Study On The Gene Engineering Subunit Vaccine Of H7 Subtype Avian Influenza

Content: The complementary DNA of 1.7kb HA gene was prepared from the viral gene RNA by RT-PCR. The amplified fragment was cloned into the plasmid vector pMD 18-T,then sequenced and subcloned into the downstream of the baculovirus transfer vector pBlueBac4.5/V5-His-TOPO and named as pH7HA. The pH7HA was sequenced and co-transfected the sf9 cell with the lined Bac-N-Blue^? DNA by the technique of cationic liposome mediated transfection. The recombinant baculovirus was purified by three cycles of plaque assay with the chromogenic substrate X-gal in the low temperature melting agarose and analyzed by PCR. By means of that we obtained several strains of recombinant baculovirus rpH7HA. We confirmed that the target gene fragment was inserted into the baculovirus genome by PCR. SDS-PAGE result showed that the HA gene was expressed in the baculovirus / insect expressing system. Additionally, the Western-blot assay proved the HA protein of H7N1 subtype avian influenza virus AIV having good biological activit.The HA protein was emulsified into the Freued' s adjuvant vaccine. Fifteen SPF chikens separate into 3 groups:vaccine one time group,vaccine two time group and Control group. H7HA vaccine used to immunize 3- week-age SPF chichens by intramuscle injection 20μ g per chicken, after two weeks, vaccine two time group immunize the H7HA protion again. The SPF chickens were challenged with H7N1 isolate of HPAI virus by the fourth week after the immunization. The result show, vaccine one time group and vaccine two time group protection rate against H7N1 are 40% and 100% . We also examine conditin of virus release, vaccine two time group virus release rate is 0% . We expectation to solve the problem and makes the contribution for our country's AIV manufactures.We expresse Avian Influenza Virus H7HA gene protion in recombinant baculovirus on large scale.The result indicated that, if we maintenance the rigt temperature 27.5°C, the rotational speed 150rpm, the increase right amount surface active agent l0u/L and suitable cell 2×10⁵～3×10⁵cell/ml may pass the insect/ baculovirus expresses the system to transfer the bottle to obtain lots of protein,which has the biology active goal. This experiment may take this vaccine industrial production the important basis, at the same time also may for next step continue to ferment the pot research to provide the important foundation.