Construction And Immune Efficacy Of Recombinant Fowlpox Virus Co-expressing M2Gene And HA Gene Of H5Subtype Avian Influenza Virus

H5subtype avian influenza (AI) has caused serious economic losses to the poultry industry in our country, which is prevented and controlled majorly by vaccine immunization. Due to the rapid variation of H5N1subtype avian influenza virus (AIV), the speed of vaccine development can’t match up the speed of antigen variation. It is necessary to develop a cross-protection vaccine. M2protein which has highly conserved antigenic epitopes is a transmembrane protein of AIV, and it is one of the ideal candidate antigens for cross-protection vaccine. Thereafter, we constructed the recombinant fowlpox virus (rFPV) co-expressing M2gene and hemagglutinin (HA) gene of H5subtype AIV and evaluated its immune response. This study, laid the foundation for the development of new avian influenza vaccines.1. Prokaryotic expression of M2gene without transmembrane region of H5N1subtype AIVTransmembrane region of M2gene was replaced with four glycines, and the shorten M2gene (sM2) was amplified by overlap PCR and inserted into prokaryotic expression vector pGEX-6p-1. The expressed protein was induced by IPTG and was purified by glutathione resin. The result of SDS-PAGE showed that sM2fusion protein is soluble with molecular weight of36kDa. The antiserum against M2protein was prepared by immunized mice with the purified sM2fusion protein. The result of Indirect Fluorescence assay (IFA) revealed that the antisera produced a specific fluorescence in MDCK cells infected with H5N1subtype AIV, indicating that sM2protien had a good immunogenicity.2. Development of monoclonal antibodies against sM2protein8-week-old Balb/c mice were immunized with100μg purified sM2fusion protein. Spleen cells of immunized mouse were fused with myeloma cells(SP2/0) after forth immunization. Four monoclonal antibodies (McAbs) were obtained by ELISA using the sM2fusion protein or GST protein as coated antigens. These McAbs were named as1A9,2D4,3E6and4E7, which isotype belonged to IgG2b, IgG1, IgG2a and IgG2a respectively. The result of IFA showed that specific fluorescence was observed when MDCK cells were infected with different clades of H5subtype of AIV and stained with McAbs, indicating that these McAbs can be used to detect M2protein of H5subtype AIV.3. Construction of the recombinant fowlpox virus (rFPV) co-expressing M2gene and HA gene of H5subtype AIVThe full length of M2gene was PCR amplified and inserted into FPV transfer vector p12LS to construct the recombinant transfer vector p12LSM2; M2gene regulated by promoter PE/L was inserted into FPV intermediate transfer vector p12LSHA, so that the two genes can reverse tandem to obtain transfer vector p12LSHAM2. Then the p12LSM2and p12LSHAM2were used to transfect by Liposomal into chick embryo fibroblast (CEF), which was pre-infected with wide type FPV. The purified rFPV-M2and rFPV-HAM2were obtained by selection of blue-white plague. The insertion of taget genes into the FPV genome DNA was confirmed by extracting of the genome DNA of rFPV and PCR amplification of the M2gene. The results of IFA and Western-blot indicated that M2gene and HA gene were expressed correctly in rFPV.4. Immune efficacy trials of recombinant virus in SPF chickens4groups of7-day-old SPF chickens were randomly assigned and inoculated subcutaneously with2×104PFU rFPV-M2, rFPV-HA, rFPV-HAM2, and wt-FPV respectively, other3groups were vaccinated with0.2mL RE-5, RE-6killed vaccines and PBS as the control. The weight gain of chickens, HI titer of antibody, and lymphocyte proliferation with peripheral blood were determinated at different days. Then these chichens were challenged with H5N1 subtype AIV of two different clades with105.5EID50/chicken at21d after immunization. The results showed that there was no significant difference in the weight gain of the chickens immunized rFPVs and normal control; lymphocyte proliferation level induced by rFPV-HAM2was higher than that induced by rFPV-M2, and both were higher than other groups. When the chickens were challenged with LK strain and HA strain, the protection rates of chickens immunized with rFPV-HAM2were50%and42%, respectively, which were a bit higher than that of chickens immunized with rFPV-HA (30%and25%), and lower than that of RE-5and RE-6immunized chickens. Also, the shedding rate of chickens in rFPV-HAM2group was a little lower than that of chickens in rFPV-HA group.