Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor

A technology of bioreactor and porcine pseudorabies, which is applied in the field of cell engineering, can solve the problems of low yield and high cost of porcine pseudorabies virus liquid culture, and achieve the effects of small space occupation, easy quality and tight connection

Inactive Publication Date: 2012-09-26
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] One of the objectives of the present invention is to overcome the shortcomings of low production and high cost of porcine pseudorabies virus liquid culture in exi...

Method used

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  • Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the processing of microcarrier

[0053] 1. Siliconization: take a few ml of silicone oil, soak the inner wall of the mixing bottle, recover the excess silicone oil, dry the mixing bottle, wash it nine times with tap water, wash it three times with double distilled water, and dry it for later use.

[0054] 2. Hydration: Take by weighing the appropriate amount of microcarriers by the final volume of the culture, so that the final concentration of microcarrier Cytodex1 is 3mg / ml, put into a stirring bottle, and use Ca-free 2+ , Mg 2+ -PBS 100ml / g (that is, every gram of microcarriers corresponds to adding Ca-free 2+ , Mg 2+ -PBS 100ml. ), soak overnight at room temperature, or 3hr at 37°C, discard the PBS, and use Ca-free 2+ , Mg 2+ -Wash once with PBS50ml / g, discard, and finally add Ca-free 2+ , Mg 2+ - PBS 50ml / g, autoclaved, 115°C, 10psi, 15min.

[0055] 3. Balance: Discard the PBS in the stirring bottle, add DMEM (high sugar) cell growth medium (p...

Embodiment 2

[0056] Embodiment 2: Bioreactor microcarrier culture ST cell

[0057] 1. Cell recovery: Take out the cryopreservation tube from the liquid nitrogen tank, quickly put it into water at 36°C-37°C, shake the cryopreservation tube, and thaw it as soon as possible; suck out the cell suspension with a straw, put it into a sterile centrifuge tube, Add 10mL of cell culture medium (DMEM culture medium with 6% calf serum), pipette to suspend the cells; centrifuge the cell suspension at 1000rpm for 10 minutes, discard the supernatant; add cell culture medium to dilute properly, and transfer to a cell culture bottle Place in a 37°C incubator for culture, replace the cell culture medium once after 6 hours, and continue the culture.

[0058] 2. Subculture and culture of cells: Take well-grown dense monolayer ST cells, suck out the cell culture medium, and wash with PBS for 1-2 times; then add an appropriate amount of trypsin digestion solution with a mass fraction of 0.25%, and digest at 37°...

Embodiment 3

[0069] The mensuration of embodiment 3 virus activity

[0070] Viral activity assays were performed using the TCID50 method. When virus TCID50 is measured, the porcine pseudorabies virus liquid obtained by cultivating is made serial 10-fold dilution with MEM culture medium, i.e. 10 -1 、10 -2 ...10 -8 , 100 μL of each dilution was added to the wells of a 96-well cell culture plate, and then the ST cell suspension digested and dispersed by trypsin-EDTA was added, 100 μL per well (the cell content was expressed as 3×10 5 / mL or so), each dilution was repeated 8 times, and a normal cell culture control was set up, placed in a 5% CO2 incubator, cultured at 37°C, and the cell lesions and controls were observed daily for a total of 2 to 4 days. The number of wells with cytopathic changes was recorded, and the TCID50 of the virus was calculated according to the Reed-Muench method; at the same time, the TCID50 of the porcine pseudorabies virus liquid cultured in the spinner bottle w...

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Abstract

The invention discloses a method for generating a porcine pseudorabies virus by culturing an ST cell in a microcarrier of a bioreactor. The method comprises the following steps: (1) selecting the ST cell as a cell line for making vaccine; (2), transferring and culturing the ST cell; (3) reproducing a porcine pseudorabies virus cell virus seed; (4) culturing the ST cell in the microcarrier of the bioreactor in a suspension way; (5) reproducing porcine pseudorabies virus liquid; and (6) treating the obtained virus liquid. The method has the advantages that the cell vitality is more than and equal to 90%, the living cell density is more than and equal to 1*10<7> cells/mL, and the virus liquid tissue culture infectious dose 50 (TCID50) is more than and equal to 10<8.0>. The production process is intensive and smooth, the dimension is easy to amplify, the production period is short, a small space is occupied, little pollution is caused to the environment and is easy to treat, the virus liquid is convenient to obtain, the quality is easy to balance and stabilize, the production cost can be reduced obviously, and the yield and the quality of the vaccines are improved.

Description

[0001] Statement of priority [0002] This application claims the priority of the provisional application No. 201110327706.5 filed with the State Intellectual Property Office of China on October 25, 2011, the specification of which is incorporated herein by reference in its entirety. technical field [0003] The invention relates to a method for cultivating viruses, in particular to a method for producing porcine pseudorabies virus by cultivating ST cells using bioreactor microcarriers, and belongs to the technical field of cell engineering. Background technique [0004] Pseudorabies (PR), also known as Aujeszky's Disease (AD), is caused by one or more pseudorabies viruses (PRV or ADV) in the Herpesviridae, Alpha-Herpesviridae subfamily Acute infectious disease of animals. Pseudorabies virus can cause pigs, cattle, sheep, dogs, cats, rabbits, mice, minks, foxes and other domestic and wild animals to become ill. The symptoms are similar to rabies. Among them, pigs are the mo...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/245A61P31/20C12R1/93
Inventor 张智明任德强戴秀莉武佳斌臧玉婷张立恒潘兴广姜力吴金
Owner 哈药集团生物疫苗有限公司
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