Cloning And Prokaryotic Expression Of Chicken IFN-γ Gene

Interferon (IFN) is a kind of cytokines that have effect on anti-virus and immunesystem. In them IFN-γ, named as typeⅡinterferon, is secreted by T lymphocytes and NKcells stimulated by antigen and mitogen and influences the cellular and humoral immuneresponse. Chicken IFN-γ was firstly reported by Lowenthal in 1995. Then at the same year,a cDNA encoding chicken IFN-γ was cloned and expressed in Escherichia coli (E.coli) byDigby. The reseach on IFN-γ has made great progress in its structure and biological andimmune function in recent years. In order to research mechanism of chicken IFN-γ inanti-virus and immune regulation, in present study this gene was cloned, expressed andidentified. Firstly, a DNA fragment of about 500bp was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from ConA-activatedspleen lymphocytes of chicken, the primer design was based on the published sequence inGenBank. After isolation and purification, the fragment was subcloned specially intopGEX-4T-1 vector. Then the recombinant plasmid was identified with PCR and EcoRⅠand SalⅠdigestion, and then sequenced. Secondly, the result of sequencing shows that the open reading frame(ORF)ofchicken interferon-gamma (ChIFN-γ) gene consists of 495bp, and encodes a protein of 164amino acids. Mature polypeptide of ChIFN-γ consists of 145 amino acids. Chicken IFN-γsequence shows 100.0% identity to the published gene in GenBank at either the nucleotideor amino acid level. ChIFN-γ gene shares 96.8%, 95.6%, 95.6% and 93.5% identities atnucleotide level, and 97.0%, 97.0%, 93.3% and 87.3% identities at amino acid level toturkey, ring-necked pheasant, Japanese quail and helmeted guineafowl gene, respectively.But it only shares 80.6% and 81.8% identities at nucleotide level, and 66.7% and 67.9%identities at amino acid level to duck and goose, respectively. By comparing of thehomology, ChIFN-γ gene shares identities to mammals from 27.1% to 39.6% at nucleotidelevel and from 24.2% to 29.7% at amino acid level, respectively. The phylogenetic treeanalysis of IFN gene and its amino acids indicated that chicken IFN-γs have much nearerevolution relationship with galliforms and mammals' IFN-γs than with chicken type ⅠIFNs at nucleotide and amino acid level. And the comparative analysis of antigenicityshowed that the IFN-γ antigenicity has high similarity among galliforms. Finally, the recombinant plasmid was transformed into E.coli BL21, and was inducedat 37℃ with 1.0mmol/L IPTG for ChIFN-γ gene expression. The expressed products weresubjected to SDS-PAGE, and the result showed that it existed as inclusion bodies, a fusionprotein about 43kD as we expected was found. In conclusion, in the present study we cloned and expressed Chicken IFN-γ gene inprokaryocyte successfully. It would provide the basic evidence for the knowledge ofinterferon sequence evolution relationship among different species, and for the furtherresearch on immunological and molecular biological properties and its amplification ofrecombinant ChIFN-γ protein.