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Method for producing porcine epizootic diarrhea CV777 strain virus by cultivating Vero cell in low serum

A porcine epidemic diarrhea and low serum technology, applied in the field of veterinary biology, can solve problems such as unexplained, and achieve the effects of reducing production costs, increasing yield, and increasing cell density

Inactive Publication Date: 2015-05-20
成都史纪生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no relevant literature report on the production of porcine epidemic diarrhea CV777 virus by culturing Vero cells in low serum

Method used

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  • Method for producing porcine epizootic diarrhea CV777 strain virus by cultivating Vero cell in low serum
  • Method for producing porcine epizootic diarrhea CV777 strain virus by cultivating Vero cell in low serum
  • Method for producing porcine epizootic diarrhea CV777 strain virus by cultivating Vero cell in low serum

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Embodiment 1

[0036] Embodiment 1: the method for producing porcine epidemic diarrhea CV777 strain virus by culturing Vero cells with low serum, it comprises the following steps: S1. passage and cultivation of cells for preparation:

[0037] Take a T75 flask and culture it to cover a monolayer of Vero cells, digest the cells with EDTA-trypsin cell dispersion, the EDTA-trypsin cell dispersion is a mixture of 0.15% trypsin and 0.02% EDTA in Hank's solution; Blow and blow dispersed cells with cell growth medium, add 20ml of cell growth medium, and culture Vero cells in a carbon dioxide incubator at a temperature of 37±2°C for 72 hours. When a good cell monolayer is formed, carry out amplified culture. DMEM culture fluid with 10% serum content;

[0038] S2. Adapting cells to low serum medium: including the following sub-steps:

[0039] S21. Domestication of first generation cells:

[0040] Inoculate the Vero cells expanded in step S1 into the first-generation low-serum medium for acclimatizat...

Embodiment 2

[0054] Embodiment 2: the method for producing porcine epidemic diarrhea CV777 strain virus by culturing Vero cells with low serum, it comprises the following steps: S1. passage and cultivation of cells for preparation:

[0055] Take a T75 flask and culture it to cover a single layer of Vero cells, digest the cells with EDTA-trypsin cell dispersion, the EDTA-trypsin cell dispersion is a mixture of 0.15% trypsin and 0.02% EDTA in Hank's solution, Blow and blow dispersed cells with cell growth medium, add 20ml of cell growth medium, and culture Vero cells in a carbon dioxide incubator at a temperature of 37±2°C for 72 hours. When a good cell monolayer is formed, carry out amplified culture. MEM medium with 10% serum content;

[0056] S2. Adapting cells to low serum medium: including the following sub-steps:

[0057] S21. Domestication of first generation cells:

[0058] The Vero cells expanded in step S1 were inoculated into the first-generation low-serum medium for acclimatiza...

Embodiment 3

[0072] Embodiment 3: the effect of low serum medium acclimatization Vero cell

[0073] Use T75 cell flasks, 37 ℃ static culture adapted to the fourth generation of Vero cells in low serum medium, the ratio of serum addition is 1%, 2%, 3%, 4%, 5% five gradients, and the serum content is set to 10% The Vero cells cultured in DMEM solution were used as the control group, and the results were as follows: figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 , Image 6 See Table 1 for the indicators during the cell culture process.

[0074] Table 1: Effects of MD series low-serum medium on acclimatization of Vero cells

[0075]

[0076]

[0077] It can be seen from Table 1 that the average cell yield, cell activity and average specific growth rate of the experimental group were higher than those of the control group. Example 4: Porcine epidemic diarrhea virus titer detection after inoculation of Vero cells obtained from MD series low-serum medium acclimatization

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Abstract

The invention discloses a method for producing a porcine epizootic diarrhea CV777 strain virus by using cultivating a Vero cell in low serum, and belongs to the technical field of veterinarian biological sciences. The method comprises the following steps: S1, preparing a culture medium for the passage and cultivation of the cell; S2, domesticating the cell to adapt to a low-serum culture medium; S3, domesticating the cell to adapt to the low-serum cultivation environment; S4, propagating cellular virus seeds; and S5, propagating the prepared virus solutions. Through the adoption of the method for producing the porcine epizootic diarrhea CV777 strain virus by using the low-serum cultivated Vero cell provided by the invention, the production cost can be obviously lowered, the downstream purification efficiency can be improved, the production scale can be fast and stably enlarged, and the balanced and stable quality can be easily realized.

Description

technical field [0001] The invention belongs to the technical field of veterinary biology, and in particular relates to a method for producing porcine epidemic diarrhea CV777 strain virus by culturing Vero cells with low serum. Background technique [0002] Since the second half of 2010, a new round of outbreaks of porcine epidemic diarrhea has occurred in my country. It first occurred in Guangxi, and then became widespread in Hunan, North China, East China, Shandong, Hebei and other places. It became a nationwide pandemic and caused a large number of deaths of suckling piglets. According to statistics from the Epidemiology Center of the Ministry of Agriculture, porcine epidemic diarrhea in 2011 ( The death toll of pigs caused by PED disease has occupied the first place of all existing pig diseases in my country. [0003] At present, the domestic porcine epidemic diarrhea vaccine production technology, whether it is the traditional spinner bottle culture technology or the ad...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071C12R1/93
Inventor 徐宏军胡来根牟和平岳丰雄任丽王洁清
Owner 成都史纪生物制药有限公司
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