CN116855503B - Stable transgenic cell strain over-expressing MRGPRX2 and construction method and application thereof - Google Patents

Stable transgenic cell strain over-expressing MRGPRX2 and construction method and application thereof Download PDF

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CN116855503B
CN116855503B CN202311101724.0A CN202311101724A CN116855503B CN 116855503 B CN116855503 B CN 116855503B CN 202311101724 A CN202311101724 A CN 202311101724A CN 116855503 B CN116855503 B CN 116855503B
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cells
mrgprx2
rbl
cell strain
cell
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CN116855503A (en
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邱云良
陈华英
徐梦婕
徐婷婷
杨彬
刘永涛
谢行舟
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Shanghai Yinuosi Biotechnology Ltd By Share Ltd
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
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    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • C12N2510/02—Cells for production

Abstract

The invention discloses a stable transgenic cell strain over-expressing MRGPRX2, and a construction method and application thereof. The stable transgenic cell strain is a stable transgenic cell strain with the preservation number of CCTCC NO: C2023150 or CCTCC NO: C2023151. The MRGPRX2 gene expression quantity of the stable transgenic cell strain is obviously improved compared with that of the starting cell before transfection, and the stable transgenic cell strain can be used as an effector cell to reflect the anaphylactic reaction-like degree of the drug; the method can be practically applied to screening of allergy-like risk medicaments, and the sensitivity is obviously improved compared with that of the starting cells before transfection.

Description

Stable transgenic cell strain over-expressing MRGPRX2 and construction method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a stable transgenic cell strain over-expressing MRGPRX2, and a construction method and application thereof.
Background
Adverse reactions are generated in the application process of the medicine, the adverse reactions are important problems seriously threatening public health, allergic reactions are common types in the adverse reactions, and researches find that some medicines are easy to cause similar allergic symptom adverse reactions, do not depend on a specific immune mechanism, do not need secondary or multiple excitation, are rapid in reaction occurrence, can occur within about 30 minutes after the first administration, and have obvious dose-effect relationship, and are called as 'anaphylactoid reaction' or 'pseudo anaphylactic reaction'.
The current research on the mechanism of anaphylactic generation is widely considered that the medicine acts on the body to activate some membrane receptors on the surfaces of effector cells (mast cells and basophils), and the receptors further activate downstream signal channels through coupling with G protein so as to promote the increase of intracellular calcium ion levels, and then the cells degranulate to release mediums such as histamine and the like to cause anaphylactic symptoms. In recent years, the research discovers that a receptor MRGPRX2 regulated by MRG family genes exists on the surface of cells, which is probably a direct target point of the action directly causing the anaphylactoid reaction, so that the formation mechanism of the anaphylactoid reaction can be revealed through further research on the receptor genes.
The current in vitro evaluation model of the anaphylactoid uses mast cells or basophils of human or mouse sources, such as LAD2, RBL-2H3, ku815 and the like, but the cells can only predict the anaphylactoid occurrence, and the detection sensitivity of the anaphylactoid reaction is poor.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a stable transgenic cell strain over-expressing MRGPRX2, and a construction method and application thereof. Compared with untransfected starting cells, the MRGPRX2 gene expression quantity of the stably transfected cell strain constructed by the invention is obviously improved, and further, the stable transgenic cell strain has higher detection sensitivity in the detection of anaphylactic reaction.
In order to achieve the aim, the invention designs two sections of genes of the rat MRPRX2 gene (r-MRPRX 2) and the mouse MRPRX2 gene (m-MRPRX 2) related to the rat cells and the mouse cells, and inserts the two sections of genes into lentiviral plasmids respectively to construct the over-expression plasmids. And respectively transfecting the constructed overexpression plasmid loaded with the target gene into RBL-2H3 and P815 cells to ensure that the target gene is overexpressed in the cells. And then, detecting the MRPRX2 gene expression quantity in the two stable transgenic cell strains through q-PCR, comparing with untransfected starting cells, and determining the construction effect of the stable transgenic cell strains. And (3) comparing and detecting the constructed stable transgenic cell strain with the starting cell, and adopting anaphylactic-like detection indexes, namely an Annexin V positive cell rate, a Fluo-4AM mark rate, beta-hexosaminidase and histamine release amount response cell degranulation.
