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Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content

A screening method and virus liquid technology, applied in animal cells, vertebrate cells, urinary tract/kidney cells, etc., can solve problems such as time-consuming, cumbersome operation, and infection

Pending Publication Date: 2021-09-17
JINYUBAOLING BIO PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the virus infection method disclosed in the above document 1 needs to inoculate the virus into the cell line to be screened in the process of screening the cell line with high virus content, and then co-culture for about 48 hours to observe the cell pathological changes, so there are cumbersome operations, Time-consuming and long-term problems, and there may be a risk of infection during the vaccination process

Method used

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  • Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content
  • Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content
  • Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Detection of CD163 receptor in Marc-145 adherent cells by indirect immunofluorescence method

[0040] This embodiment uses the indirect immunofluorescence method to detect the CD163 receptor on the cell surface in the Marc-145 polyclonal adherent cell population (multiclonal adherent cell population formed by the propagation of different Marc-145 adherent cell lines), and utilizes this The polyclonal adherent cell population is cultured and proliferated in vitro to measure the virus content in the obtained virus fluid, which specifically includes the following operations.

[0041] 1.1, the cultivation of Marc-145 adherent cells: Marc-145 adherent cells (preserved by the National Engineering Laboratory of Veterinary Vaccines of Jinyu Baoling Biopharmaceutical Co., Ltd.) were cultivated in cell culture flasks, and the growth solution contained 10% (volume percent) DMEM medium (commercially purchased) of newborn bovine serum, at 37°C 5% CO 2 Cultured in a const...

Embodiment 2

[0051] Embodiment 2: Can be used for cultivating PRRSV to obtain the screening of the Marc-145 monoclonal adherent cell line of the virus liquid of high virus content

[0052] This embodiment uses indirect immunofluorescence to detect the CD163 receptor on the cell surface in the Marc-145 adherent cell population (a monoclonal adherent cell population formed by the propagation of a single Marc-145 adherent cell line), and uses the monoclonal The parietal cell population is cultivated and proliferated in vitro to measure the virus content in the obtained virus fluid, which specifically includes the following operations.

[0053] 2.1. Obtaining of Marc-145 monoclonal adherent cell population: Digest Marc-145 adherent cells covered with monolayer and in good condition without exogenous virus contamination in step 1.1 of Example 1 into single scattered cells with trypsin. Dilute the cells in DMEM medium containing 10% newborn bovine serum, inoculate 1-2 cells / well in a 96-well cel...

Embodiment 3

[0061] Example 3: Detection of CD163 receptor in Marc-145 suspension cells by indirect immunofluorescence method

[0062] This example uses the indirect immunofluorescence method to detect the CD163 receptor on the cell surface in the Marc-145 polyclonal suspension cell population (polyclonal suspension cell population formed by the propagation of different Marc-145 suspension cell lines), and uses the polyclonal suspension The PRRSV is cultivated and proliferated outside the cell population to measure the virus content in the obtained virus liquid, which specifically includes the following operations.

[0063] 3.1. Culture of Marc-145 suspension cell population: Marc-145 suspension cells (preserved by National Engineering Laboratory of Veterinary Vaccines of Jinyu Baoling Biopharmaceutical Co., Ltd.) were cultured in cell culture flasks, and the growth medium was CD293 medium (commercially purchased ), at 37°C 5% CO 2 Cultured in a constant temperature shaking incubator, cul...

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Abstract

The invention discloses a screening method of a Marc-145 cell strain capable of being used for culturing PRRSV to obtain a virus liquid with high virus content, and belongs to the technical field of bioengineering. The method provided by the invention comprises the steps: detecting the percentage of cells with CD163 receptors on the cell surfaces in a Marc-145 monoclonal cell population formed by breeding a candidate Marc-145 monoclonal cell strain in the Marc-145 cell population by utilizing an indirect immunofluorescence method, and screening out the Marc-145 cell strain which can be used for culturing PRRSV to obtain the virus liquid with high virus content according to the percentage. The method has the advantages of rapidness, convenience, objectiveness and accuracy, can predict the virus content in the virus liquid obtained by culturing PRRSV by the Marc-145 cell population, and can guide vaccine production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a screening method for Marc-145 cell strains that can be used to cultivate PRRSV to obtain a virus liquid with high virus content, and in particular to a method for detecting CD163 receptors on the cell surface by using an immunofluorescence method to quickly, A convenient, objective and accurate method for screening Marc-145 cell lines that can be used to cultivate PRRSV to obtain high virus content virus liquid, and using immunofluorescence to detect CD163 receptors on the surface of Marc-145 cells to predict the effect of using the cells to culture and proliferate PRRSV in vitro Method for obtaining virus content in virus liquid and its application in vaccine preparation. Background technique [0002] Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS, commonly known as blue ear disease) is an exposure caused by...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577C12N5/071
CPCG01N33/6872G01N33/577G01N33/56966C12N5/0686G01N2333/70596
Inventor 孔彩平朱旭俎红丽赵丽霞李敏乌日罕刘燕军孙慧王娥娥郝苏云韩冬张海宝乔煜婷郝鹏
Owner JINYUBAOLING BIO PHARM CO LTD
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