Expression Of H5 Subtype Of Avian Influenza Virus Hemagglutinin Protein By Insect Cells And The Research Of Its Immunogenicity

H5N1 subtype highly pathogenic avian influenza viruses (AIV) not only cause disaster to poultry industry, but also threaten to human public health security. Immunization of inactive vaccine is one of the effective strategies to prevent and control this disease. The process of screening of vaccine candidate is time-costing and labor-consuming procedures. The production of vaccine is involving live virus and restricted by suppling of chicken eggs. The AIV subunit vaccine based on hemagglutinin (HA) gene can avoid the disadvantages of inactivated vaccine. Hemagglutinin protein which is one of the domainant surface antigens, can elicite antibody in animal and is the primary target protein of development of subunit vaccine. Previously studies showed that the biological activity, antigenicity and immunigenecity of HA protein and Matrix protein were expressed by baculovirus system are similar to natural HA protein in virions. But there are only few reports abou the biological activity, antigenicity and immunigenecity of HA protein were expressed by baculovirus system are similar to natural HA protein in virions The current study adopted baculovirus system to expression of recombinant HA derived from H5 subtype of AIV, and characterized the hemagglutinin activity, probability of assembles of virus-like particles and immunigenecity of the recombinant HA protein.Construction of HA contained clone vector. Two stratigies werte were employed to get HA gene. One of the strategies was cloned HA gene from H5 subtype AIV (Re-6) detection antigen with RT-PCR, and named as L-HA. In another method, the consensus HA gene was selected from the results of aligment of HA gene from GenBank, named as H-HA. The selected HA genes were synthesized after optimization of codon based on codon usage bias of insect cell. The intergrity HA gene (1857 bp) or lack of signal peptide and transmembrane of HA gene (1701 bp) was amplified by PCR with templates of synthesized plasmid(HHAc-T and LHAc-T) containing the HA gene, and further inserted into the pMD-19 T vector. The recombinant plasmid wrer named as L-NSTHA-19T, L-HA-19T, H-NSTHA-19T, and H-HA-19T, respectively.Construction of recombinant baculovirus vectors to express HA gene and charaterization of recombinant HA protein. The recombinant baculovirus HA genes were constructed as following briefly description. The target HA genes in pMD-19 T vector were treated by EcoR Ⅰ and Hind Ⅲ enzyme and inserted into pFastBac1 vector (pF-H-HA, pF-H-NSTHA, pF-L-HA, and pF-L-NSTHA), and then transformated into DH10 Bac competent cell with three round of blue/white screen tests to get series of recombinant bacmids (rBac-H-HA, rBac-H-NSTHA, rBac-L-HA, and rBac-L-NSTHA). These rBacmids were transfection into Sf9 cell to product recombinant baculovirus. The supernatant of lysis recombinant baculovirus Sf9 cell cultures were collected at 96h after infection. The supernatant solutions were characterized by HA test, SDS-PAGE, Western-Blot and IFA. Results showed that the HA activity and immunogenicity of of these recombinant HA proteins were similar to that of the inactivated H5 viruses. The virus-like particles (VLPs) were observed by detection of tranmision electron microscope.Characterization of imunogenecity of recombinant HA proteins in chickens. Fouteen days old specific pathogen free chickens were vaccinated with oil emusion vaccine which contain the H-HA and L-HA recombinant protein derived from the Sf9 cell. Serum were collected via wing veins at 14-,21-, and 28-day post vaccination, and the antibody against HA were tested by hemagglutinantion inhibition test and ELISA, respectively. Results showed that both L-and H-HA recombinant proteins succefully elicited higher antibody in chickens.The FACS was employed to test dynamic changes of subgroups of CD3+/CD4+ and CD3+/CD8+T lymphocytes at time point of 3-,5-,7-, and 10-day post vaccination. Lymphocyte transformation test was also adopted to test the total lymphocytes activities at the same time point of FACS. Results showed that the rates of subgroups of CD3+/CD4+ and CD3+/CD8+ T lymphocytes in the two groups of chickens injection with vaccine which was made of L-HA or H-HA recombinant proteins containing vaccine were slightly higher than of the chickens immunized with inactivated H5 subtype vaccine. The results of lymphocyte transformation test were similar to those of FACS detection. These results indicated that the HA recombinant prtoteins based vaccine were effectively induced humoral antibody response and cellular immune response. The recombinant HA prtoteins showed good immunigenecity and the overall effect of immune reponse elicited by recombinant HA prtoteins slightly better than commercial inactivated H5 subtype vaccine.Taken together of all results in this study, Althought the recombinant HA proteins expressed in a baculovirus espression system always has been reported, the low titer of the recombinant HA protein. the recombinant H5 subtype HA prtoteins were successfully expressed in Sf9 cell and the higher HA titer of recombinant protein,these proteins showed good immunigenecity in chikens. The current study also provided technical database and biomaterial supports for future H5 subunit vaccine development.