A porcine circovirus type 3 virus strain and its application

A technology of porcine circovirus and type 3 virus, applied in antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve problems such as vaccine immunization failure

Active Publication Date: 2021-07-06
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, there are also reports that PCV3 infection may cause the failure of PCV2 vaccine immunity, so the detection and vaccine research for PCV3 is very necessary for the prevention and control of PCV3

Method used

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  • A porcine circovirus type 3 virus strain and its application
  • A porcine circovirus type 3 virus strain and its application
  • A porcine circovirus type 3 virus strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Isolation and identification of porcine circovirus type 3 ZM-PCV3-14CGMCC No.17290.

[0031] 1.1 Experimental materials

[0032] The disease samples used in the experiment came from pig farms with porcine circus disease in Guangdong Province, and the collection time was January 2018; the porcine kidney cell line (Porcine Kidney, PK-15) was purchased from the American Culture Center ATCC.

[0033] 1.2 Primer design

[0034]Referring to the PCV3KY965242.1 gene series published in GenBank, two pairs 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:

[0035] PCV3-F-1: 5'-TAGTATTACCCGGCACCTC-3';

[0036] PCV3-R-1: 5'-GCCCGGCACCAAAATGAGAC-3';

[0037] PCV3-F-2: 5'-TTAGAGAACGGACTTGTAAC-3';

[0038] PCV3-R-2: 5'-ACAGCTGGCACATACTACAC-3';

[0039] 1.3 Collection and processing of disease materials

[0040] Collect samples o...

Embodiment 2

[0057] Embodiment 2: the mensuration of porcine circovirus type 3 virus strain ZM-PCV3-14 virus titer

[0058] The isolated virus liquid was inoculated on the PK15-B1 cell monolayer, adsorbed at 37°C for 30 minutes, added DMEM cell maintenance solution containing 2% calf serum, cultured at 37°C for 60 hours, continued to pass to the 20th passage, and stored at -20°C. Use DMEM cell maintenance solution to make 10 for PCV3 -1 ~10 -8 Serially dilute and inoculate a monolayer of PK15-B1 cells in a 96-well plate, inoculate 4 wells for each dilution, and inoculate 100 μl in each well. At the same time, a negative control was set up, placed in a CO2 incubator for 12 hours at 37° C., treated with 300 mM D-glucosamine hydrochloride, and incubated at 37° C. for 48 hours. Fix the cells with absolute ethanol, measure the number of wells containing green fluorescent substance cells in each dilution by the IFA method, and finally calculate the TCID of the virus by the Reed-Muench method ...

Embodiment 3

[0059] Embodiment 3: the preparation of porcine circovirus type 3 ZM-PCV3-14 strain inactivated vaccine

[0060] 3.1 Virus culture

[0061] Use low-sugar DMEM medium containing 10% fetal bovine serum, 100U / ml penicillin, 100g / ml streptomycin, T225cm 2 Cell culture flask, 37°C, 5% CO 2 Cultivate PK-15 cells adherently in an incubator; when the fusion degree reaches about 80%, discard the culture medium, inoculate porcine circovirus type 3 ZM-PCV3-14 strain, and then use 2% fetal bovine serum, 100U / ml penicillin, 100g / ml streptomycin low-sugar DMEM medium, cultured at 37°C, 5% CO2 for 12h, treated with 300mM D-glucosamine hydrochloride, continued to culture at 37°C for 48h, and harvested the virus culture; freeze-thawed repeatedly Centrifuge at 10,000×g for 10 minutes three times to remove cell debris and collect the supernatant to obtain porcine circovirus type 3 virus liquid, which is stored at -80°C for later use.

[0062] 3.2 Inactivation of virus liquid and inactivation...

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Abstract

The invention discloses a porcine circovirus type 3 virus strain and application thereof. The name of the porcine circovirus strain of the present invention is porcine circovirus type 3 ZM-PCV3-14, and the preservation number is CGMCC No.17290. This strain was isolated from a pig herd that recently broke out of the disease in China. It represents the prevailing domestic strain and has a good strain background. It can be used as an inactivated vaccine production strain and a virus species for testing. Related experimental research provided materials and laid the material foundation.

Description

technical field [0001] The invention relates to the field of pathogenic microorganisms, belongs to the field of veterinary biological products, and relates to the isolation and identification of porcine circovirus type 3 virus and its application in inactivated vaccines. Background technique [0002] In June 2015, a porcine reproductive disorder, porcine dermatitis nephrotic syndrome (PDNS), broke out on a pig farm in North Carolina, USA, and research showed that the disease was caused by a new type of virus infection, namely porcine ring Virus type 3-PCV3, porcine circovirus type 3 (Porcine cirovirus 3, PCV3). Further studies have shown that PCV3 is widely distributed in major farms in the United States, South Korea, Italy, Poland and other countries. [0003] Porcine circovirus type 3 (Porcine cirovirus 3, PCV3) belongs to the family Circoviridae and belongs to the genus Circovirus. It is dodecahedral in shape and has no envelope. Genetic analysis of PCV3 shows that the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/06A61K39/12A61P31/20C12R1/93
Inventor 张文亮栗利芳刘新月李鹏宇肖进齐鹏宋芳李玲杨祎
Owner CHINA ANIMAL HUSBANDRY IND
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