Study On DNA Vaccine Which Expresses HA Gene Of H5 And H7 Subtype AIV

A successful avian influenza vaccine should provide wide protection against the challenge of several subtypes of AIV. But it is very difficult to develop an ideal vaccine providing cross protection against all subtypes of AIV, for the highly frequent antigenic variation of HA and NA gene of AIV. As long as the high mutation is concerned, it is a risk to use traditional attenuated vaccine which may drift to HPAIV, but inactivated vaccines also have some disadvantages such as pool efficacy, high cost et al. So, many researchers have been exploiting new influenza vaccines. In this study, a double-valence mammalian Expressing plasmid which encodes H5 and H7 subtype AIV HA gene was constructed, and the immunogenicity and protective efficacy was tested. 　　 Haemagglutinin is one of the surface proteins of AIV, and it is also the important protective antigen. To gain a DNA vaccine which can provide protection not only against H5 subtype of Avian Influenza Virus but also H7 subtype, the recombinant plasmid co-expressing H5HA and H7HA protein of AIV have been designed and constructed. H7HA gene of AIV A/FPV/Rostock/34(H7N1),which derived from recombinant plasmid PCI-H7HA,was subcloned into plasmid PCI-H5HA and a double-valence DNA vaccine which was named PCI-H5HA-H7HA have been constructed. Both H5HA and H7HA gene were promoted by CMV, and were followed by Poly(A) sequence. In order to testify the expression in mammalian cell of this plasmid, the DNA vaccine was transfected into 293-T cell, and indirect-- immunofluorescent test was conducted 48h after the transfection. The result indicate that H5HA and H7HA can all be expressed in mammalian cell, in addition, the expression quantity of H5HA protein is higher than H7HA. This study lay the foundation for developing immune test on chickens and the assessment of immune efficacy .After the indirect-immunofluorescent test, the outcome of immune protection trial was promising. And the plasmid was used to immunize 4-week-age SPF chichens by intramuscle injection, for comparison, another group of SPF chickens were vaccinated with PCI-H5HA and PCI-H7HA jointly .The Haemagglutinin inhibition(HI) titer induced by DNA vaccine were tested weekly for 6 weeks and the protection ratio were calculated. The SPF chickens were challenged with H5N1 or H7N1 isolate of HPAI virus by the sixth week after the immunization. And the result show, 50% and 40% protection rate against H5N1 and H7N1 were obtained prospectively from PCI-H5HA-H7HA vaccinated groups, while 70% and 68.4% of protection rate were obtained from the groups immunized with PCI-H5HA and PCI-H7HA jointly .The study suggested the possibility that the double-gene plasmid can induce protection more or less, but the titer is yet unsatisfactory, inferring that the ingestion of DNA and its expression level may play an important role. In order to improve the protective efficacy ,the further study on various influencing factors still need.