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Porcine circovirus type 3 strain, and preparation method and application thereof

A technology of porcine circovirus and strains, applied in biochemical equipment and methods, viruses, antiviral agents, etc., can solve the problems of easy contamination of exogenous viruses, etc., and achieve the effect of strong pathogenicity

Inactive Publication Date: 2019-01-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using conventional methods to isolate PCV3 virus is very easy to contaminate exogenous virus

Method used

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  • Porcine circovirus type 3 strain, and preparation method and application thereof
  • Porcine circovirus type 3 strain, and preparation method and application thereof
  • Porcine circovirus type 3 strain, and preparation method and application thereof

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Effect test

Embodiment 1

[0044]In the research process of the present invention, through a large number of experiments, the following method is finally selected to prepare the cloned virus with PCV3 virus characteristics, and optimize it through a large number of different cells to screen out the cells that are most suitable for the effective replication of the cloned virus. The screening process will not be repeated here, and the preparation and cultivation methods of the novel PCV3 virus will be described below.

[0045] Step 1: Construction of PCV3 double-copy full-gene infectious molecular clone

[0046] According to the PCV3 whole genome PCV3 / CN / Hebei-LY / 2015 sequence (sequence number: MF318451) submitted to the NCBI database for this project, two copies were artificially synthesized by Beijing Zhongmei Taihe Biotechnology Co., Ltd. through nucleic acid synthesis technology The whole genome of PCV3 / Hebei-LY was connected in an end-to-end manner. Subsequently, the synthesized double-copy gene, th...

Embodiment 2

[0070] Embodiment 2 animal regression experiment

[0071] Ten 4-week-old healthy SPF piglets negative for PCV1, PCV2, PRV, PRRSV, CSFV, PPV, SIV, JEV, and PEDV were screened and randomly divided into two groups. The first group of 5 pigs was the negative control pig, and the second group of 5 pigs was the challenge group. Each pig was inoculated with 2 ml of PCV3-LY virus liquid rescued by infectious cloning technology through the nasal cavity, and the body temperature of the pigs was recorded every day before and after the challenge And other clinical manifestations, changes in anal body temperature. Pigs were weighed at 0, 7, 14, 21, and 28 days after challenge, and the body weight changes of the pigs were recorded. Twenty-eight days after the challenge, the surviving piglets were sacrificed and the heart, liver, spleen, lung, kidney, inguinal lymph node, hilar lymph node, and tonsil were collected. Diseased organs and tissues were visible to the naked eye.

[0072] The re...

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Abstract

The invention provides a porcine circovirus type 3 strain PCV3-LY and application thereof. The virus is the porcine circovirus type 3 rescued by infectious cloning technology at home and abroad for the first time. After the virus is inoculated to monolayer porcine kidney cells PK15 for adherent culture, D-glucosamine treatment is carried out, then culture is continued, a cell culture is harvestedand used as an inoculum of a next round of passage, and finally a cell culture-adapted strain is obtained by successive passages. During the adaptation process, significant cytopathic changes can be observed after 15 passages. Indirect immunofluorescence confirms that the cell is able to support efficient replication of porcine circovirus type 3. The titer of the virus is lgTCID[50]=10<6.53> / mL. An animal regression test finds that two pigs are killed after five 28-day-old pigs are challenged with the virus, and the virus is more pathogenic to piglets. The virus is the porcine circovirus type3 rescued by infectious cloning technology at home and abroad for the first time, and provides direct support for further research on pathogenesis of PCV3 and development of vaccine research for effectively preventing and controlling the disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine circovirus type 3 strain, a preparation method and application thereof. Background technique [0002] Porcine circovirus (PCV) is a member of the genus Circovirus in the family Circoviridae. Porcine circovirus particles are icosahedral symmetric, non-enveloped, single-stranded circular DNA viruses. Under the electron microscope, porcine circovirus is a non-enveloped, icosahedral, round, small-particle virus with a diameter of 17 nm and a single-stranded circular DNA genome. Porcine circovirus is insensitive to organic solvents such as chloroform, iodine, and alcohol, and can survive stably at 70°C for 15 minutes, but it is sensitive to reagents such as phenol, quaternary ammonium compounds, sodium hydroxide, and oxidizing agents. Before 2016, PCV was divided into PCV1 and PCV2 according to the differences in pathogenicity, antigenicity and nucleic acid sequenc...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/10A61K39/12A61P31/20
CPCA61K39/12A61K2039/552A61P31/20C12N7/00C12N2750/10021C12N2750/10034C12N2750/10051
Inventor 刘爵姜海军王丹韦莉王菁朱珊珊全荣侯磊褚军李子璇郭雨欣奚衍洋宋慧琦袁枫
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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