[Cloning, high level expression and purification of porcine IFN gamma].
Peripheral blood lymphocytes from a single swine were stimulated with Concavadin A for 17 h, and the total RNA was isolated from it. Then, the mRNA specific for porcine IFN gamma was amplified by reverse transcription polymerase chain reaction. After sequencing, the IFN gamma gene has been successfully inserted into vector pJLA-503 and highly expressed in E. coli. Recombinant porcine IFN gamma expressed as inclusion body, which was dissolved in 7 mol/L guanidine chloride and subsequently renatured by dilution in refolding buffer containing 0.5 mol/L L-arginine. In order to obtain pure protein, the renatured IFN gamma was purified by the chromatographies of SP-Sepharose FF and Sephacryl S-200 HR. As a result, the final pure product can been seen as a single band in SDS-PAGE, and the cytokine activity was verified by inhibiting the cytopathic effect.
Guo
Y J
YJ
Department of Medical Genetics, The Second Military Medical University, Shanghai 200433, China. gyjz268@yahoo.com
Wu
D
D
Chen
R W
RW
Sun
S H
SH
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013552:Swine
D011994:Recombinant Proteins; D007371:Interferon-gamma