Construction And Identification Of A Recombinant Canine Type 2 Adenovirus Expressing NP Protein Of H5N1 Subtype Tiger Influenza Virus

[Objective]Construct and identify a recombinant of canine type 2 adenovirus expressing NP protein of H5N1 subtype tiger avian influenza virus,it has offered the foundation of theory and technology to the research of recombinant live vector vaccine for mammalian, especially the H5N1 subtype tiger avian influenza virus.[Methods]With DNA recombinant technology,to construct eukaryotic expression vector pVAX-NP expressing NP gene of avian flu virus;using indirect immunofluorescence and indirect ELISA method detect which expression in the mammalian cells BHK-21 cells;NP expression cassette fragment of pVAX -NP was cloned into the E3 area of the CAV-2 vector pPoly2-CAV2,obtain the recombinant plasmid pCAV-2-NP which NP expression cassette was inserted in missing E3 zone.Using the method of liposome-mediated,import the recombinant plasmid pCAV-2-NP into mammalian cells of DK cells;observed cell changes,and when it be like a typical cytopathic effect of adenovirus,to observe the morphology of recombinant virus using electron microscopy,to identify the hemagglutinin characteristics of recombinant virus through hemagglutinin experiment,to detect NP expression cassette fragment whether or not missing and transcription of the NP gene mRNA through application of PCR and RT-PCR,using indirect immunofluorescence and ELASA indirect method to detect of the expression of NP gene in mammalian cells of DK, the application of conventional virus cultured in vitro to detec the genetic stability of recombinant virus.[Results]The eukaryotic expression vector pVAX-NP can express NP protein in mammalian cells correctly and possess its corresponding biological activity,recombinant virus of CAV-2-NP has the typical morphological characteristics and blood coagulation properties of CAV-2,the fragment of NP expression cassette is not missing in the process of reproduction,and NP gene transcription to mRNA,ELISA and immunofluorescence analysis showed that the product of recombinant expression can be idemified by the NP monoclonal antibody of avian flu virus,has a good in vitro genetic stability.[Conclusion]Construct and obtain the recombinant virus of canine type 2 adenovirus expressing NP protein of H5N1 subtype tiger avian influenza virus successfully.