Construction Of H5N1 Vaccine And The Establishment Of The Vaccine Production Process

Avian influenza is caused by the species influenza A virus adapted to bird. The highly pathogenic avian influenza virus subtype H5N1 cross species barriers to infect humans and other mammals, often with fatal outcomes. Today, the only effective way to prevent the spread of the disease is the inoculation of vaccine, But the traditional production technique cannot meet the demand when the Avian influenza outbreak.A recombinant Bombyx mori baculovirus, vBmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. We constructed the baculovirus transfer vector pFastBacHTb- gp64-HA. The recombinant transfer plasmid was transformed into the BmDH10Bac competent cells, and inserted into the Bacmid DNA by transposition recombination. Then we constructed the recombinant virus vBmgp64-HA. The BmN insect cells were infected with the recombinant virus discernly. The result of Western blotting, SDS-PAGE and haemoglutination test (HA) showed that it was successfully expressed in infected BmN cell.We could get large-scale recombinant baculovirus from the infected bombyx mori pupa, because the production was up to 1 mg per pupa. It provided a solid foundation to study and optimize the production technique to produce good vaccine in future.In the initial production technique, we used low-speed centrifugation, high-speed centrifugation, ultracentrifugation and zone centrifugation to purify enveloped virus .The specific activity of semi product was 206 u/mg, and the HA activity recovery was 6.8%, but it failed to find the target protein in the SDS-PAGE and Western- Blotting. It was the silkworm pupa oil whose content was 20% in the silkworm pupa seriously affect the separation of the virus, but also caused a false positive in the hemagglutination test. Then we improved the production technique by steps of ammonium sulfate precipitation, it played a good role in separeting the silkworm pupa oil from the taeget protein, and with 60% saturation ammonium sulfate solution deposition virus particles instead of ultracentrifugation, we could see higher activity recovery(88.9%). In SDS-PAGE and Western-Blotting analysis, now there was an obvious target protein band. The hemagglutination titer of semi product which produce from improved production technique was 28, but its protein concentration reached 103 mg/mL because there was too much other protein, not only caused low speed of ultrafiltration, but also caused the failue of removing bacteria by membrane filtering. It was necessary to improve the production technique, so we used 10μm,0.45μm filter to remove other proteins, added one zone centrifugation, and used desugarization by dialysis instead of desugarization by ultrafiltration to purify the recombinant virus. With these methods, the specific activity of the sample from the second zone centrifugation increased to 100 u / mg, and hemagglutination titer of the samples recycled from zone centrifugati reached1: 24, the protein concentration was only 3.2 mg / mL, and its specific activity was up to 100 u / mg. It laid a good foundation of further studies of production technique.