The first aspect of the invention provides a stable transgenic cell strain which overexpresses MRGPRX2, wherein the stable transgenic cell strain is a stable transgenic cell strain with a preservation number of CCTCC NO: C2023150 or CCTCC NO: C2023151.
The invention provides a stable transfer cell strain which overexpresses MRGPRX2, wherein the MRGPRX2 is derived from a mouse, and the starting cell of the stable transfer cell strain is a P815 cell;
or the MRGPRX2 is derived from a rat, and the starting cells of the stable transfer cell strain are RBL-2H3 cells;
the MRGPRX2 expression quantity in the stable transfer cell strain is at least 60 times of that of the starting cell.
In the present invention, the amino acid sequence of MRGPRX2 derived from a mouse may be the amino acid sequence of NCBI accession No. NP-001030040.2, and the coding nucleotide sequence is preferably the nucleotide sequence of NCBI accession No. NM-001034868.3.
In the present invention, the amino acid sequence of MRGPRX2 derived from rat may be the amino acid sequence of NCBI accession No. NP-001002280.1, and the encoding nucleotide sequence is preferably the nucleotide sequence of NCBI accession No. NM-001002280.1.
In the present invention, the MRGPRX2 gene expression level in the stable transgenic cell line is preferably at least 65 times, at least 70 times, at least 75 times or at least 80 times that of the starting cell.
In a third aspect, the present invention provides a method for constructing a stable transgenic cell line according to the first or second aspect, comprising the steps of transfecting an outgoing cell with the overexpression plasmid of MRGPRX2 and selecting.
In the present invention, the backbone plasmid of the over-expression plasmid may be pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro.
In a fourth aspect, the present invention provides a method of screening for an allergy-like risk drug, the method comprising: and (3) contacting the drug to be tested with the stable transgenic cell strain according to the first aspect or the second aspect to obtain a detection index result.
In the present invention, the detection index may be selected from the group consisting of intracellular calcium ion level change, the degree of exocytosis of the cells, and the release rate of inflammatory mediators released by degranulation of the cells.
In the present invention, the intracellular calcium ion level change is detected by a calcium ion fluorescent probe, for example, by a Fluo-4AM calcium ion fluorescent probe.
In the present invention, the degree of exocytosis of the cells can be detected by an Annexin V positive rate.
In the present invention, the release rate of inflammatory mediators released by degranulation of cells can be detected by the release rate of histamine and beta-hexosaminidase.
In a fourth aspect, the invention provides the use of a stable transgenic cell line according to the first or second aspect for screening for an allergy-like risk drug.
In a fifth aspect, the present invention provides the use of a stable transgenic cell line according to the first or second aspect for the preparation of an ananaphylaxis detection reagent.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the invention respectively transfects the over-expression slow virus plasmids containing the rat and mouse MRGPRX2 genes into RBL-2H3 cells and P815 cells, screens to obtain the stable transgenic cell strain which over-expresses MRGPRX2, and can obviously improve the expression quantity of the MRGPRX2 genes, and can also be used as a new effector cell to reflect the anaphylactic reaction degree of the medicine, so that the sensitivity is obviously improved compared with the starting cell before transfection, and the MRGPRX2 is definitely verified to be the action target point of the medicine when being practically applied to screening of the anaphylactic risk-like medicine.
Biological material preservation information
The stable transgenic cell strain MRGPRX2-P815 of the invention is preserved in China Center for Type Culture Collection (CCTCC) in 2023, 5 and 31 days, and the preservation address is as follows: the Chinese university of Wuhan, the post code 430072, the preservation number is CCTCC NO: C2023150, and the culture name is MRGPRX2-P815 of the mouse mast cell tumor cell with MRGPRX2 over-expression.
