[Detection for the antiviral activity and primary application of the chicken gamma interferon in yeast Pichia pastoris].
To obtain recombinant chicken interferon gamma (ChIFN-gamma) with natural antivirus bioactivity in yeast Pichia pastoris eukaryotic expression system, the cDNA of chicken interferon gamma mature protein was synthesized by reverse transcription-polymerase chain reaction (RT-PCR)from the total mRNA of the lymphocyte from chicken blood, which stimulated with Con A for 4 to approximately 10 hours. The 493 bp nucleotide sequence of chicken interferon gamma mature protein was cloned into expression vector pPICZa-A, which had been cleaved between EcoR I and Xba I . After linearized by BstX I, the recombinant vector was transferred into yeast Pichia pastoris, strain X33. The recombinant strain was isolated and identified by polymerase chain reaction. After induced by methanol, the recombinant protein was examined by SDS-PAGE. The result of SDS-PAGE analysis in the concentrated fermentation supernatant showed that the molecular weight of the recombinant protein was approximate 16kDa, two recombinants were conformed to secrete chicken interferon gamma(16kDa). The classical experiment of detection for interferon activity (cell pathological Effect Inhibition Assay)and the preliminary result of therapy proved that the recombinant protein is of good antivirus activity. So far chicken interferon gamma had been expressed highly in E. coli, but it tend to form biologically inactive inclusion bodied combined with difficulties in refolding, so the recombinant chicken gamma interferon with natural antivirus bioactivity produced in yeast expression system is the best way, the recombinant interferon gamma have great practical value and application foreground.
Cai
Mei-hong
MH
College of Biological and Environmental Engineering, Jiangsu University, Zhenjiang 212013, China. caimeihong@ujs.edu.cn
Zhang
Su-fang
SF
Cao
Rui-bing
RB
Guo
Wei-ling
WL
Chen
Pu-yan
PY
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002645:Chickens; D015870:Gene Expression; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D009521:Newcastle Disease / Q000188:drug therapy*; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma