Soluble expression of chicken interferon-γ and antiviral activity of purified expression product

Abstract:

[Objective] We cloned and expressed chicken interferon-gamma gene (chIFN-γ), and detected the bioactivity of chIFN-γ expressed in Escherichia coli (E.coli) . [Methods] The chIFN-γ mature protein gene was amplified by RT-PCR from spleen lymphocytes of chicken which were stimulated with concanavalin A (ConA) and then cloned into the prokaryotic expression vector pET-32a (+). Recombinant expression plasmid of pET-32a (+)-chIFN-γ was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression with IPTG induction. Purified soluble rchIFN-γ proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The antiviral activity was detected by MDCK-VSV system. [Results] A 456 bp cDNA encoding chIFN-γ mature protein gene was cloned and successfully expressed in E.coli with approximate molecular weight of 31.0 kDa, which could be recognized by anti-His mAb and rabbit polyclonal antibody against chIFN-γ. The recombinant chIFN-γ proteins were expressed to form inclusion bodies with a portion of soluble protein. The soluble rchIFN-γ proteins were purified by Ni-NTA column under a native condition with the yield of 3.0 mg/L. The purified recombinant chIFN-γ (rchIFN-γ) proteins by 1:32 dilution could resist 100 TCID50 VSV virus attack. [Conclusion] The purified rchIFN-γ proteins by Ni-NTA column under a native condition had better antiviral activity, which will establish a basis for further developing new type antiviral interferon praeparatum.