| Avian influenza virus (AIV) has caused huge losses in poultry industry. One of which H5subtype AIV not only can cause high mortality in poultry, but also can be transmitted to humans by the poultry, and results in threaten on public health. The development of AIV vaccines is important to prevent and control infection of H5subtype AIV. In order to improve the immune efficacy of vaccine or avoid interference on differentiating infected from vaccinated animals, new engineering vaccine was developed. The hemagglutinin (HA) of AIV is the major protective antigen that it can induce protective immunity, and chicken interleukin6(ChIL-6) can improve the immune efficacy of vaccines. So the recombinant fowlpox virus co-expressing HA gene and ChIL6can induce more protective immunity. In this study, H5subtype AIV ha strain(clade2.3.2.1) which is a prevalent strain now were selected as the parent virus, Then, the recombinant fowlpox virus (rFPV) expressing HA gene of AIV ha strain (rFPV-haHA) and co-expressing HA and ChIL-6genes (rFPV-haHAIL6), and their immune response in SPF chickens were evaluated. This study laid the foundation for the development of rFPV vaccines against avian influenza.1. Construction of recombinant fowlpox virus (rFPV) expressing HA gene of H5subtype AIV (rFPV-haHA) and co-expressing HA and ChIL-6genes (rFPV-haHAIL6) To eliminate BamHI site in HA gene of AIV ha strain, HA gene with synonymous mutation was amplified by overlap PCR. Then, the HA gene was inserted into transferring vector p12LS under the control of promoter Ps to construct the recombinant transferring vector p12LShaHA. Further, ChIL-6gene regulated by promoter PE/L was inserted into the transferring vector p12LShaHA, so that the two genes can reverse tandem to obtain transfer vector p12LShaHA-IL6. The transferring vector p12LShaHA and p12LShaHA-IL6were used to transfect by Lipofectin.reagent. The rFPV-haHA and rFPV-haHAIL6were obtained by selection of blue-white plaque. DNA of recombinant fowlpox viruses were extracted and subjected to PCR to confirm the insertion of HA gene and IL-6gene in the chromosomal DNA of PFV. The results of IFA confirmed that the HA gene and IL-6gene were expressed in rFPV. The growth curves of rFPVs were similar as that of wt-FPV.2. Immune efficacy of rFPV-haHA and rFPV-haHAIL6in SPF chickens10-day-old SPF chickens were grouped randomly. The chickens were immunized with rFPV-IL6, rFPV-haHA, rFPV-haHAIL6and wt-FPV at a dosage of105PFU/0.2mL, respectively.. The chickens was also immunized with oil adjuvant inactivated vaccines of AIV ha strain (clade2.3.2.I) and AIV lk strain (clade2.3.4) as positive control, and PBS as negative control. The weight gains of chickens were recorded on days0,6,13and20postimmunization. HI titer of antibody, lymphocyte proliferation and T cell subgroup analysis with peripheral blood on days7,14,21postimmunization were determined. The results showed that the rFPVs had no effect on weight gain of chicken. Although HI antibody level induced by rFPV-haHA and rFPV-haHAIL6in chickens were significantly lower than that by oil adjuvant inactivated vaccines, the lymphocyte activation and ratio of CD4+and CD8+induced by rFPV-haHA and rFPV-haHAIL6were increased,, indicating a stronger cellular immunity. When the vaccinated SPF chickens were challenged with virulent AIVs, the protection rates against the parent virus induced by rFPV-haHA and rFPV-haHAIL6were91.6%, respectively. The protection rates against the heterologous AIV lk strain induced by rFPV-haHA and rFPV-haHAIL6were50%and58.3%, respectively. The rate of virus shedding in chickens immunized with rFPV-haHAIL6was lower than that with rFPV-haHA. |
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