The Clone, Prokaryotic Expression And Function Study Of Human IL-16

Human 1L-16 gene encodes a 631aa pro-protein, after cleavage by caspase-3, the released C-terminal 121 a.a. peptide is its biological functional fragment. IL-16 can induce the chemo action, activation and proliferation of CD4+ T cells. As a new cytokine, its feature keeps unknown. And the natural concentration of IL-16 is pretty low in blood, only about 10-9M; it is unfeasible to purified IL-16 from peripheral blood. To break through the restriction, prokaryotic expression and harvest rich, is necessary for further research.The human IL-16 biological active fragment (783-1187bp) was obtained by RT-PCR from PBMCs, and was cloned into pBluescriptSK (+) vector. After the sequence was confirmed, it was cloned into the prokaryotic expression vector pT7-7His. The expressed product issoluble, MW 18kD, which was purified by Ni+ metal chelate affinity chromatography. Its antigen activity was assayed by Western blotting, and its ability to induce the proliferation of CD4+ cell was confirmed by FACS assay.Both of the eukaryotic and prokaryotic expression IL-16 was assayed the ability to inhibit HIV-1 promoter. The CAT report gene can measure the expression level of HIV-1 promoter. The results show that, both IL-16, can inhibit the HIV-1 promoter, but not dominate.As a cytokine to increases proliferation, its high concentration (10-5 M) can induce apoptosis in Jurkat cells. The cell count, MTT assay and FACS results all confirm the fact.34We further investigate the apoptosis related gene. Using different concentration of IL-16 treat Jurkat cells in 1640 without serum, The expression level of Bid and c-Myc increase together with the increase IL-16 concentration. These suggest that Bid and c-Myc may take a part in the apoptosis induced by high concentration of IL-16. In the contl'ast, FasL doesn't increase but decrease with the 100 fold gradually increasing of IL-16 concentration.Several signal transduction pathway was triggered the binding of IL-16 and CD4. So we study for several signal pathways and assay 5 molecular in them. They are PKC, PKA, MEK1 in ERK pathway, MEKK1 in SAPK/JNK, p38 in p38 pathway, the three latter pathways all belong to the MAP kiiiase pathway.The inhibition of PKC decreases the proliferation after the IL-16 treatments, Which hint its role in proliferation signal transduction. The inhibition of PKA has different effect. Its regulation pathway still keeps unknown.After the addition of PD98,059, which is high specific inhibitor of MEK1, the cell proliferation increased much high in every concentration rhIL-16 treatment. We deduced that MEK1 pass down an apoptosis signal in the experiment system.MEKK1 expression level was assayed by RT-PCR, After IL-16 treat target cells for 24 hours, all treatment has me equal quantity expression of MEKK1, including the control; while, when the time up to 48 hours, the induction effect eliminate.p38 is another member of MAPK family. It has no effect on the IL-16 induced Jurkat cell death.