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Objective: To construct the Fas ligand (FasL) and inducible nitric oxide synthase (iNOS) coexpressed PCA13 plasmid used for packing of adenovirus, so as to observe the immunoprotective effect of FasL on the adenovirus vector., Methods: By way of the internal ribosome entry site (IRES), gene engineering techniques such as preparation and transformation of competent cells, plasmid extraction, agarose gel electrophoresis and restriction enzymolysis were used for the construction of the polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS, which could coexpress FasL and the iNOS gene after multisubcloning steps., Results: FasL and iNOS connected with the IRES were successfully cloned to the PCA13 plasmid and verified by enzymolysis (600 bp FasL, 1000 bp IRES and 4000 bp iNOS) and the gene sequence was concordant with the gene bank., Conclusions: The polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS was successfully constructed.