The stable transgenic cell strain MRGPRX2-RBL-2H3 of the invention is preserved in China Center for Type Culture Collection (CCTCC) in year 2023, month 5 and 31, and the preservation address is as follows: the preservation number of the Chinese university of Wuhan is CCTCC NO: C2023151, and the culture name is rat basophilic cell MRGPRX2-RBL-2H3 with over-expression of MRGPRX 2.
Drawings
FIG. 1 shows a map of the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector.
FIG. 2 is a map of r-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid.
FIG. 3 is a diagram showing the sequencing result of r-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid.
FIG. 4 is a map of the m-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid.
FIG. 5 is a diagram showing the sequencing result of m-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid.
FIG. 6 is a graph showing comparison of r-MRGPRX2 gene expression levels in RBL-2H3 cells and MRGPRX2-RBL-2H3 cells, wherein P is less than 0.001.
FIG. 7 is a graph showing comparison of m-MRGPRX2 gene expression levels in P815 cells and MRGPRX2-P815 cells, wherein P < 0.001.
FIG. 8 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cells Annecin V-FITC positive rate after administration of atracurium besylate, for one-way analysis of variance, representing RBL-2H3 cells with P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents that the MRGPRX2-RBL-2H3 cell is P < 0.05 and # is P < 0.01 compared with the RBL-3H3 cell.
FIG. 9 is a bar graph of positive rate of Annexin V-FITC for P815 and MRGPRX2-P815 cells following administration of atracurium besylate, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to RBL-3H3 cells.
FIG. 10 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cells Fluo-4AM markers following administration of atracurium besylate, for one-way anova, representing RBL-2H3 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents that the MRGPRX2-RBL-2H3 cell is P < 0.05 and # is P < 0.01 compared with the RBL-3H3 cell.
FIG. 11 is a bar graph of Fluo-4AM labeling rate of P815 and MRGPRX2-P815 cells following administration of atracurium besylate, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to RBL-3H3 cells.
FIG. 12 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cell histamine release after atracurium besylate administration, for one-way anova, representing RBL-2H3 cells with P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
Fig. 13 is a bar graph of histamine release from P815 and MRGPRX2-P815 cells following administration of atracurium besylate, for one-factor analysis of variance, representing P815 cells, P < 0.05, P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 14 is a bar graph of β -aminohexosaminidase release rates from RBL-3H3 and MRGPRX2-RBL-2H3 cells following administration of atracurium besylate, for one-way anova, representing RBL-2H3 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 15 is a bar graph of P815 and MRGPRX2-P815 cell beta-hexosaminidase release rates following administration of atracurium besylate, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 16 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cells Annecin V-FITC positive rates following rocuronium bromide dosing, for one-way anova, with x representing RBL-2H3 cells, P < 0.05, P < 0.01 compared to the placebo group; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents that the MRGPRX2-RBL-2H3 cell is P < 0.05 and # is P < 0.01 compared with the RBL-3H3 cell.
FIG. 17 is a bar graph of positive rate of Annexin V-FITC for P815 and MRGPRX2-P815 cells following rocuronium bromide administration, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to RBL-3H3 cells.
FIG. 18 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cells Fluo-4AM markers following rocuronium bromide dosing, for one-way analysis of variance, representing RBL-2H3 cells, P < 0.05, P < 0.01 compared to the blank control; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents that the MRGPRX2-RBL-2H3 cell is P < 0.05 and # is P < 0.01 compared with the RBL-3H3 cell.
FIG. 19 is a bar graph of P815 and MRGPRX2-P815 cell Fluo-4AM markers following rocuronium administration, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the blank control; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to RBL-3H3 cells.
