Construction And Expression Of Interleukin-24 Recombinant Expression Vector And Study Of Its Antitumor Effects

Interleukin-24 (IL-24), originally designated as the melanomadifferentiation-associated gene-7(MDA-7), is stably expressed in human tissues associated with the immune system such as the spleen, thymus, peripheral blood leukocytes and normal melanocytes. The MDA-7 gene was identified by subtraction hybridization from the cDNA library of human melanoma cell line, HO-1 treated with the combination of IFN-β and mezerein. The expression of MDA-7 is elevated in terminally differentiated human melanoma cells. Current information based on structural and sequence homology, has led to its renaming as IL-24 at 2002. A notable property of IL-24 is its ability to induce apoptosis in a large spectrum of human cancer derived cell lines, but whether it could induce apoptosis in HIC cells has not been reported. The relationship between N-linked glycosylation of IL-24 and its function is not known. For these we study in three aspects as shown below.In this study a recombinant expression vector of human IL-24 cDNA was constructed and expressed in E. coli. The hIL-24 cDNA fragment was amplified from plasmid TRAP-hIL-24 by PCR, then cloned into the prokaryotic vector pGEX-KG, and expressed inductively as a fusion protein in E. coli. The expressed IL-24-GST fusion protein was purified through GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot analysis. Restriction enzyme digestion analysis showed that the recombinant prokaryotic expression vector pGEX-KG-IL-24 was successfully constructed and was expressed stably in E. coli. The relative molecular mass(Mr) of the expression product was identical with the predictive size, identified by Western blot. The recombinant expression vector pGEX-KG-IL-24 has been constructed successfully and expressed as a fusion protein in E. coli BL21 (BlysS), which will be helpful to the production of IL-24 antibody and the further study of the biological function of IL-24.An eukaryotic expressing plasmid of IL-24 was constructed, and its inhibitory effects on the growth of tumor cells in vitro and in vivo was studied. The eukaryotic expressing plasmid of IL-24 (pEGFP-IL-24) was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into HIC cells with Lipofectamine 2000 Reagent and the expression levels were determined by LSCM; the proliferation of HIC cells was measured by MTT assay; and apoptosis rate and cell-cycle distribution of HIC cells were measured with Flow Cytometry. Mice were inoculated with B16 cells, which had been transfected with pEGFP-IL-24 or empty vector, and the tumor size in mice was detected. The inhibitory effect of IL-24 transfection in mice solid tumor was observed and measured. The expression of pEGFP-IL-24 in HIC cells was determined by LSCM after the transfection of pEGFP-IL-24. Comparing to the control group, the proliferation of HIC cells was inhibited by transfection with pEGFP-IL-24 and the G2-M phase of the transfected cells was also increased. Moreover the percentage of mice with detectable tumor decreased after being inoculated with B16 cells transfected with pEGFP-IL-24. Growth rate of tumor in mice model was significantly inhibited in IL-24 gene therapy group as compared with the control group (p<0.05). From these results we may conclude: proliferation of HIC cells was inhibited by pEGFP-IL-24 transfection.; the intratumor injection of pEGFP-IL-24 can remarkably inhibit the growth of solid tumor in mice.N-linked glycosylation is important for glycoprotein function. In some cases, glycosylation has been shown to be important for folding, protein stability, immune function , or receptor usage. In addition, it can alter protein-protein interactions, either directly or by altering protein structure. IL-24 has three N-linked glycosylation sites(N85lT, N99VS and N126RT). In order to identify the relationship between the structure and the function of N-linked protein glycosylation of IL-24, four eukaryotic expression vectors encoding IL-24 mutants were constructed in which the three N-glycosylation sites(Ng5lT, N99VS and N126RT) were mutated separately or in combination by site-specific mutagenesis. In each mutant, Asn codon in each NXS/T glycosylation sequon was mutated into Asp codon. Each IL-24 mutant and wild typeIL-24 were expressed in B16 cells individually and confirmed by LSCM. The inhibition effects on B16 proliferation by IL-24 and each mutant were examined by MTT methods. We found that all of IL-24 mutants decreased the inhibition effects on B16 proliferation comparing with that of wild type IL-24. The IL-24 M3 mutant, in which three of N-glycosylation sites (NgsIT, N99VS and N126RT) were mutated, induced lowest anti-tumor effects among all the mutants. We conclude that N-glycosylations of IL-24 are required for its proliferation inhibition effects and are important for its anti-tumor function.