Porcine seneca valley virus strain and application thereof

A technology of virus and pig plug, which is applied in the field of application of swine Senega Valley virus strains in the preparation of swine Senega Valley virus inactivated vaccines, can solve problems such as backward management of pig farms and complex and diverse pig raising modes, etc. Achieve strong product competitive advantages, broad application prospects, and rapid preparation methods

Active Publication Date: 2017-12-26
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the prevention and control of swine Senega Valley disease depends on the effective management of pig farms. However, domestic pig farming models are complex and diverse, and pig farm management is relatively backward. In view of the pathogenic characteristics of Senega Valley virus, it is necessary to develop corresponding biological products to prevent and control the disease. The outbreak of the disease is very urgent

Method used

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  • Porcine seneca valley virus strain and application thereof
  • Porcine seneca valley virus strain and application thereof
  • Porcine seneca valley virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a porcine seneca valley virus strain and an application thereof. In the invention, the seneca valley virus strain CH-FJZZ-2017 is isolated from pathological materials of porcine idiopathic vesicular disease in a pig farm in Fujian Province, with the preservation number of CGMCC No. 12160. The isolated porcine seneca valley virus CH-FJZZ-2017 in the invention is isolated from swinery newly suffering from the epidemic disease within Chinese territory, represents the current epidemic predominant virus strain in China, has a good virus strain background, and can be used as an inactivated vaccine production virus strain and a virus seed for testing, thereby providing a material for subsequent relevant experimental study, and laying a material foundation.

Description

technical field [0001] The invention relates to the field of pathogenic microorganisms, belongs to the field of biological products, and in particular relates to the application of a porcine senega virus strain in preparing porcine senega virus inactivated vaccines. Background technique [0002] Senecavirus A (SVA), also known as Seneca Valley virus (SVV), is a non-enveloped single-stranded positive-sense RNA virus belonging to the family Picornaviridae Senecavirus Viruses. The total length of the SVA genome is about 7.3kb, including an open reading frame and non-coding regions at both ends. SVA encodes a polyprotein with a size of 2181aa, which can be hydrolyzed by the proteases of the host cell and the virus itself to form 12 proteins, namely L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B , 3C and 3D etc. Among them, VP4, VP2, VP3, and VP1 are structural proteins of SVA, and VP2, VP3, and VP1 proteins are located on the surface of the virus capsid, and can induce a good immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02C07K16/10A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C07K16/1009C12N7/00C12N2770/32021C12N2770/32034C12N2770/32051
Inventor 张晓战马洁巴利民张国栋肖进齐鹏
Owner CHINA ANIMAL HUSBANDRY IND
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