Construction of the Bi-cistronic Co-expression Plasmid of mouse IL-12 and the Expression in Vitro and in Vivo

Abstract: Interleukin-12 (IL-12) is a disufide-linked heterodimeric cytokine which is secreted by antigen presenting cell (APC), such as monocytes/macrophages and B cells. It has been showed that IL-12 can stimulate the proliferation and activity of T cells and NK cells. This cytokine also has the ability to augment lytic activity of CTL. NK cells and macrophages. IL-12 can induce the production of many cytokines and regulate the development of Thl cells from ThO cells, which result in the improvement of cellular immune response. These results indicate that IL-12 plays an important role in anti-tumor immune response. The therapeutic potential for IL-12 in the treatment of malignancies has been evaluated in many animal models, such as the B16F10 melanoma, the Renca renal carcinoma, the Lewis lung carcinoma and so on, and protective effects have been demonstrated. However, the toxicities caused by systemic delivery of IL-12 protein-limit the application of IL-12 in clinic. It has been a new alternative way for dissolving this problem after Wolff et al offered a new method for gene therapy棗injecting naked DNA in vivo in 1990. After local delivery of IL-12 plasmid, IL-12 can be expressed and secreted by the cells of local tissues. A lager amount of IL-12 in local tissues canimprove the micro-circumstance of tumor and enhance anti-tumor immuneresponse in local. Moreover, the level of IL-12 in serum is so low that can prevent the toxicities caused by systemic delivery of IL-12 protein.ObjectiveWe use bi-CMV promoter to construct a bi-cistronic co-expression plasmid for murine interleukin-12梡CmIL-12 which can express in eukaryotic cells. We also observe the plasmid expression in vitro or in vivo to determine whether it can express mIl-12 with biological activity in vitro and has the potential ability to augment the activity of NK cells in vivo. If it works, it will provide the basis of IL-12 tumor gene therapy.MethodThe full-length cDNA encoding mIL-12 p35 and p40 were digested from the plasmids of pBluescript SK/mp35 and pBluescript SK/mp40. They were cloned into eukaryotic cells expression vector pcDNA3.1(+) respectively. So pC35 and pC40 plasmid were formed. Subsequently, the p35 expression unit (CMV-p35-BGHPA) was digested from pC35 and inserted into pC40 to produce the bi-cistronic co-expression plasmid in which the p35 and p40 gene were controlled by their own promoter. Therefore, a bi-cistronic co-expression plasmid pCmIL-12 was constructed.pCmIL-12 was transfected into COS-7 cells in Vitro and the supernatant of COS-7 was collected to determine the content of mIL-12 and the influence on the activity of murine NK cells. Furthermore, the plasmid was intradermal injected to observe whether it affect the activity of murine NK cells in vivo.ResultpCmIl-12 plasmid was verified by enzymes digesting and DNA sequences analysis. The results were consistent with those expected.pCmIL-12, pC35+pC40 and-pcDNA3.1 were transfected into COS-7 cells for transient expression and their expression supernatant was cultivated with the splenocytes of mice. The NK cells activity of the former two reached 42.9 + 3.78% and 28.6 + 3.82% respectively. Compared to the mean level of NK cells activity induced by pcDNA3.1 expression supernatant (21.4 + 3.89%), there is significant difference between them (p<0.05). The activity of pCmIL-12 expression supernatant was significant elevated compared with pC35 and pC40 co-expression supernatant (PO.05) . The mIL-12 in the supernatant was detected by ELISA. The expression supernatant of pCmIL-12 contained 270~350pg/ml mIL-12 proteins while that of pcDNA3.1 didn't contain mIL-12.The plasmids of there groups were intradermal injected in mice. The NK cells activity was significant enhanced and reached 52.6 + 5.6% by intradermal delivery of pCmIL-12. The activity was 20.0 + 8.8% and 18.4 + 5.4'% respectively by intradermal delivery of pC35+pC40 and pcDNA3.1. There is significant difference between the former group and the later two groups (p<0.01)….
Key words: Helicobacter pylori; recombinant plasmid; CagA; CTB

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