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Porcine parvovirus proliferation method

A parvovirus and cell technology, applied in the direction of microorganism-based methods, viruses, virus/bacteriophage, etc., can solve problems such as difficult culture titers, achieve the effects of less passage times, simple process, and wide application prospects

Active Publication Date: 2018-11-13
苏州良辰生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the virus removal / inactivation validation, the titer of the indicator virus needs to reach 10 6 TCID 50 As mentioned above, but porcine parvovirus has different proliferation effects on different cells, and the time of lesion appearance, lesion characteristics, difficulty of lesion observation, virus content and hemagglutination value are all different. If you can't grasp the law, choose the best It will be difficult to cultivate high titer if the best cell line is used for virus culture and culture technology

Method used

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  • Porcine parvovirus proliferation method

Examples

Experimental program
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Effect test

Embodiment 1

[0025] 1. Cell subculture: Take out the frozen ST cells from liquid nitrogen, melt them quickly in a 37°C water bath, transfer them to a centrifuge tube with a sterile pipette, and add MEM medium containing 10wt% FBS drop by drop to 5mL , blow and aspirate several times, mix well, centrifuge immediately (3000rpm, 5min), and remove the supernatant. Then add 10mL of 10wt% FBS MEM medium, blow and aspirate several times, mix well, transfer to a T25 culture bottle with 10mL of 10wt% FBS MEM medium, place at 37°C, 5% CO 2 Cultivate in an incubator for 24h to 48h, and when the cells cover a single layer, they are used for virus amplification and virus titer determination.

[0026] 2. Amplification of PPV: Take out the frozen PPV primary virus species from the -70°C refrigerator (working virus bank), thaw at room temperature, dilute 10 times with MEM medium without FBS, and then inoculate 1mL in the already Store in a cell flask full of monolayer ST cells at 37°C, 5% CO 2 In the in...

Embodiment 2

[0029] It is basically the same as Example 1, except that bovine serum albumin is added to the supernatant to a final concentration of 2wt%, mannitol to a final concentration of 3wt%, and L-arginine to a final concentration of 3wt%.

Embodiment 3

[0031] It is basically the same as Example 1, except that bovine serum albumin is added to the supernatant to a final concentration of 1 wt%, glycine to a final concentration of 1 wt%, and L-arginine to a final concentration of 0.5 wt%.

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Abstract

The invention relates to a porcine parvovirus proliferation method. The method includes: performing inoculation of porcine parvovirus in ST cells, and performing cell culture until 90%-100% of cells have cytopathic effects; repeatedly freezing and thawing to enable breakage of host cells, centrifugally removing cell debris, and collecting supernatant; well mixing the supernatant, bovine serum albumin, mannitol and / or glycine and L-arginine, and performing freeze drying, wherein BSA (bovine serum albumin) accounts for 1-2% of the total mass of mixed liquor, mannitol and / or glycine accounts for1-3% of the total mass of the mixed liquor, and L-arginine accounts for 0.5-3% of the total mass of the mixed liquor. The porcine parvovirus proliferation method is convenient and simple in process and few in passage number and has a promising application prospect in virus removal / inactivation verification. After proliferation, the virus titer is not lower than 106.0 TCID50 / 0.1ml and remarkably higher than that of porcine parvovirus cultured by the prior art.

Description

technical field [0001] The invention belongs to the virus culture technology in the field of microorganism application, and in particular relates to a method for multiplying porcine parvovirus. Background technique [0002] The medium is the basic solution for maintaining the survival and growth of cells in vitro. The synthetic medium generally contains essential amino acids, which have an impact on cell growth and virus proliferation. Amino acids are a class of substances that can increase virus titers. Under the same culture conditions, a small amount of amino acids can be added to obtain higher titer viruses. [0003] Freeze drying or "lyophilization" is a technical method used to remove water. Therein, the aqueous solution is cooled below its eutectic point until it is completely frozen. Afterwards, the atmospheric pressure is reduced to vacuum, allowing the water to sublimate and come out of solution. The dissolved factors remain as porous solids, which can then be r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2750/14051
Inventor 吴娟缪汝娉鲍大为
Owner 苏州良辰生物医药科技有限公司
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