The Expression Of Murine IL-18 By Eukaryotic System And Preliminary Study Of Its Bioactivities

Objective: establishing the eukaryotic expression system mimic the natural expression of murine IL-18.Methods: The cDNA encoding the precursor of murine IL-18(mpro-IL-18) was obtained by RT-PCR from total RNA of murine splenocytes, and cloned into T-vector pCR4-TOPO, then, subcloned into eukaryotic expression vector pVR1012 (pVR1012-mpro-IL-18). Meanwhile, the eukaryotic expression vector encoding murine IL-1 ?converting enzyme pEGFP-Nl-mlCE was constructed successfully. After the transfection of pVR1012-mpro-IL-18 alone or together with pEGFP-Nl-mlCE into COS-7 cells, the mRNA expression were analyzed by RT-PCR respectively and the expression of IL-18 in the cell lysates was proved by Immunoblot Assay. The secreted IL-18 (sIL-18) in the supernatants of transfected COS-7 cells was measured by ELISA. The proliferation of murine splenocytes, stimulated by supernatant containing sIL-18 togermer with IL-2, was measured with 3H-TdR incorperation assay. The CTL activity of splenocytes against syngenic tumor cells from healthy mice injected intramuscularly with triple vectors pVR1012 -mpro-IL-18,pEGFP-Nl-mlCE and PVR1012-hIL-2 was assayed by JAM-TEST.Results: Correct recombinant eukaryotic expression vector encoding mpro-IL-18 and mICE were constructed. The expression of these two genes at the level of mRNA and protein were detected respectively. The IL-18 secreted from the transfected cells was also found in the supernatants and was able to enhance the proliferation and cytotoxicity of murine splenocytes.Conclusion: The eukaryotic expression system mimic the natural expression of murine IL-18 was established successfully, and this expression system has the potentials such as gene therapy of cancer.