Expression And Bioactivity Assay Of Mature Chicken Interleukin-18 Protein Mutant Using The Pichia Pastoris Expression System

Chicken interleukin-18(ChIL-18) has multiple biological activities including induction of IFN-γfrom NK cells and antigen- or mitogen-stimulated Th1 cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NK cell cytotoxicity. The Pichia pastoris is one of the most successful freign protein expression systems until now. It offers economy, ease of manipulation, the aility to perform complex post-translational modifications, and high expression levels. So it is important to direct the expression of a bioactive mature chicken interleukin-18 (mChIL-18) in P.pastoris. The mChIL-18 gen was reconstructed by using the technique of site-specific mutagenesis based on the P.pastoris-preferred codons, and the recombinant plasmid pPIC9K-mChIL-18 was constructed. The objective of this research was to transform the cloned mChIL-18 gen into P.pastoris, and then cultured the P.pastoris, expressed the mChIL-18 and determined the bioactivity, which provides a good foundation for the research and development of mChIL-18. Main results of this research are as followed:To get a high expression and activity of recombinant mature chicken interleukin-18 (mChIL-18), the mChIL-18 gen was reconstructed by using the technique of site-specific mutagenesis based on the P.pastoris-preferred codons. The pPIC9K-mChIL-18 of expression vector was constructed by inserting mChIL-18 fragment. Sequencing results of plasmid pPIC9K-mChIL-18 indicated that structural mChIL-18 gene was integrated into the correct reading frame.Sal I-linearized recombinant plasmid pPIC9K-mChIL-18 was transformed into GS115 by electroperation. Multi-copy recombinant strains were screened by G418. Specific Pichia clony PCR products showed that foreign mChIL-18 gene was integrated into the host cell. 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(MTT)assays and chicken embryo fibroblasts-vesicular stomatitis virus(CEF-VSV)system. The results showed that 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-γinduced 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.By using the technique of site-specific mutagenesis and optimizing expression conditions, the high expression of bioactive recombinant mature chicken interleukin-18(mChIL-18) in Pichia pastoris had been achieved, which could be produced in large-scale fermentation.