Abstract: ISG15 gene was cloned from MDCK cells using RT\|PCR， and the alignment of ISG15 with the reported sequence （GenPept databank: XM_0036390532） revealed 99 % nucleotide sequence identities. The ISG15 was cloned into expression vector pET\|28a to construct the expression plasmid p28a\|M15， and was expressed in Escherichia coli. The protein expression in E. coli induced by IPTG was secreted in the supernatant with a recombinant protein of about 22 kD. The recombinant protein was purified using Ni\|chelate affinity chromatography and then added into MDCK cells quantitatively with and without AIV H9N2 subtype infection. The results showed that ISG15 protein could inhibit AIV replication in normal MDCK cells， however， it could promote AIV replication in MDCK cells infected by AIV H9N2 subtype. This study will provide theoretical basis for ISG15 antiviral function in vitro.

Key words: ISG15, MDCK cell line, antiviral activity, H9N2