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Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV)

A spleen-kidney necrosis virus, infectious technology, applied in the field of proliferation of mandarin fish infectious spleen-kidney necrosis virus ISKNV, can solve the economic loss of mandarin fish farming industry and other problems, and achieve the effect of benefiting virus proliferation, large surface area, and good condition

Active Publication Date: 2015-06-10
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002]Mandarin fish (Siniperca chuatsi) is one of the important freshwater cultured species in my country. ISKNV) caused serious economic losses to the mandarin fish farming industry

Method used

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  • Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Measure the number of CPB subcultured to 65 subcultures at 0h, 24h, 48h, 72h, and 96h by a Countstar cell counter, and make a cell growth curve (see figure 1). Select the cells that were cultured for 48 hours and in the middle of the logarithmic growth phase, digest them with trypsin, pour off the trypsin after 1 to 2 minutes, add L15 culture medium containing 6% v / v fetal bovine serum to resuspend CPB, and at the same time, ISKNV The stock solution was diluted, and the CPB was inoculated synchronously according to the MOI of 0.85-2.3. The inoculated mandarin fish brain tissue cell line CPB was cultured at a constant temperature of 28°C, and the poison was collected when the CPE reached more than 80%.

[0035] Determination of viral content, data processing and statistical analysis.

[0036] Result analysis: the number in the mid-logarithmic growth phase is 1.08×10 5 CPB cells per ml are most suitable for inoculating ISKNV. After culturing and collecting the virus, t...

Embodiment 2

[0038] CPB cells passaged to passage 65 were selected and cultured at a constant temperature of 28°C. Select the cells that were cultured for 24 hours and in the early logarithmic growth phase, digest them with trypsin, pour off the trypsin after 1-2 minutes, add L15 culture medium containing 10% v / v fetal bovine serum to resuspend CPB, and ISKNV The stock solution was diluted, and the CPB was inoculated synchronously according to the MOI of 30-45. The inoculated mandarin fish brain tissue cell line CPB was cultured at a constant temperature of 28°C, and the poison was collected when the CPE reached more than 80%.

[0039] Determination of viral content, data processing and statistical analysis.

[0040] Result analysis: the number in the early logarithmic growth period is 0.92×10 5 CPB cells per ml are most suitable for inoculating ISKNV. After culturing and collecting the virus, the virus copy number contained in 400ul cell fluid can reach up to 1.62×10 8 copies. After c...

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Abstract

The invention discloses a proliferation method of a siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV). According to the proliferation method, synchronous virus inoculation is adopted, the virus and a siniperca chuatsi brain tissue cell line (CPB) are in a suspended state, and the mutual contact surface area between the virus and the siniperca chuatsi brain tissue cell line is large so that the virus enters cells through more space receptors; and the virus infects the cells while the cells differentiate and proliferate, thus obtaining the high content ISKNV. After the CPB is cultured for 30 hours to 60 hours, the CPB is transferred to 5%-8% v / v of fetal calf serum culture medium, and the ISKNV is synchronously inoculated at the MOI (Multiplicity of Infection) value of 0.85-2.3, thus obtaining a low-cost ISKNV proliferation method. After the CPB is cultured for 20 hours to 40 hours, the CPB is transferred to 8%-10% v / v of fetal calf serum culture medium, and the ISKNV is synchronously inoculated at the MOI (Multiplicity of Infection) value of 30-45, thus obtaining a short ISKNV proliferation method. Cultivation methods can be selected according to existing conditions. The proliferation method provides theoretical basis for production of low-cost viruses.

Description

technical field [0001] The invention relates to a propagation method of mandarin fish infectious spleen and kidney necrosis virus ISKNV. Background technique [0002] Mandarin fish (Siniperca chuatsi) is one of the important freshwater characteristic aquaculture species in my country. In recent years, the iridescent virus disease of mandarin fish caused by Infectious spleen and kidney necrosis virus (ISKNV) has caused serious damage to the mandarin fish aquaculture industry. serious economic loss. In 1997, Wu Shuqin and others first discovered a spherical virus with a diameter of 150nm in sick mandarin fish, and considered it to be the pathogen of the fulminant infectious disease of mandarin fish. In 1998, He Jianguo and others further proved the pathogenicity of the virus through recurrent infection. Through histological studies, it was found that the spleen and kidney of mandarin fish were the main organs infected by it, and it was named infectious spleen and kidney necros...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
Inventor 罗霞付小哲李宁求黄志斌林强
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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