The Construction Of Eukaryotic Expression Vectors Of Avian Influenza Virus H5N2 HA Gene And Immune Protection Test

Avian influenza(AI) is a number of influenza A virus and can cause fatal disease for avian specises. HPAIV was placed the class A infectious disease by OIE. Since ,It was reported in 1878 and has beening happened all over the world .It has not only led to the seriously loss of economy of the poultry enterprise all over the world, but also threated human health seriously. Avian Influenza Virus(AIV)H5Nl in 1997,H9N2 in 1999,H7N7 in 2003 and H5N1 in 2004 infected human beings successively so that people understand the public sanitation significance of avian influenza.AIV is round shape with envelope membrane. There are two main glycoproteins on the surface of AIV, One of which is the Hemagglutinin(HA)with the important function. HA gene is an important gene of protective antigen of Avian Influenza Virus(AIV),It can bind to receptor of host cells and lead body to produce antibody against HA.In this study, HA gene was to amplified from H5N2 AIV isolate by RT-PCR,and cloned ,sequenced and analyzed, then a recombinant pVAX1-HA was constructed and expressed in chicken embryo fibroblast (CEF).Protective test of the recombinant was carried out against with highly pathogenic avain influenza virus in chicken. This can provide with scientific test dates for AIV prevention.Based on published HA gene sequences of H5 subtype AIV in Genbank, a pair of specific primers for HA gene of H5N1 was designed and synthesised. The HA cDNA was amplified from H5N2 AIV isolate by reverse transcription-polymerase chain reaction(RT-PCR),then the HA gene was cloned into pMD18-T.The recombinant, named pMD18-T-HA, was converted to Ecoli JM109,and pMD18-T-HA was identificated by enzyme digestion and PCR, the DNA fragments were sequenced subsequently and compared with Genbank data. The result showed that the cloned some 1695bp fragment covered the HA gene of the open reading frame encoding 564 amino acids of the HA gene. The HA gene sequence of the AIV isolate was highly homologous to that of H5N1 AIV(above 94%). The single basic amino acid at the cleavage site of HA implies the low pathogenic characteristics of the virus isolates.The HA gene of the AIV isolate that was cloned into pMD18-T system were subcloned into eukaryotic expression vector pVAX1 at the downstream of CMV directedly . The recombinant, named pVAXl-HA, was identified by HindIII and XhoI digestion and PCR confirmation. Then the recombinant plasmids were transfected into Chicken embryo fibroblast (CEF) cells with liposome, these...