H3 subtype canine influenza virus strain, inactivated vaccine and preparation method thereof
A technology of canine influenza virus and inactivated vaccine, which is applied in the field of H3 subtype canine influenza virus strain, its inactivated vaccine and its preparation, can solve the problems restricting the development of vaccines, and achieve good protection, good safety and stability sex high effect
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Embodiment 1
[0043] Separation and identification of viruses:
[0044] (1) Virus separation: In 2019, the nasopharyngeal swabs were collected from Beijing Yanqing, a pet hospital respiratory symptom, and after the collected nasopharyngeal sterile treatment, inoculated 10 days of age SPF chicken embryo, each embryo 0.2 ml, 37 ° C hatched, abandoned within 24 hours of death embryo, 72 hours, collected chicken embryo cargo liquid, measure blood coagulation, and prepare for -70 ° C.
[0045] (2) Split of the virus: Extract the separated dog influenza HB strain to extract viral RNA, performing the sequencing analysis of the sequence of RT-PCR amplification HA and NA fragments. The results show that the separation plant is a dog flu virus H3N2 subtype strain, named A / CANINE / Huabei / 2019 (H3N2), figure 1 and figure 2 . The virus strain was stored in the ordinary microbiological center of China's Microbial Square Safety Management Committee on April 2021, and the number is CGMCC.NO.20649.
[0046...
Embodiment 2
[0050] Toxic culture conditions and inactivation conditions
[0051] (1) The optimal vaccination dose, temperature and time selection: Take 10 days old chicken embryo randomly divided into 15 pieces / group, virus virus virus content 10 3.0 EID 50 / 0.1ml, 10 4.0 EID 50 / 0.1ml, 10 5.0 EID 50 The production versus, 0.2 ml / embryo, which was placed under 37 ° C and 35 ° C, respectively, 48 hours, 64 hours, 72 hours, respectively, and the HA titer of different groups of chicken embryo-liquid , Harvest and virus content, determining the vaccination dose of 10 4 EID 50 / 0.1ml, the optimum culture temperature is 35 ° C, and the optimum harvest time is 48 hours.
[0052] (2) Best Inactivation Time: Add the formaldehyde solution at the end concentration, mixed with a constant temperature of 4 ° C, 1 to 7 days after inactivation, etc., samples are inoculated in 10 days age SPF chicken embryos 5 Enum, 0.2ml / embryo, 37 ° C for 120 hours after 120 hours, Errrrocytocystone liquid HA titer,...
Embodiment 3
[0054] Preparation of Dog Flu Virus H3 subtype inactivated vaccine
[0055] (1) Inoculation: H3 subtypeless influenza virus is 2 × 10 in the diaper cavity pathway 4 EID 50 The dose of the enumeration is inoculated at 10-day-old SPF chicken embryo, incubated at 35 ° C;
[0056] (2) Incubation and observation: 48 hours after inoculation, 1 time per day, 48 hours within 48 hours, 48 hours after inoculation, the active embryo is taken out of 2 ~ 8 ° C cooling overnight 6 to 18 hours;
[0057] (3) Harvest: Take the cooled chicken embryo, disinfectantly peeled gas chamber, sterile stripping gas chamber, absorburial liquid, centrifugally, the supernatant is stored in 2 ~ 8 ° C is a virus stock solution; According to the "People's Republic of China", the number of infection measurements specified in the three-year version of the 19th year of the Chinese Pharmacopoeia conducted by the virus content; image 3 reward)
[0058] (4) Concentration and Washing Virus Rate: The viral stock soluti...
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