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A strain of classical swine fever virus with high reproductive capacity and its construction method

A swine fever virus and capability technology, applied in the directions of viruses, antiviral agents, viruses/phages, etc., can solve the problems of slow growth of swine fever attenuated vaccine strains, low virus prices, and high vaccine production costs, so as to improve the success rate of virus rescue. , reduce production costs, improve the effect of cell adaptability

Active Publication Date: 2020-06-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the problems of slow growth and low toxicity in cells of existing attenuated swine fever vaccine strains and the high cost of vaccine production, and provide a strain of swine fever virus with high reproductive capacity and its construction method

Method used

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  • A strain of classical swine fever virus with high reproductive capacity and its construction method
  • A strain of classical swine fever virus with high reproductive capacity and its construction method
  • A strain of classical swine fever virus with high reproductive capacity and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Specific 8 site mutations on the C strain genome

[0031] Using the infectious clone of attenuated CSFV strain C as a template and C1802A-fwd / C1802A-rev as primers, a linearized plasmid containing the 1802-site mutation was amplified by PCR. Using a one-step directional cloning kit (Vazyme), under the action of recombinase, the linearized plasmid containing the 1802-site mutation was recombined into a circle in vitro to obtain an infectious clone pRecC-C1802A containing the 1802-site mutation.

[0032] Using the plasmid pRecC-C1802A containing the 1802-site mutation as a template and T3310G-fwd / T3310G-rev as primers, a linearized plasmid containing 1802 and 3310-site mutations was amplified by PCR. The linearized plasmid was recombined into a circle in vitro using the recombinase in the one-step directional cloning kit to obtain infectious clones pRecC-C1802A, T3310G containing mutations at 1802 and 3310.

[0033] Using the same method, mutations at position...

Embodiment 2

[0034] Example 2: Rescue of C-strain virus containing specific 8 site mutations.

[0035] Add 1 mL of the overnight cultured bacterial solution into 200 mL of LB medium, add 200 μL of Ampicillin at a concentration of 50 mg / mL at the same time, and expand the culture at 28°C for 16 hours. After the bacterial liquid was collected, the plasmid was extracted with a plasmid purification kit (Promega). After the concentration was determined, the plasmid was digested overnight with restriction endonuclease XhoI (TaKaRa) to obtain a linearized plasmid. Using the purified linearized plasmid as a template, viral genomic RNA (Ambion) was transcribed in vitro.

[0036] After PK-15 cells were digested, they were washed twice with serum-free medium Opti-MEM (Thermo). Mix 2 μg genomic RNA with 2 × 10 6 After the cells were mixed, they were added to a 2mm electroporation cup (Bio-Rad), and electroporation was performed with an electroporation instrument Gene Pulser Xcell (Bio-Rad), paramet...

Embodiment 3

[0038] Example 3: Detection of genetic stability of CSFV-Cmut8.

[0039] After infecting PK-15 cells with CSFV-Cmut8, they were passed on for 30 generations. During this period, each generation of venom was collected and stored in a -80°C refrigerator. After extracting RNA (AXYGEN) from the 1st, 10th, 20th and 30th generation virus and inverting it into cDNA (Vazyme), the genome was divided into 5 segments for full-length sequencing. The sequences of the primers for sequencing are SEQ ID NO.17~SEQ ID NO.26, specifically as shown in Table 2 below. The sequencing results showed that no mutations occurred in the 1st, 10th, 20th and 30th generation poisons, and no back mutations appeared in the specific 8 sites ( image 3 ), indicating that the virus has good genetic stability.

[0040]

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Abstract

The invention relates to construction and application of a swine fever virus and aims at providing a swine fever virus with high reproductive capacity and a construction method of the swine fever virus. A strain of the virus is collected in China Center for Type Culture Collection (CCTCC) under the name of a site-directed mutation strain CSFV-Cmut8 of low virulent strain of the swine fever virus,with a collection number of CCTCC No (number): V201744. CSFV-Cmut8 is obtained by modifying 8 specific sites on C strain genome by a reverse genetic method. Compared with a female parent C strain, thevirus can generate higher titer in in-vitro culture cells, keeps a stable hereditary character in a continuous passage process and has no pathogenicity to a natural host, namely a swine. The virus can increase a yield of a vaccine, lowers the cost and serves as a candidate vaccine virulent strain for swine fever immunity. The virus can be used for improving cell adaptability of other vaccines taking the C strain as a framework, increases a virus rescue success rate and lowers the production cost of swine fever cell vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a strain of swine fever virus with high reproductive capacity and a construction method thereof. Through the establishment of this technology, the growth ability of CSF attenuated vaccine C strain in in vitro cultured cells can be improved, and it can also be used for in-depth research on the replication and pathogenic mechanism of CSF virus. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease characterized by acute onset, persistent high fever, systemic generalized spotting and splenic infarction caused by capillary wall degeneration. Although it has already attracted the attention of various countries, due to the intensification of the pig industry and the globalization of the pig trade, swine fever still poses a major threat to the pig industry. The International Organization for Animal Health (OIE) listed the disease as a class A infectious dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/187A61P31/14
CPCA61K39/12A61K2039/5254A61K2039/552C12N7/00C12N2770/24321C12N2770/24334
Inventor 方维焕李肖梁
Owner ZHEJIANG UNIV
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