title: [Expression and bioactivity assay of mature chicken interleukin-18 protein mutant using Pichia pastoris expression system].
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 degrees C, pH 6.5, methanol concentration 2% and expression time 120 h. The obtained mChIL-18 protein could stimulate T lymphocytes proliferation. IFN-gamma induced by mChIL-18 could directly inhibit the growth of VSV in CEF,and its antiviral activity was about 1.7 x 10(4) U/mL which was produced by 400 microg/L of mChIL-18.
  
  
  CONCLUSIONS
  The high expression of bioactive recombinant mature chicken interleukin-18 (mChiL-18) in Pichia pastoris had been achieved.
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Shandong Taian 271018, China. wangxi_hui@163.com
authors.forename: Xihui
authors.initials: X
authors.lastname: Wang
authors.forename: Shousong
authors.initials: S
authors.lastname: Yue
authors.forename: Jingdong
authors.initials: J
authors.lastname: Hu
authors.forename: Yijun
authors.initials: Y
authors.lastname: Huang
authors.forename: Jinlong
authors.initials: J
authors.lastname: Chen
authors.forename: Tingting
authors.initials: T
authors.lastname: Wang
authors.forename: Zhongling
authors.initials: Z
authors.lastname: Fan
authors.forename: Hongkun
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D020382:Interleukin-18 / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*
chemical_list: D020382:Interleukin-18; D050505:Mutant Proteins