A kind of attenuated canine distemper strain and its prepared vaccine composition and application
A vaccine composition and canine distemper virus technology, which can be applied in the directions of vaccines, veterinary vaccines, biochemical equipment and methods, etc., can solve the problems of canine distemper epidemic that cannot be effectively controlled, and the antigenic targeting is not strong.
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Embodiment 1
[0034] Example 1 Acquisition of canine distemper virus HL001-M3 strain
[0035] 1. Take the well-grown Vero cells, digest with trypsin, inoculate in a cell flask, and use a cell growth medium ( The pH was adjusted to 6.8-7.2) and the culture was continued at 33°C to 37°C to form a good monolayer for virus inoculation.
[0036] 2. The canine distemper virus HL001 strain is inoculated into the well-grown passaged cell monolayer, and the cell growth solution (pH) containing 95% to 99% by volume of DMEM medium and 1% to 5% by volume of newborn bovine serum is used. Adjusted to 6.8-7.2) Continue to culture at 33°C to 37°C. After 72h to 96h, when more than 80% of the cells become diseased, harvest the cell culture virus solution.
[0037]3. Repeat the above steps for continuous subculture to obtain the attenuated canine distemper virus strain, and the obtained attenuated canine distemper virus strain is sequenced. The results show that the nucleotide at 1430 of the H gene is stably...
Embodiment 2
[0038] Example 2 Biological characteristics of canine distemper virus HL001-M3 strain
[0039] 1. Pathogenicity test
[0040] Fifteen healthy susceptible antigen-antibody-negative dogs aged 2-3 months were randomly divided into 3 groups, with 5 dogs in each group.
[0041] Table 1 Grouping of pathogenic test animals
[0042] group Inoculation strain Inoculation dose 1 HL001-M3 strain Nasal 2ml, abdominal cavity 4ml (10 5.5 FAID 50 / ml)
2 HL001 strain Nasal 2ml, abdominal cavity 4ml (10 5.5 FAID 50 / ml)
3 DMEM medium Nasal 2ml, abdominal cavity 4ml
[0043] Observed for 21 days after the virus inoculation, the dog's body temperature, spirit, appetite, feces, eye and nasal secretions were observed and recorded every morning and evening. The specific results are shown in Table 2.
[0044] Table 2 Pathogenicity of HL001-M3 strain to dogs
[0045]
[0046] The results showed that the canine distemper virus HL001 strain coul...
Embodiment 3
[0072] Example 3 Preparation of canine distemper virus HL001-M3 strain attenuated live vaccine
[0073] 1. Virus proliferation
[0074] The canine distemper virus HL001-M3 strain prepared in Example 1 was synchronously inoculated with the Vero cell suspension, added with DMEM medium containing 2% newborn bovine serum, and placed at 37° C., 5% CO 2 nourish. After 80% of the cells were damaged, the virus was harvested, the virus titer was determined, and the cells were stored at low temperature.
[0075] 2. Preparation of protective agent
[0076] Add 40 g of sucrose and 8 g of gelatin to each 100 ml of deionized water, and after fully melting, set it to high pressure steam sterilization (121° C. for 30 min).
[0077] 3. Preparation of the vaccine
[0078] The virus liquid prepared and stored above was mixed with protective agent at 1:1 (volume ratio), and lyophilized. The specific ratio of vaccine content is shown in Table 4.
[0079] Table 4 Canine distemper virus HL001-...
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