Abstract: 【Objective】 To construct multi-copy integrative pichia pastoris for expression and assembly of virus-like particles(VLPs) to support the research of genetically engineered vaccine of avian influenza virus(AIV) H5N1subtype. 【Method】 Partial 18S rRNA gene (rDNA) of Pichia pastoris GS115 strain was amplified out and inserted into enzymatic XbaⅠ and Bsp1407Ⅰ sites of plasmid pPIC9K to construct multi-copy integrative vector p8K. And, structural protein HA, M and NA genes of AIV H5N1 subtype were amplified by RT-PCR to construct expression plasmids p8K-HA/M/NA, respectively. Subsequently, those expression plasmids were linearized,, mixed proportionally and electro-transformed into Pichia pastoris GS115 strain, and screened on Geneticin G418 plates. Later, positive recombinants carrying multiple-copied HA, M and NA genes identified by PCR were picked out, enrichment cultured and induction expressed, harvested, broken by High speed homogenation and confirmed by Western-blot, respectively. 【Result】 Cell lysates of positive recombinant yeasts confirmed by PCR method c be identified out by immunoblotting with three bands corresponding to HA, M₁ and NA. And, 80~120nm virus-like particles (VLPs) be observed by electron microscopy.chickens injected by VLPs produce specific neutralization antibodies against AIV. 【Conclusion】 Recombinant pichia pastoris can multi-integrate, express and assemble AIV VLPs.