decreased (PPα in the GDD low were decreased (PConclusion: Spleen of TX mice shows the cellular immunity hyperfunction, which is mainly dominated by the negative immunoloregulation. GDD has a certain effect in regulating cellular immunity hyperfunctional state of TX mice, but it's difficult to thoroughly change the negative immune regulation.
ABSTRACT
<p><b>OBJECTIVE</b>To express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen.</p><p><b>METHODS</b>The Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%.</p><p><b>RESULTS</b>Western-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual.</p><p><b>CONCLUSION</b>Gag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.</p>