Secreted expression of the VP2 protein of very virulent infectious bursal disease virus in the methylotrophic yeast Pichia pastoris.
The VP2-encoding gene of very virulent infectious bursal disease virus (vvIBDV) was amplified using reverse transcription (RT)-polymerase chain reaction (PCR) and inserted into pPICZalphaA vector. Recombinant plasmid DNA was integrated into the chromosome of the transformed Pichia pastoris by electroporation and expressed protein identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut+ phenotype and secreting multi-copy integrants in the recombinant yeast. A recombinant protein of approximately 67 kDa was secreted into the supernatant from the yeast when induced with methanol. The expressed supernatant was bound with chicken anti-IBDV polyclonal antibodies. Western blotting with antibodies against vvIBDV indicated that the recombinant VP2 protein retained its antigenicity. High-level production (10 mg/100 ml) of the recombinant VP2 protein indicated that P. pastoris was an efficent expression system for vvIBDV VP2 protein.
Wu
Pei C
PC
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Su
Hung Y
HY
Lee
Long H
LH
Lin
Der T
DT
Yen
Pei C
PC
Liu
Hung J
HJ
D015870:Gene Expression; D007243:Infectious bursal disease virus / Q000235:genetics*; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins; D000432:Methanol