Expression And Assembly Of Virus-like Particles Of Avian Influenza Subtype H5N1

Avian Influenza（AI）is a respiratory disease of birds caused by influenza A virus.So far,a main prevention and control strategy for avian influenza is vaccination.At present,whole-virus inactivated vaccine is widely used,but the respiratory tract cannot induce effective mucosa immunity to the vaccine,although the diffusion of viruses can be suppressed,and the effect of alleviating symptoms is achieved.However,defects of the inactivated vaccine cannot be ignored,such as antigen variations,allergic reactions to egg components.Therefore,it is especially important to further study and develop efficient and safe new vaccines.Virus-Like Particles（VLPs）doesn’t contain nucleic acids,and have whole antigenic epitopes similar to natural viruses,is a safe and efficient vaccine.1.In the early stage of the experiment,sick poultry feces,poultry cloacal swabs,and wild bird feces,a total of 109 samples were collected from distinct areas of Xinjiang,and prepared for RT-PCR analysis of HA gene of avian influenza virus（AIV）subtype H5.Previously,subtype-specific primers for amplification of HA gene had been designed for identification of AIV H5 subtype.Finally,4 strains of AIV were isolated out and categorized into Clade 8 by genetic evolutionary analysis.2.To study the genetic engineering vaccine of the avian influenza virus（AIV）subtype H5N1,a recombinant Pichia pastoris with multiple integrative copies of structural protein genes of avian influenza virus.At first,partial 18S r RNA（r DNA）sequence of strain GS115 was Inserted into between of Xba I and Bsp1407I sites in the p PIC9K vector to construct the p8K vector.Subsequently,the multi-copy integrated expression plasmids,p8k-HA,p8k-M,and p8k-NA,were constructed by the HA,M and NA genes inserted into p8K vector,respectively.Before that,the HA,M and NA genes were amplified from genomic RNA of AIV subtype H5N1 by RT-PCR.Then,expression plasmids were linearized by enzyme digestion respectively,mixed proportionally,were electro-transformated to GS115 competent cells.After enlarge culture,the recombinant cells were screened on the geneticin G418 resistance plate,identified by PCR,and copies of HA,M and NA genes in the genome were estimated.Those positive recombinants were scale-up cultivated for inducible expression,high pressure homogeneous crushing and Western-blot.In the cell lysate of positive recombinants can be detected out protein strips identical with the molecular weight of HA,M1 and NA,and virus-like particles with diameter 80～120 nm approximately can be found out by electron microscope.Furthermore,immunized chicken can produce anti-AIV neutralization antibody.Those results suggest that Pichia pastoris can express and assemble AIV VLPs effectively,which provides a foundation for a genetic engineering vaccine of AI based on yeast.3.A lentiviral expression vector with green fluorescent protein（EFP）was inserted into HA,M1 gene of AIV in multiple clone sites（MCS）to construct expression vectors,p CDH-HA and p CDH-M1,respectively.Those expression vectors p CDH-HA and p CDH-M1,and packaging plasmids p LOX and ps PAX2 were co-transfected to BHK₂₁cells,afterwards,adaptive passaged continuously and harvested according fluorescence intensity.The cells were precipitated by 25%ammonium sulfate,western-blot was chosen to detect immunogenicity of target proteins.The test results suggest that the immuno-blot showed two protein strips with a molecular weight of HA and M1,and VLPs with a diameter of about 80～120 nm can be observed out by electron microscope.The recombinant proteins were detected with HA blood coagulation test,which showed that blood agglutination titer can reach 2⁵.The above all tests suggest that the lentiviral vector-mediated BHK₂₁cells can successfully express and assemble AIV VLPs,which is expected to provide a basis for the research of the future avian influenza vaccine based on mammalian cells.