Valproic Acid Exerts Differential Effects On Activated Human Peripheral Lymphocytes And Expression And Identification Of Recombinant Human Interleukin 7 Product In Escherichia Coli

Aim:To investigate the effect of valproic acid (VPA) on the activation, apoptosis and cytokine syntheses of activated human peripheral lymphocytes. To construct a prokaryotic expression vector for human interleukin7 (IL-7), identify its expression product and optimize its expression condition.Methods:1. The expression levels of CD69 on T lymphocytes stimulated with phorbol 12,13-dibutyrate (PDB) and ionomycin (Ion) were evaluated with flow cytometry after staining with fluorescent monoclonal antibodies. Mitochondrial membrane potential was determined by flow cytometry using the probe DiOC6(3). Intracellular cytokine staining was used to detect cytokine syntheses, including IL-2, IL-4, IFN-y and TNF-α, within CD3+ T lymphocytes stimulated with PDB plus Ion in the presence of VPA.2. The cDNA fragment encoding human IL-7 was amplified by RT-PCR from the total RNA of human PBMC, and then the IL-7 gene was cloned into pET-3d plasmid to construct a prokaryotic expression vector. The positive clone was analyzed by DNA sequencing. The recombinant protein was expressed in Escherichia coli (E. coli) BL21(DE3) strain and identified with Western blotting.Results:1. Our results showed that VPA enhanced lymphocyte activation under low concentrations while inhibited at high doses (P<0.01). Treatment with VPA at 1 mmol/L or 5 mmol/L reduced the activation-induced cell death (AICD) while high dose of VPA promoted apoptosis of lymphocytes stimulated with PDB plus ionomycin. The ratio of IL-2+ cells was significantly enhanced by 1 mmol/L and 5 mmol/L VPA whereas 25 mmol/L VPA decreased the ratio of IL-2+ cells. However, VPA (from 1 mmol/L to 25 mmol/L) dose-dependently increased the proportion of IFN-γ+ and TNF-α+ cells. The effect of VPA on IL-4 synthesis was not so marked.2. The prokaryotic expression vector for IL-7 was constructed and confirmed by DNA sequencing. The expression vector was transformed into E. coli and was expressed after induction with IPTG. The relative molecular weight of the protein was 17500 daltons, which is consistent with theoretical value. Western blotting showed that the recombinant protein was mainly presented in inclusion bodies. The optimized expression condition was 4 h induction at 37℃with 0.4 mmol/L IPTG.Conclusion:1. VPA at low doses (1 mmol/L,5 mmol/L) promoted CD69 expression on activated lymphocytes whereas it turned to inhibit the expression of CD69 at high dose (25 mmol/L). In the mean time, VPA of low dose increased the mitochondrial membrane potential while high dose of VPA decreased it in activated lymphocytes. Furthermore, interleukin 2 (IL-2) synthesis was enhanced by low doses of VPA but inhibited by high dose. However, interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) synthesis were dose-dependently enhanced by VPA when compared with those of PDB plus Ion-treated cells.2. The prokaryotic expression vector for human IL-7 has been constructed successfully and the expression condition has been optimized, which has paved the way for further study on human IL-7.