Construction of murine IL-12 eukaryotic expression plasmid of single chain fusion gene and its potential in gene therapy of cancer

Objective To construct a single chain fusion gene of murine IL-12(msIL-12) explore its application in gene therapy primarily. Methods By RT-PCR the p40cDNA and p35cDNA were respectively obtained from the total RNA of mouse peritoneal macrophages stimulated with LPS and after introducing a linker by second PCR respectively they were successively cloned into pcDNA3 to construct the eukaryotic expression plasmid of msIL-12, pmsIL-12. The pmsIL-12 purified by PEG was transfected into COS-7 cells. The protein concentration of IL-12 in supernatant of the transfected COS-7 cells was measured by ELISA. Injecting purified pmsIL-12 intradermally, its effect on NK activity of normal mouse splenocytes was detected by MTT, and impact on survival period of H22 (a hepatoma cell line)-bearing mice was observed. Results The sequence of p35cDNA obtained was identical to that of M86672 in GenBank. The sequence of p40cDNA obtained was identical to that of AH004859 in GenBank and also to that of M86671 in GenBank except that the 1000th base is G instead of A. The eukaryotic expression plasmid of msIL-12 and pmsIL-12 was successfully constructed. The IL-12 protein concentration in supernatant of COS-7 cells transfected by pmsIL-12 was 2.55±0.60ng/ml. After injecting pmsIL-12 intradermally, NK activity was enhanced for normal mouse splenocytes, and the survival period of H22-bearing mice was evidently lengthened. Conclusion Mouse eukaryotic expression plasmid of IL-12 single chain fusion gene was constructed, and the plasmid could be used in gene therapy.