Expression and bioactivity assay of mature chicken interleukin-18 protein mutant using Pichia pastoris expression system

Abstract:

Abstract：[Objective] We expressed mature chicken interleukin-18 (mChIL-18) in Pichia pastoris. [Methods] The mChIL-18 gen was reconstructed by using site-specific mutagenesis based on the Pichia pastoris-preferred codons. The recombinant plasmid pPIC9K/mChIL-18 was constructed and transformed to Pichia pastoris GS115 by electroperation. Multi-copy recombinant strains were screened by Geneticin (G418). The expression of mChIL-18 protein was induced by methanol. SDS-PAGE and Western-blot were used to analyze the expressed products. The bioactivity of mChIL-18 was measured by methyl thiazolyl tetrazolium assays and chicken embryo fibroblasts-vesicular stomatitis virus (CEF-VSV) system. [Results] The protein of mChIL-18 could be secreted by GS115. The optimum expression conditions, a rate of 480 mg/L, were obtained as follows: temperature 28 ℃, pH 6.5, methanol concentration 2% and expression time 120 h. The obtained mChIL-18 protein could stimulate T lymphocytes proliferation. IFN-γ induced by mChIL-18 could directly inhibit the growth of VSV in CEF, and its antiviral activity was about 1.7×104 U/mL which was produced by 400 ng/mL of mChIL-18. [Conclusion] The high expression of bioactive recombinant mature chicken interleukin-18(mChIL-18) in Pichia pastoris had been achieved.