Cloning and secretory expression of HA and NA gene of H5N1 avian influenza virus in Pichia pastoris

Objective To clone and expresshem agglutinin(HA), and neuramid inase(NA) prote in of H5Navian influenza virus for the preparation of antibodies and to lay a foundation for genetic engineering vaccine.Methods H5A and N1Agene of avian virus were amplified with PCR and then cloned into Pichia pastoris expression vector pPIC 9k containing the AOX1 promoter and factor signal sequence bewteen Not and SnaB site.The recombinant plasmid pPIC9k H5A and pPIC9k N1A were transformed into Pichia pa storis stra in GS115 with electroporation.Multi copy recombinants were screened and ferented inflasks and inducted with 1% methanol.Sodium dodecyl sulfate polyacry lamide gelelectrophoresis(SDS PAGE) and Western blot were used to detect the prote in expression in induced upernatant.Results Ago-Gelelectrophoresis and sequence certificate ind icated that H5A and N1A gene were cloned into pPIC 9K successfully.SDS PAGE showed that the gene could express product with consistent molecular weight as estmiated(64KD and 52KD).The highest volume of protein expression was observed at the 3rd and 4th day.Western blot showed that the target protein could bind with H5 subtype AIV serum.Conclusion The HA and NA genes of avian influenza virus H5N1 were successfuly cloned and expressed, which could be useful for dev eloping diagnose reagent or vaccine of H5N1.