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Reporter gene cell strain and establishment method and application thereof

A reporter gene and construction method technology, applied in the biological field, can solve the problems that cannot be used to evaluate the drug activity of IL-4/IL4Rα antibody, and achieve a significant specific effect

Pending Publication Date: 2019-12-10
DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method cannot be used to evaluate the activity of IL-4 / IL4Rα antibody drugs

Method used

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  • Reporter gene cell strain and establishment method and application thereof
  • Reporter gene cell strain and establishment method and application thereof
  • Reporter gene cell strain and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 constructs reporter gene cell line

[0063] 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:

[0064] (1) Plasmid construction

[0065] (a) Construction of reporter gene plasmid

[0066] The whole gene synthesis STAT6 cis-acting element sequence SEQ ID NO: 2 was inserted into the luciferase reporter gene vector pGL4.23[luc2 / minP] through the restriction endonuclease sites Acc65 and Hind III, and transformed into Escherichia coli, challenged Bacteria, sequenced to obtain the reporter gene system carrier pGL4.23[STAT6 / luc2 / minP] as shown in Figure 1 (A), wherein, STAT6sequence is the STAT6 cis-acting element sequence SEQ ID NO:2, and other elements are pGL4.23 Intrinsic elements on [luc2 / minP];

[0067] (b) Construction of type I IL-4R expression plasmid

[0068] The NCBI reference sequence number of the...

Embodiment 2

[0081] Example 2 IL-4 stimulates monoclonal cell 10B5 to express luciferase experiment

[0082] (1) Prepare medium: preheat analysis medium (2% FBS+F12) and complete medium (10% FBS+F12) before experiment;

[0083] (2) Cell collection: Take out the cell culture dish, observe the cell density under an inverted microscope to ensure that the cells are not contaminated; discard the old medium, rinse the cells with sterilized PBS, trypsinize for 2 minutes, and stop the digestion with complete medium; 1000rpm After centrifugation for 5 minutes, add F12 medium containing 2% FBS and resuspend the cells for cell counting;

[0084] (3) Cell plating: Resuspend the cells to 4E5 cells / mL, spread 50 μL / well in a 96-well plate, add PBS (100 μL / well) to the surrounding wells, and temporarily place in a 37°C incubator;

[0085] (4) Add IL-4: Take a 96-well plate, dilute IL-4 to 50ng / mL, and dilute it by 3 times, that is, pipette 60 μL of the mixture from the previous well and add it to 120 μL...

Embodiment 3

[0089] Example 3 Evaluation of Dupilumab (Dupilumab) and K10-4 Biological Activity Experiment

[0090] (1) Prepare medium: preheat analysis medium (2% FBS+F12) and complete medium (10% FBS+F12) before experiment;

[0091] (2) Cell collection: Take out the cell culture dish, observe the cell density under an inverted microscope to ensure that the cells are not contaminated; discard the old medium, rinse the cells with sterilized PBS, trypsinize for 2 minutes, and stop the digestion with complete medium; 1000rpm After centrifugation for 5 minutes, add F12 medium containing 2% FBS and resuspend the cells for cell counting;

[0092] (3) Cell plating: Resuspend the cells to 4E5 cells / mL, spread 50 μL / well in a 96-well plate, add PBS (100 μL / well) to the surrounding wells, and temporarily place in a 37°C incubator;

[0093] (4) Add IL-4, Dupilumab and K10-4: Take a 96-well plate, dilute dupilumab and K10-4 to 600 μg / mL, and dilute it at a ratio of 3.5 times, that is, pipette 40 μL ...

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Abstract

The invention provides a reporter gene cell strain and an establishment method and application thereof. The reporter gene cell strain comprises reporter gene plasmid, wherein the reporter gene plasmidcomprises an STAT6 (signal transducer and activator of transcription 6) signal response element; the STAT6 signal response element comprises at least one copied STAT6 binding motif; and the nucleotide sequences of the STAT6 binding motif are shown in SEQ ID NO:1. The reporter gene cell strain provided by the invention is applicable to evaluation on specific combination of IL-4 and a receptor IL-4R, meanwhile is applicable to an IL-4 / IL4R alpha antibody to interrupt combination of IL-4 and IL-4R, is used for monitoring and evaluating activity of the IL-4 / IL4R alpha antibody, is remarkable in specificity, short in detection time and high in sensitivity and has great significances in monitoring and evaluating the activity of a monoclonal antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a reporter gene cell line and its construction method and application. Background technique [0002] In recent years, antibody drugs have been widely used in the clinical treatment of various diseases such as tumors, transplant rejection, bacterial infections, and autoimmunity due to their safety, effectiveness, and specificity. In the development of antibody drugs, it is an urgent problem to establish an efficient and rapid antibody drug screening and evaluation system. At present, antigen binding activity experiments and cell proliferation inhibition experiments are mainly used to evaluate the activity of antibody drugs in vitro. Although the antigen binding activity test can directly reflect the binding ability of the antigen and the antibody, it cannot reflect the inhibitory effect of the antibody drug on cell proliferation; although the cell proliferation inhibition test directly...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N5/10C12Q1/02
CPCC12N5/0682C12N15/65C12N15/85C12N2503/02C12N2510/00C12N2800/107C12N2840/203G01N33/5008G01N2500/10
Inventor 李文英甄滢滢殷伟邵喆黄应峰
Owner DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD