Construction And Identification Of A Recombinant Canine Type 2 Adenovirus Expressing HA Gene Of H5N1 Subtype Tiger Avian Influenza Virus

[Objective] As far as, AIV already spreads worldwide.Every disastrous outbreak event of AIV will make great economic loss.Especially the Highly Pathogenic Avian Influenza Virus, which can cause up to 100% outbreak rate with/or death rate and even can infect mankind and kill them. AIV's ecological and common healthy meaning already acquired mankind's highly concern. AIV already exists all over the China, and result in different degree economic loss.In order to reduce the loss, to provide strong foundation in vaccine study, construct and identify a recombinant of canine type 2 adenovirus expressing HA protein of H5N1 subtype tiger avian influenza virus, which can be used as the normal routine immunization, reduce the occurrence of avian flu, increase the capacity of our country to prevent and control avian flu.[Methods] With molecular biology technology, to construct eukaryotic expression vector pCAGGS-HA expressing HA gene of avian flu virus;using indirect immunofluorescence and indirect ELISA method detect which expression in the mammalian cells BHK-21 cells; HA expression cassette fragment of pCAGGS-HA was cloned into the E3 area of the CAV-2 vector pPoly2-CAV2, obtain the recombinant plasmid pCAV-2-HA which NP expression cassette was inserted in missing E3 zone. Using the method of liposome-mediated, import the recombinant plasmid pCAV-2-HA 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 HA expression cassette fragment whether or not missing and transcription of the HA gene mRNA through application of PCR and RT-PCR, using indirect immunofluorescence and ELASA indirect method to detect of the expression of HA gene in mammalian cells of DK, the application of conventional virus cultured in vitro to detect the genetic stability of recombinant virus.[Results] The eukaryotic expression vector pCAGGS-HA can express HA protein in mammalian cells correctly and possess its corresponding biological activity, recombinant virus of CAV-2-HA has the typical morphological characteristics and blood coagulation properties of CAV-2, the fragment of HA expression cassette is not missing in the process of reproduction, and HA gene transcription to mRNA, ELISA and immunofluorescence analysis showed that the product of recombinant expression can be identified by the HA monoclonal antibody of avian flu virus, has a good in vitro genetic stability.[Conclusion] In this experiment, construct the recombinant vector pCAV-2-HA of canine type 2 adenovirus expressing HA gene of A/Tiger/Harbin/132/2007 successfully; transfect and obtain the recombinant virus CAV-2-HA in mammalian cells successfully which can express biologically active recombinant HA protein.