Screening On Adjuvants Of AIV H9N2 Virus-like Particles Vaccine

To make the H9N2 virus-like particle vaccine into market as quickly as possible, and to combat H9N2 avian influenza effectively,this subject plans to screen out one of the most suitable adjuvant for avian influenza H9N2 virus-like particle vaccine among claasical white oil adjuvant, ISA 206 adjuvant and the microspheres adjuvant. The main content of this subject includes the following sections.1 The preparation of the H9N2 subtype avian influenza virus-like particles In this study, we first synthesized the HA gene, NA gene and M1 gene of the H9N2 avian influenza virus.And the HA gene, NA gene, M1 gene were inserted into a baculovirus expression vector forming a recombinant plasmid named p Fast Bac-Dua L-HA-NA-M1. The recombinant plasmid was introduced into E.coli competent cells, then we identified the plasmids after blue-white scanning. Qualified recombinant plasmids containing HA gene, NA gene, M1 gene were screened out. The qualified recombinant plasmids were transfected into sf9 insect cells, gaining P1, P2, and P3. And the virus-like particles finally obtained, which were the materials for the next test.2 The preparation and screening of H9N2 virus-like particles vaccine with with oil adjuvant, ISA206 adjuvant and the microspheres adjuvant Making prepare of vaccines with oil adjuvant with different oil-water ratio and different concentrations of aluminum stearate, vaccines with ISA 206 adjuvant, vaccines with microspheres,through the results of the viscosity testing, the stability testing, the sterility testing and the safety testing, qualified vaccines were screened out.The best oil-water ratio was 3:1, and the best concentration of aluminum stearate was 0.5%. The optimum time of screened microspheres adsorbing virus-like particles was 3 hours, and the optimum volume of 5mg screened microspheres adsorbing virus-like particles was 8m L. The loading capacity of the screened microspheres was89.04% and the loading efficiency was 38.45%.3 The evaluation of the VLP vaccines with different adjuvants The lysozyme content level is an indicator of animal non-specific immunity. The study showed that the group of the VLP vaccine with microspheres had better performance that other groups of VLP vaccine with adjuvants. This test also measured the humoral immunity level and the cells immunity level of each testing groups, and the results showed that virus-like particles without adjuvants produced an effective humoral immunity into the body, but it had short duration and low antibody levels. Virus-like particle vaccines with different adjuvants all producted effectively antibody levels and their duration and antibody levels were longer and higher.The oral microspheres group had longest duration and highest antibody level than the other vaccines with white oil adjuvant and ISA 206 adjuvant. The three kinds of adjuvants did not produce an effective cellular immunity.The study showed that The microspheres adjuvant is most suitable adjuvant used in H9N2virus-like particle vaccine, compared the other two adjuvants: the white oil adjuvant and th ISA 206 adjuvant. The oral immunization route of H9N2 virus-like particle vaccine with microspheres is more convenient than injections, and can obtain a higher level of humoral and duration.