A Porcine Circovirus Type 2 Virus Strain and Its Application

A porcine circovirus, type 2 virus technology, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the mixed infection and secondary infection of multiple bacteria and viruses, economic losses in the breeding industry, and decreased body resistance and other problems, to achieve the effect of good vaccine immunization, strong product competitiveness, and prevention of epidemic and transmission.

Active Publication Date: 2020-11-20
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, PCV1 is non-pathogenic and widely exists in pigs and pig-derived cell lines, while PCV2 is currently one of the most harmful pathogens to the world's pig industry and has brought huge economic losses to the global pig industry. , can cause postweaning multisystemic wasting syndrome (Postweaning multisystemicwasting syndrome, PMWS), piglet congenital tremor, porcine dermatitis and nephropathy syndrome (Porcine dermatitis and nephropathy syndrome, PDNS), porcine respiratory disease syndrome (Porcine respiratory disease complex, PRDC) , sow reproductive disorders and intestinal diseases, collectively known as porcine circovirus diseases (PCVDs), studies have shown that PCV2 proliferates in the lymphatic system of pigs, can cause immune cell apoptosis, cause immune suppression, and lead to body resistance decline, thus inducing the mixed infection and secondary infection of various bacterial viruses, causing huge economic losses to the breeding industry

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  • A Porcine Circovirus Type 2 Virus Strain and Its Application
  • A Porcine Circovirus Type 2 Virus Strain and Its Application
  • A Porcine Circovirus Type 2 Virus Strain and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Isolation and identification of porcine circovirus type 2 ZM-PCV2-13CGMCC No.17291.

[0030] 1.1 Experimental materials

[0031] The disease samples used in the experiment came from pig farms with porcine circus disease in Fujian Province, and the collection time was October 2017; the porcine kidney cell line (Porcine Kidney, PK-15) was purchased from ATCC, the American Culture Center.

[0032] 1.2 Primer design

[0033] Referring to the PCV2MF616427.1 gene series published in GenBank, two pairs of specific primers were designed using Primer5, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The nucleotide sequence of the specific primer is as follows:

[0034] PCV2-F-1: 5'-ACCAGCGCACTTCGGCAG-3';

[0035] PCV2-R-1:5'-AATACTTACAGCGCACTTC-3';

[0036] PCV2-F-2: 5'-TCACTTAGGGTTAAGTGG-3';

[0037] PCV2-R-2: 5'-AATGGCATCTTCAACACC-3';

[0038] 1.3 Collection and processing of disease materials

[0039] Collect samples of lymph n...

Embodiment 2

[0055] Embodiment 2: the mensuration of porcine circovirus type 2 virus strain ZM-PCV2-13 virus titer

[0056] 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 48h, continued to pass to the 25th passage, and stored at -20°C. Use DMEM cell maintenance solution to make PCV2 10 -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 set up a negative control, put CO 2 Cultivate at 37°C for 12h in an incubator, treat with 300mM D-glucosamine hydrochloride, and continue to cultivate at 37°C for 48h. 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 50 . The...

Embodiment 3

[0057] Embodiment 3: the preparation of porcine circovirus type 2 ZM-PCV2-13 strain inactivated vaccine

[0058] 3.1 Virus culture

[0059] 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 2 ZM-PCV2-13 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 3 times at 10000×g for 10 min to remove cell debris and collect the supernatant to obtain porcine circovirus type 2 virus liquid, which is stored at -80°C for later use.

[0060] 3.2 Inactivation of virus liquid and inactivation test

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Abstract

The invention discloses a porcine circovirus II virus strain and application thereof. The name of the porcine circovirus II virus strain is porcine circovirus II ZM-PCV3-13, and the preservation number is CGMCC No.17291. The virus strain is separated from swinery in which the porcine circovirus II newly breaks out and is endemic and represents a current domestic epidemic predominant virus strain,the virus strain has a good virus strain background, can be used as a inactivated vaccine to produce virus strains and virus seeds for inspection, a material is provided for subsequent related experiment research, and a material basis is laid.

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 a porcine circovirus type 2 virus strain and its application in the preparation of inactivated vaccines. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is the smallest animal virus that has been discovered so far, and it is one of the important members of the Circovirus genus in the family Circoviridae. According to its antigenicity, nucleotide sequence and pathogenicity, it can be divided into two genotypes: type 1 and type 2, namely PCV1 and PCV2. Among them, PCV1 is non-pathogenic and widely exists in pigs and pig-derived cell lines, while PCV2 is currently one of the most harmful pathogens to the pig industry in the world, and has brought huge economic losses to the global pig industry. , can cause postweaning multisystemic wasting syndrome (Postweani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K16/08C07K16/06A61K39/12A61P31/20C12R1/93
Inventor 张文亮刘新月栗利芳李鹏宇肖进齐鹏宋芳李玲张淼
Owner CHINA ANIMAL HUSBANDRY IND
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