Construction And Identification Of Adenovirus Vector Containing EGFP And Cytokine Interleukin 23

The cholangiocarcinoma is one of the highest malignant tumor with increasing incidence in recent years. Unfortunately , most patients who suffer from cholangiocarcinomas are advanced stage when diagnosed ,in which only 10% of them can be treated with complete resection ,but its prognosis is still very poor, 5-year survival rate is only 5% -13% in the western countries. Doctors and researchers make great efforts to find the best combinating therapy for several decades, and now, gene and immune therapy are considered the fourth antitumor prospective method following opreation and radiochemical therapy.The same with other tumors, the basic mechanism for its recurrence and metastasis is the lose of protective immune response against tumor invasion. The failing to present effect tumor antigen and induce antitumor immune response is the key for host's immune escaping, because tumor cells with weak antigenicity are absent with major histocompatibility complex, co-stimulating factors ,as well as, adhensive molecules. Nowadays, the great advances in antitumor immune research is the understanding of antigen presenting cell. The dendritic cell the most potential APC in body is the irritant for antitumor immune response and is considered as'guard'of body immune system.IL-23 a newly discovered cytokine is essential for antitumor cell immune, interleukin 23 can enhance not only the DC survival ability in tumor environment but also the presenting ability, IL-23 can promote the proliferation of active T lymphocyte and memory T lymphocyte and induce active T cells and DC to Produce TH1 type cell factor such as, IFN-γ,IL-12 and so on. IL-23 has strong antitumor function according to the animal experience, for example, the tumor size was obviously regressed when CT26 colon carcinoma cell line was transfected with recombinated IL-23 by retrovirus vectors,and 70% mice showed strong preventativeimmunities against colon carcinoma implanted again. Therefore, IL-23 is a very nice candidate gene and immune adjuvant for antitumor immunities.Adenovirus vectors with characteristics including high titers, large insertional capacity and high transduction efficiencies are commonly used in the research of gene therapy.This test is going to clone gene encoding P19 and P40 subunits of IL-23, construct copy disable Adenovirus vectors with Admax method. EGFP gene is also inserted to monitor exogenous gene expression. The further research will include antigen presenting and secreting abilities of DC modified with IL-23.Objective: To construct copy disable adenovirus vector containing EGFP and recombinated interleukin 23 gene after cloning P19 and P40 dual subunits in order to study the antitumor therapy induced by dendritic cell vaccine genetically modified with active cytokin IL-23.Methods: first,cloning P19 by Designing P19 Probes to acquire a positive clone from Person's marrow cell cDNA bank, and cloning P40 by obtaining P40 total RNA from the human leukemia cell K562 ,the cDNA was amplified by RT-PCR .the recombinated plasmid pAdTrack-mCMV-P19-IRES-P40-GEFP was obtained after P19 , P40 and EGFP genes were connected together with IRES sequence and inserted into shuttle vector PDC-316, HEK293 cells were infected by the plasmid and framework adenovirus vectors. IL-23 adenovirus vector was abtained and identified by PCR, restrict enzymes,and the expression of EGFP. Adenovirus titer and its infectionability wrer finally tested by TCID50 method.Result: the sequence of P19 and P40 gene is completely consisten t with the reported sequence of the genebank, the contructed PDC-316-P 19-IRES-P 40-IRES- GEFP plasmid was proved to have P19-IRES-P40 slice of 1387bP by restriction endonuclease analysis. EGFP protein well expressed in 3 days and the phagocytizing blot appear in 11 days after infection and package of virus. The adenovirus vector containing human IL-23 and EGFP genes was constructed successfully, the titer of purified ade-IL-23-EGFP is as high as 6.5×109 Pfu/ ml.Conclusion: we successfully contructed the copybisable adenovirus vector ade-IL-23-EGFP, it is the basic work for the construction of DC vaccine transfected with IL-23.