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Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells

A technology of DBN-SX07 and PK-15, which is applied in the field of veterinary biology, can solve problems such as unexamined, and achieve the effects of reducing production costs, expanding production scale, and improving quality

Inactive Publication Date: 2015-05-06
成都史纪生物制药有限公司
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  • Summary
  • 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 circotype II DBN-SX07 strain virus by culturing PK-15 cells in low serum

Method used

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  • Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells
  • Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells
  • Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the method for cultivating PK-15 cells with low serum to produce porcine circular type II type DBN-SX07 strain virus, it may further comprise the steps:

[0037] S1. Subculture and culture of cells for preparation:

[0038] Take a T75 flask and culture a monolayer of PK-15 cells, digest the cells with EDTA-trypsin cell dispersion solution, the EDTA-trypsin cell dispersion solution is a mixture of 0.15% trypsin and 0.02% EDTA in Hank's solution solution; use cell growth solution to blow and disperse cells, add 20ml cell growth solution, and place PK-15 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. The above-mentioned cell growth medium is DMEM culture medium with 10% serum content;

[0039] S2. Adapting cells to low serum medium: including the following sub-steps:

[0040] S21. Domestication of first generation cells:

[0041] Inoculate the PK-15 cells expan...

Embodiment 2

[0055] Embodiment 2: the method for culturing PK-15 cells with low serum to produce porcine circotype II type DBN-SX07 strain virus, it may further comprise the steps:

[0056] S1. Subculture and culture of cells for preparation:

[0057] Take a T75 flask and culture a monolayer of PK-15 cells, digest the cells with EDTA-trypsin cell dispersion solution, the EDTA-trypsin cell dispersion solution is a mixture of 0.15% trypsin and 0.02% EDTA in Hank's solution Blow and disperse cells with cell growth medium, add 20ml of cell growth medium, place PK-15 cells in a carbon dioxide incubator at a temperature of 37±2°C for 72 hours, and carry out amplified culture when a good cell monolayer is formed. The above cell growth medium is 10% serum content MEM culture medium;

[0058] S2. Adapting cells to low serum medium: including the following sub-steps:

[0059] S21. Domestication of first generation cells:

[0060] The Marc-145 cells expanded and cultivated in step S1 were inoculat...

Embodiment 3

[0074] Embodiment 3: low serum medium acclimates PK-15 cell effect

[0075] Use T75 cell flasks, 37 ℃ static culture adapted to the fourth generation of PK-15 cells in low serum medium, the ratio of serum addition is 1%, 2%, 3%, 4%, 5% five gradients, and the serum content used is set to The PK-15 cells cultured in 10% DMEM was used as the control group, and the results were as follows: figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 , Figure 6 See Table 1 for the indicators during the cell culture process.

[0076] Table 1: Effects of MD series low-serum medium on acclimatization of PK-15 cells

[0077]

[0078] 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.

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Abstract

The invention discloses a method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells, and belongs to the technical field of biology of veterinaries. The method comprises the following steps: S1. performing passage and culture of cells; S2. domesticating the cells to enable the cells to be adapted for low seroculture bases; S3.domesticating the cells to enable the cells to be adapted for low seroculture environment; S4. propagation of cell virus seeds; S5. propagation of a prepared toxin liquid. The method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells, provided by the invention, can reduce the production cost obviously, besides, can improve the downstream purifying efficiency, and can rapidly and stably extend the production scale. Through the adoption of the method, balancing and stabilizing the quality is easy to realize.

Description

technical field [0001] The invention belongs to the technical field of veterinary biology, and in particular relates to a method for producing porcine circotype II DBN-SX07 strain virus by culturing PK-15 cells with low serum. Background technique [0002] PK-15 pig kidney cells (Pig Kidney), in vitro cultured as epithelial-like adherent cells, the cells are sensitive to a variety of viruses, such as porcine circovirus (PCV), porcine parvovirus (PPV), classical swine fever virus ( CSFV), etc., can be applied to the preparation of porcine circovirus vaccine, porcine parvovirus vaccine, swine fever virus vaccine, etc. Currently, the spinner bottle process is mainly used to cultivate PK-15 cells for vaccine preparation, and the culture medium is generally DMEM, 199, and MEM supplemented with 10% serum. [0003] At present, whether it is the traditional spinner bottle culture technology or the advanced bioreactor suspension culture technology, the domestic porcine circular vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071C12R1/93
Inventor 徐宏军胡来根牟和平岳丰雄田伟
Owner 成都史纪生物制药有限公司
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