Porcine rotavirus G4-G5-G9 type trivalent inactivated vaccine as well as preparation method and application thereof
The technology of porcine rotavirus and inactivated vaccine is applied in the field of porcine rotavirus G4\G5\G9 trivalent inactivated vaccine and its preparation, which can solve the problems of dead pig breeding industry economy, piglet disease, loss and the like, To achieve the effect of good immunogenicity, high safety and convenient operation
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Embodiment 1
[0041] Example 1 Isolation, identification, cloning, purification and biological characteristics of porcine rotavirus
[0042] 1.1 Isolation and identification of viruses
[0043] The disease material was obtained from the small intestine of pigs with clinical symptoms of diarrhea in large-scale pig farms in my country. The positive small intestine tissue was taken, cut into pieces with scissors, added with 10.0ml of PBS, ground with a tissue grinder or mortar, and then transferred to a 15ml centrifuge tube. Repeat freezing and thawing 3 times below -70°C, take four 1.5ml centrifuge tubes, add 1.0ml small intestine tissue fluid to each centrifuge tube, centrifuge at 12000r / min for 5 minutes, filter and sterilize with 0.22μm microporous membrane, and inoculate MA104 cells that have grown into a monolayer (drain the cell culture medium before inoculation and wash three times with serum-free DMEM medium), adsorb at 37°C for 1 hour, aspirate the inoculum, add 5ml of cell maintenance...
Embodiment 2
[0085] Example 2 Preparation and inspection of porcine rotavirus G4\G5\G9 trivalent inactivated vaccine
[0086] A preparation method of porcine rotavirus G4\G5\G9 trivalent inactivated vaccine, comprising the following steps:
[0087] 1 Culture technology of porcine rotavirus (G9-CH09, G5-KQ06 and G4-HN01 strains) virus liquid on PK-15 cells
[0088] In order to facilitate the industrial production of porcine rotavirus, the culture process of suspension PK-15 cells in the shake flask was determined by exploring the passage density and shaking speed of the suspension type PK-15 cells in the shake flask: 1.0× 10 6 Dilute passages at an initial density of cells / ml at 37°C, 130r / min, 5%CO 2 After 72 hours of culture in a horizontal shaker, the cell density can reach 6.0 to 6.5 × 10 6 When the porcine rotavirus was inoculated in a shake flask, the cell density, trypsin concentration, inoculation amount, inoculation time and other process conditions were explored, and the porcin...
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