Cloning And Expression Of Interleukin-24 Gene And Study On The Action Of RhIL-24 Induction THP-1 Cells Secretion Inflammatory Cytokines And Tumour Cells Apoptosis

Objective:In order to provide experimental basis for exploring tentative molecular machanism of human mature peptides interleukin-24 (IL-24) gene,the gene was amplified by RT-PCR and the recombinant plasmid pQE30/hIL-24 was constructed, and recombinant hIL-24 fusion protein (rhIL-24) was expressed and purified to study on the effect of rhIL-24 induction THP-1 cells secretion inflammatory cytokines including TNF-αand IL-6 , simultaneously THP-1 cells apoptosis.Methods:A pair of primers of human IL-24 gene were designed and the human IL-24 gene was amplified from health adult peripheral blood mononuclearcell by RT-PCR.The gene was directly ligated into pQE30 vector .After identifying by PCR , restriction enzyme analysis and sequence analysis, the recombinant plasmid pQE30/hIL-24 was induced by IPTG, the fusion protein was expressed in E. coli M15 and purified with Ni-NTA affinity chromatography. Human histomonocyte leukemic cells (THP-1 cells) were stimulated by different concentrations of rhIL-24 and different time point, THP-1 cells secreting cytokines of TNF-αand IL-6 were tested by ELISA and apoptosis was identified by Annexin V-FITC Apoptosis Detection Kit. Results:The human mature peptides IL-24 gene (474bp) was successfully amplified. The recombination plasmid pQE30/hIL-24 was constructed and the molecular weight about 19.2kDa fusion protein rhIL-24 was high level expressed in E.coli and was analyzed by SDS-PAGE.The purity of rhIL-24 by nickel-nitrilotriacetic acid-agarose gel (Ni-NTA) affinity chromatography is higher than of 95%. That rhIL-24 stimulated THP-1 cells to produce inflammatory cytokines including TNF-αand IL-6 had a dose- and time-dependent manner. There was an optimal concentration of rhIL-24 at 28μg/mL in the induction of TNF-αand IL-6 (278.63±8.03pg/mL and 49.48±6.92pg/mL, respectively). The THP-1 cells secreted TNF-αand IL-6 reached peak levels at 18h with rhIL-24 stimulation. Otherwise, THP-1 cells apoptosis was detected at 18h with 28μg/mL rhIL-24 treating .Conclusion:1. The recombination plasmid pQE30/hIL-24 was successfully constructed and the fusion protein rhIL-24 which molecular weight was about 19.2kDa was expressed high level in E.coli.2. rhIL-24 could stimulate THP-1 cells to produce inflammatory cytokines TNF-αand IL-6;3. rhIL-24 could induce the apoptosis of THP-1 cells in vitro.