Construction of recombinant adenoviral vector containing gene of EWS-FLI1 and antitumor immunity of its modified dentritic cell in vitro / 北京大学学报(医学版)

ABSTRACT

Objective: To construct an adenoviral vector containing cDNA of EWS-FLI1 and detect its expression in peripheral blood mononeuclear cell(PBMC).To Investigate the antitumor immunity in vitro of the EWS-FLI1 gene modified-dendritic cells.Methods: The EWS-FLI1 cDNA in plasmid Pec1/ EWS-FLI1 was digested and subcloned into the shuttle plasimid padtrack-cmv.The shuttle plasmid and the bone plasmid pADeasy-1 were cotransformed into BJ5183 cells.The recombinant plasmid was generated by homologous recombination in BJ5183 cells.The positive clone was obtained by digestion and electrophoresis.Transforming the recombinant plasmid into "293 cells" by lipofectamine method.Adenoviruses with high titer and purity were obtained by amplifying in the"293 cells" on a large scale and ultra-centrifugation in CsCL step gradient solutions.The cytotoxic activity of stimulated T cells to Ewing sarcoma cells was detected by ~(51)Cr release assay.Results: PCR showed that the adenovirus contained EWS-FLI1(cDNA.) After the PBMC were transfected by Ad EWS-FLI1,the EWS-FLI1 mRNA was detected by(RT-PCR.) The antigen-specific CTL was induced successfully by the EWS-FLI1 gene modified-DC.The vigorous antigen-specific CTL response against A673 cells was detected by ~(51)Cr release assay.The killing percentage was 35.18%?0.0128% at effector-target ratio 40∶1,which was more efficient than that of the control.Conclusion: The recombinant adenovirus was successfully constructed and could efficient express EWS-FLI1 in PBMC.After T lymphocytes were stimulated by DCs modified with EWS-FLI1 gene,the specific CTL response against Ewing's sarcoma cell line A 673 in vitro was observed successfully.~(51)Cr release assay showed that there was significant difference between the experimental group and the control group.