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CSF-1R reporter gene cell line and preparation method and application thereof

A technology of CSF-1R and reporter gene, which is applied in the field of CSF-1R reporter gene cell line and its preparation, can solve the problems of long experimental cycle, large variability of experimental results, inapplicability of rapid evaluation of antibody activity, etc., and achieve remarkable specificity Effect

Pending Publication Date: 2019-11-26
DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has a long experimental cycle and large variability in experimental results, and is not suitable for rapid evaluation of antibody activity in large quantities.

Method used

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  • CSF-1R reporter gene cell line and preparation method and application thereof
  • CSF-1R reporter gene cell line and preparation method and application thereof
  • CSF-1R reporter gene cell line and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of NFκB reporter gene cell line

[0042] In this example, reporter gene cell lines were constructed through the steps of plasmid construction, cell transfection, stable cell line screening, and monocloning. The specific methods are as follows:

[0043] (1) reporter gene plasmid

[0044] In this example, the commercially available luciferase plasmid pGL4.32 (luc2 / NF-κB-RE / Hygro) (Promega Company) was used as the reporter gene plasmid to prepare reporter gene cell lines;

[0045] (2) Cell transfection

[0046] Resuscitate HEK293 cells two weeks in advance to ensure that the cells are in the logarithmic growth phase when used; collect the cells one day before transfection, inoculate in a 6-well plate, 4E5 cells / well, and the medium is 10% FBS+DMEM;

[0047] On the day of transfection, replace the medium of the 6-well plate with fresh 10% FBS+DMEM (2mL / well), and prepare the mixture of reporter gene plasmid pGL4.32(luc2 / NF-κB-RE / Hygro) and Lipofecta...

Embodiment 2

[0053] Example 2 Construction of NFκB reporter gene cell lines expressing CSF-1R

[0054] (1) Construction of CSF-1R plasmid

[0055] Design primers for PCR amplification to obtain the full-length CSF-1R gene fragment, agarose gel electrophoresis, and use pcDNA after gel recovery TM 3.3-TOPO TM TA Cloning TM Kit kit connection and transformation, the CSF-1R gene fragment was inserted into the pcDNA3.3-TOPO vector, transformed into Escherichia coli cloning strains, the single clone was picked and sent for sequencing verification, and the CSF-1R expression plasmid was obtained;

[0056] (2) Cell transfection

[0057] Resuscitate the monoclonal NFκB-HEK293 12-9E cells two weeks in advance to ensure that the cells are in the logarithmic growth phase when used; collect the cells one day before transfection, inoculate in a 6-well plate, 1E6 cells / well, and use a medium of 10% FBS+DMEM;

[0058] On the day of transfection, replace the medium of the 6-well plate with Opti-MEM (2mL / ...

Embodiment 3

[0064] Example 3 CSF-1 / IL-34 stimulates monoclonal cell 1-4G to express luciferase experiment

[0065] (1) The day before the experiment, prepare the medium: preheat the analysis medium (2% FBS+DMEM) and complete medium (10% FBS+DMEM) before the experiment;

[0066] (2) Cell collection: Take out the cell culture dish, observe the cell density under an inverted microscope to ensure that the cells are free of contamination; discard the old medium, rinse the cells with sterilized PBS, digest with sodium citrate for 2 minutes, and stop the digestion with complete medium; After centrifugation at 1000rpm for 5min, add DMEM medium containing 2% FBS and resuspend the cells for cell counting;

[0067] (3) Cell plating: Resuspend the cells to 5E5 cells / mL, spread 100 μL / well in a 96-well plate, add PBS (100 μL / well) to the surrounding wells, temporarily place in a 37°C incubator, and incubate overnight;

[0068] (4) On the day of the experiment, the medium in the white 96-well plate wa...

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Abstract

The present invention provides a CSF-1R reporter gene cell line and a construction method and an application thereof. The reporter cell line comprises NF-kappa-B reporter gene plasmids; the NF-kappa-Breporter gene plasmids comprise a luciferase reporter gene; and the reporter gene cell line also comprises CSF-1R expression plasmids. The reporter gene cell line expresses human-derived CSF-1R on cell membrane and intracellularly expresses luciferase in response to NF-kappa-B signals, CSF-1 / IL-34 can specifically activate the expression of the luciferase of the reporter gene cell line in a dose-dependent manner, at the same time target antibody drugs of the CSF-1R to block the CSF-1 / IL-34 and bind the CSF-1R on surfaces of the reporter gene cell line, and have important applications in detection of antibody drug activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a reporter gene cell strain and its preparation method and application, in particular to a CSF-1R reporter gene cell strain and its preparation method and application. Background technique [0002] CSF-1 was originally considered as a hematopoietic growth factor, which plays the biological function of promoting the proliferation, differentiation and survival of monocytes, macrophages and myeloid progenitor cells by binding to the only cell surface receptor CSF-1R. CSF-1R is encoded by the proto-oncogene c-Fms and is a receptor tyrosine kinase. When CSF-1 or IL-34 binds to CSF-1R, CSF-1R dimerizes and induces transphosphorylation of the receptor and activation of downstream signaling molecules MAPK and Akt. [0003] CN201180017294 discloses a method for measuring the biological activity of CSF-1R antibody drug, the method comprising adding CSF-1 and CSF-1R antibodies to the NKM-1 tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/65C12N15/85C12Q1/66C12Q1/02C12R1/91
CPCC07K14/715C12N5/0603C12N5/0686C12N15/65C12N15/85C12N2510/00C12Q1/66G01N33/5008G01N33/5044G01N2500/10
Inventor 甄滢滢李文英殷伟邵喆黄应峰
Owner DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD
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