[High level secretion expression of PoIFNalpha in Pichia pastoris].
The porcine alpha interferon gene was inserted into the Pichia pastoris expression vector of pPICZalphaA which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid was transformed into host cell E.coli JM109 and then was extracted for analysis of restriction enzymes. It was confirmed that heterogeneous gene spliced into vector pPICZalphaA was IFNalpha gene. The recombinant plasmid of pPICZalphaA-IFNalpha was linearnized by Sac I and transformed into KM71 by electroporation. SDS-PAGE and Western blot analysis showed that IFNalpha product was observed in the supernants with a little larger molecular weight size than the natural IFNalpha. The rIFN gene has the same antigenicity as natural one. The expressed rIFN accumulated up to about 0.45mg/mL. The cytokine activity of the supernantants was vertified by WISH/VSV system,which is about 2.1x10(4)IU/mL.
Huang
Hai
H
School of Life Science, Nanjing Normal University, Nanjing 210097,China. oswald1115@163.com
Xie
Pei
P
Yu
Rui-Song
RS
Liu
Hui-Li
HL
Zhang
De-Fu
DF
Cao
Xiang-Rong
XR
Li
Zhen
Z
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D034421:Sus scrofa / Q000235:genetics*; D014170:Transformation, Genetic
D016898:Interferon-alpha; D011994:Recombinant Proteins