Seneca valley virus vaccine and its preparation method and application
A virus vaccine, Seneca's technology, applied in the field of biotechnology and biological products, can solve problems such as loss of control and severe forms of prevention and control
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Embodiment 1
[0059] Embodiment 1: Isolation, cultivation and screening of SVV / FJ / 001 strain
[0060] 1.1 Virus isolation
[0061] Collect the vesicle skin, vesicle fluid and other diseased tissues of SVV-infected pigs, add PBS to grind, after repeated freezing and thawing 3 times, centrifuge at 4°C, 5000rpm for 10min, collect the supernatant, filter through a 0.22μm filter, extract RNA, and use RT-PCR And QRT-PCR and other methods to identify that the disease material does not contain other pathogens such as foot-and-mouth disease virus that can cause vesicular lesions in pigs except for SVV.
[0062] Inoculate the above-mentioned identified disease material into newly formed BHK-21 cells at 10% of the virus culture solution, incubate at 37°C for 1 hour, add DMEM culture solution, culture at 37°C for 5 days, harvest the virus solution, and pass through 3 times After repeated freezing and thawing, the second generation was inoculated, cultured for 4 days, and the toxic medium was harvested...
Embodiment 2
[0077] Example 2: Identification of Seneca Valley Virus
[0078] 2.1 Identification of virus by RT-PCR
[0079] The supernatant of cells such as BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells infected with the stably passaged SVV / FJ / 001 strain was used with RNAeasy Mini Kit (Qiagen ) to extract total RNA, after reverse transcription, amplify the P1 gene, purify and recover it, and then send it for sequencing. The results show that the P1 gene can be amplified.
[0080] 2.2 Identification of viral antigens by indirect immunofluorescence
[0081] After the culture of BHK-21 cells infected with SVV / FJ / 001 strain was repeatedly frozen and thawed, it was inoculated into a six-well plate with slides placed at the bottom and grown with BHK-21 cells (monolayer cells grew to 60%-70 %), placed in 5% CO 2 In a 37°C incubator, indirect immunofluorescence was performed according to the conventional method after 24 hours. The primary antibody w...
Embodiment 3
[0082] Example 3: Establishment of SVV / FJ / 001 strain to pig challenge model
[0083] Six test pigs were screened from non-epidemic areas, and tested by neutralization experiments, the SVV neutralizing antibody titer was 9 TCID 50 , at the same time set up grinding tissue disease material virus as a control, the challenge dose is 2mL / head, observe continuously for 15 days, observe and record the disease situation, judge the disease situation of animals according to the symptoms such as hooves, nose mirror and lips blisters, and count Points (5-point system, the judgment standard is formulated according to the foot-and-mouth disease scoring standard, the higher the score, the more serious the clinical symptoms). The results of the challenge showed that the SVV / FJ / 001 strain showed typical clinical symptoms from the second day after the challenge, with blisters appearing on the hooves, and some blisters appeared on the nose mirror later. After the tissue virus was challenged, cl...
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