Construction Of Conditionally Replicative Adenovirus Expressing Interleukin-18 And Investigation Of Its Antitumor Activity In Vitro

Objective To construct conditionally replicative adenovirus (CRAd) expressing Interleukin(IL)-18. To investigate the antitumor activity of replication competent adenovirus expressing IL-18 on malignant melanoma A375 cells in vitro.Methods One pair of specific primers was designed and used to get human IL-18 cDNA sequences and change its restriction enzyme sites of BglⅡby polymerase chain reaction using pJW-IL-18 as the template. The PCR product was inserted into pCA13 plasmid to construct the recombinant plasmid (pCA13-IL-18). The pCA13-IL-18 plasmid was cut with BglⅡto get the expression cassette of IL-18 gene and then the expression cassette was cloned into pZD55 which has been cut with BglⅡand dephosphated to form pZD55-IL-18 plasmid. It was identified that the pZD55-IL-18 had been constructed successfully. The plasmid pZD55-IL-18 was transfected in HEK293 cells together with plasmid pBHGE3 to obtain the recombined CRAd, ZD55-IL-18. The viral plaques appeared 9~12 days after infection. The recombined adenoviruses, extracted DNA, were verified by PCR. Viruses were plaque purified, propagated on HEK293 cells and purified by CsCl gradient according to standard techniques, and functional PFU titers were determined by plaque assay on HEK293 cells. The antitumor potential of ZD55-IL-18 to A375 cells was evaluated by MTT assay and crystal violet dye method. The effect of ZD55-IL-18 on the IL-18 expression in mRNA levels of A375 cells was detected by RT-PCR. The expression of IL-18 and E1A protein was also detected by Western-blotting.Results CRAd expressing IL-18 had been constructed successfully. The analysis of PCR indicated the recombinant adenovirus ZD55-IL-18 containing IL-18 gene and without wild adenovirus. Functional PFU titers of ZD55-IL-18 were 1×1011PFU/ml. Western-blotting analyses indicated ZD55-IL-18 can express E1A and IL-18 in adenovirus infected A375 cells. ZD55-IL-18 is more effective to inhibit the proliferation of A375 cells than Ad-IL-18 in vitro.Conclusion CRAd ZD55-IL-18 can enhance the expression of IL-18 and inhibit the proliferation of malignant melanoma A375 cells. CRAd expressing IL-18 may be used for further investigation of Gene-Viro therapy of cancer.