A Porcine Senega Valley Virus Strain and Its Application
A virus and pig plug technology is applied in the application field of pig Seneca Valley virus strain in the preparation of pig Seneca Valley virus inactivated vaccine, which can solve the problems of backward management of pig farms, complex and diverse pig raising models, etc. Achieve strong product competitive advantages, broad application prospects, and rapid preparation methods
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Embodiment 1
[0040] Example 1: Isolation and identification of Senega Valley virus CH-FJZZ-2017 CGMCC No.12160
[0041] 1.1 Experimental materials
[0042] The disease samples used in the experiment came from pig farms where non-foot-and-mouth disease, porcine vesicular disease, and vesicular stomatitis epidemics occurred in Fujian Province, and the collection time was June 2017; the porcine kidney cell line (Porcine Kidney, PK-15) was purchased from From the American Culture Center ATCC.
[0043] 1.2 Primer design
[0044] Referring to the SVA CH-HN-2017 (KY747511) gene series published in GenBank, a pair of specific primers were designed using Primer 5, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The nucleotide sequence of the specific primer is as follows: SVA F: 5'-GCCCTCATGCCCAGTCCTTC-3'; SVA R: 5'-GTTCAGTGATCCGAGGTGG-3'.
[0045] 1.3 Collection and processing of disease materials
[0046] Collect the vesicular skin samples from the coronal part ...
Embodiment 2
[0053] Embodiment 2: the method for plaque purification of porcine Senega Valley virus
[0054] 2.1 Preparation of cell monolayer
[0055] T75cm 2 PK-15 cells that grew well in the cell culture flask and had a confluence of more than 98% were washed once with PBS, digested with 3 mL of 0.25% trypsin (Gibco), and used 70 mL of 10% fetal bovine serum, 100 U / mL penicillin, Suspend cells in 100g / mL streptomycin low-sugar DMEM medium to make a suspension, inoculate 3mL / well in a six-well cell culture plate, place at 37°C, 5% CO 2 Culture in an incubator, and when the cells grow into a monolayer, they will be used for subsequent plaque purification experiments.
[0056] 2.2 Virus dilution and infection
[0057] Use the low-sugar DMEM medium that contains 2% fetal bovine serum, 100U / mL penicillin, 100g / mL streptomycin to do 10-fold serial dilution to 10 times the SVA virus liquid that propagates stably in step 1.5. -8 , put it aside. Aspirate and discard the original cell cultur...
Embodiment 3
[0067] Embodiment 3: the mensuration of porcine Senegal valley virus strain CH-FJZZ-2017 virus titer
[0068] Inoculate the digested and blown PK-15 cell suspension into a 96-well cell culture plate, 150 μL per well, and culture overnight; take the cell culture maintenance medium of CH-FJZZ-2017 strain cloned and purified three times in step 2.4 Carry out 10-fold dilution, dilution from 10 -1 to 10 -10 ; Discard the cell culture medium in the 96-well cell culture plate, wash the PK-15 cells once with sterile PBS, add cell maintenance solution, 100 μL per well; Add 100 μL to each well of a 96-well plate, make 8 replicates for each dilution, and set up a negative control group without virus inoculation on each cell culture plate; culture in a 37°C, 5% CO2 incubator. Continuously observe for 4 days, observe and record the lesions every day. Calculation of TCID according to the Reed-Muench method 50 . The results showed that the virulence of porcine Senega Valley virus CH-FJZ...
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