Cloning and expression of bovine adiponectin gene in Pichia pastoris.

The BovADPN gene was amplified by RT-PCR with the specific primers,cloned into the vector pMD18-T,and confirmed by sequencing and BLAST.The BovADPN gene fragment which was recovered after the digestion with EcoRⅠ and NotⅠ was subcloned into the pPICZαA vector to construct the pPICZαA-BovADPN Pichia pastoris expression vector.Then the expression plasmid was transformed into GS115 cells using LiCl method after it was linearized with SacⅠ.The multicopy transformants were selected with 0.1% Zeocin,MDH∕MMH culture plate identified by PCR,and induced with 1% methanol.The selected strain could specifically secret the molecular weight of about 40 000 BovADPN proteins which was identified by SDS-PAGE and Western-blot.