Construction of Recombinant Plasmid with Multi-copy Expression Cassette for High Expression of HBsAg in Pichia pastoris

Objective To establish a recombinant Pichia pastoris strain with multi-copy expression cassette for high expression of HBsAg.Methods Insert multiple copies of expression cassette 5′ AOX-HBsAg-TT into single copy expression vector.Transform the constructed recombinant plasmid with multi-copy expresion cassette to Pichia pastoris GS115 strain by electroporation,screen positive clones and inoculate to BMGY medium for expression under induction of methanol.Purify the expressed product by hydrophobic and ion exchange chromatography.The procedure was further scaled up to a 15 L fermentation reactor.Analyze the specificity and immunnogencity of expressed product as well as the relationship between expression level and copy number.Results ELISA,SDS-PAGE and Westem blot showed that HBsAg with good specificity was highly expressed in Pichia pastoris.Electron microscopy proved the purified expressed product as virus-like particles at a mean diameter of 22 nm.The expression level was positively related to the copy number.However,the increase of copy number showed no significant influence on the nomal growth of recombinant Pichia pastoris strain.The expressed HBsAg induced protective antibody in mice effectively.Conclusion The construction of recombinant plasmid with multi-copy expression cassette increased the expression level of HBsAg significantly.