| Avian Influenza (AI) is an infection and or disease syndrome caused by type A influenza virus. It is widespread in many countries and areas throughout the world. Not only AI brings about a lot of losses to poultry industry, but also threaten to human health. The major characteristic of the highly pathogenic avian influenza(HPAI) is sudden incidence and high mortality. Although inactivated virus vaccine and attenuated vaccine play a great role to control AI, the disadvantage of hardly differentiation vaccinated from infected chickens still exist. However, researches on gene engineering vaccine is a good solution to the problem, especially on virus vector vaccine.Herpesvirus of turkey(HVT) which are used for virus vector vaccine has many merit : no pathogenesis to chicken and other animals , persistent existence in host which can stimulate immune system effectively. It has a good application prospect. In the study with homologue recombination ,AIV H5N1 HA cassette was integrated into nonessential region US2 and US10 of HVT genome respectively, and recombinant HVTs expressing AIV HA antigen were constructed. Comparison of recombinant HVT with HA cassette in the locus of US2 or US10 region(designed rHVT-US2-HA & rHVT-US10-HA) on virus replication and immune protect efficiency was carried.First, gpt selection marker and AIV H5N1 HA cassette were subcloned into pGAB orderly and US2 region transfer vector was constructed and named pGAB-gpt-HA. US10 gene flanking sequences, gpt selection marker and HA cassette were subcloned into pUC119 vector, the pUAB-gpt-HA was constructed.Then these transfer vectors were co-transfected with herpesvirus of turkey (HVT) total DNA into the chicken embryo fibroblast cells (CEF) respectively to generate recombinant HVT by homologous recombinantion. The recombinant HVT with the HA gene integrated in the diverse loci of US2 and US10 region of HVT (designated as rHVT-US2-HA and rHVT-US10-HA) were obtained by gpt selection and identified by PCR. The expression of HA gene was verified by indirect immunofluorescence assay, western blot and hemagglutination test in infected CEF. The two recombinant HVTs can both express biological AIV HA antigen and rHVT-US2-HA has higher HA expression level than rHVT-US10-HA. rHVT-US2-HA showed the same with parental virus in in vitro kinetics, neither dose rHVT-US10-HA. rHVT-US10-HA has a new figure of plague with obvious cell fusion .At last, rHVT-US2-HA and rHVT-US10-HA were immunized into chickens to evaluate their immune protective effects. In AIV challenge immune protective assay, the HA antibody level of rHVT-US2-HA group reached 4.05log2, which is higher than rHVT-US10-HA group(3.15log2). And rHVT-US2-HA group has a better immune protective effect than rHVT-US10-HA group. In MDV challenge immune protective assay, the protective index of rHVT-US2-HA and rHVT-US10-HA group were 65.3%和78.9% respectively, which was no obvious difference. It is demonstrated that recombinant HVT with nonessential US2 gene integrated with AIV HA gene has better immune protective effect.In summary, the recombinant HVT with US2 or US10 gene as recombination region expressing AIV HA gene was evaluated in vitro and in vivo, which provided useful information of choosing nonessential region of HVT to construct recombinant virus and laid a foundation for develop AI live vaccine.
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