FIG. 20 is a bar graph of RBL-3H3 and MRGPRX2-RBL-2H3 cell histamine release following rocuronium bromide dosing, for one-way anova, representing RBL-2H3 cells with P < 0.05 and P < 0.01 compared to the placebo group; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 21 is a bar graph of histamine release from P815 and MRGPRX2-P815 cells following rocuronium bromide administration, for one-way analysis of variance, representing P815 cells, P < 0.05, P < 0.01 compared to the placebo group; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 22 is a bar graph of the β -hexosaminidase release rates of RBL-3H3 and MRGPRX2-RBL-2H3 cells following rocuronium bromide dosing, for one-way analysis of variance, representing RBL-2H3 cells, with P < 0.05 and P < 0.01 compared to the blank control; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
FIG. 23 is a bar graph of P815 and MRGPRX2-P815 cell beta-hexosaminidase release rates following rocuronium bromide dosing, for one-way anova, representing P815 cells with P < 0.05 and P < 0.01 compared to the blank control; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01; # represents MRGPRX2 cells with P < 0.05 and # P < 0.01 compared to unlabeled cells.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Detection index of anaphylactoid reaction
C48/80 is taken as a positive medicine, and the following three cell degranulation detection indexes are designed to establish an in-vitro anaphylactoid detection method.
1. Annexin V positive cell Rate
(1) Detection purpose of Annexin V positive cell rate
The cell membrane phosphatidylserine has strong affinity with protein Annexin V, the Annexin V protein can participate in cell degranulation exocytosis transport particles, a flow cytometry is applied to a FITC channel to detect the positive rate of the Annexin V, and the exocytosis degranulation condition after the drug action is detected.
(2) Annexin V fluorescent probe source
Annexin V-FITC fluorescent probe Binding Buffer was all from BioLegend Inc.
(3) Method for detecting Annexin V positive rate
1000 r min after cell collection -1 Centrifuging for 5 min (20deg.C), discarding the waste liquid after centrifuging, and counting cells and using modified table liquid according to 1×10 6 ·mL -1 To prepare cell suspensions, prepare 1.5 mL centrifuge tubes, add 500. Mu.L of cell suspension to each 1.5 mL centrifuge tube, and place into incubator (37 ℃ C., 5% CO) 2 ) Culturing 1-3 h. After the culture is finished, 500 mu L of medicines with different concentrations are added into the centrifugal tube, 3 repeated groups are arranged for each administration concentration group, and the action time of the medicines is 30 min. After the medicine is acted for 30 min, the centrifuge tube is put into crushed ice for ice bath for 10min to stop the reaction, and then the centrifuge tube is taken out for 3000 r min -1 Centrifuging for 5 min (4deg.C), removing supernatant, washing cells with cold PBS (crushed ice for 20 min), and removing residual medicine. Again 3000 r min -1 Centrifuge for 5 min (4 ℃), discard supernatant, add 100. Mu.L Annexin V solution (5. Mu.L FITC Annexin V label per 100. Mu. L Annexin V Binding Buffer) to centrifuge tube and react for 15 min in the dark. After the reaction is finished, the centrifuge tube is put into a high-speed refrigerated centrifuge for 3000 r min -1 Centrifugation for 5 min (4 ℃ C.)The supernatant was discarded, and 500. Mu. L Annexin V Binding Buffer suspension cells were added to each of the separation tubes, and the resulting cell suspension was tested for positive rate on the FITC channel using a flow cytometer.
2. Changes in cellular calcium ion concentration
(1) Detection purpose of calcium ion concentration variation
The change of the calcium ion concentration is one of reasons for promoting the exocytosis to cause the cell degranulation, and Fluo-4AM is one of the most commonly used calcium ion probes, and the experiment uses a flow cytometer to detect the marking rate of Fluo-4AM and the change of the calcium ion concentration of the cell.
(2) Fluo-4AM fluorescent probe source
Fluo-4AM fluorescent probes were from Beyotime Inc.
(3) Fluo-4AM mark rate detection method
The early cell treatment process and detection method of positive rate of Annexin V are carried out in groups, and after sample addition of each group, the sample is continued in an incubator (37 ℃,5% CO) 2 ) Incubation for 30 min,3000 r.min -1 Centrifuging at 4℃for 5 min, and removing the supernatant. Washing with calcium-free D-Hanks buffer for 2 times, 100. Mu.L of Fluo-4AM (final concentration 5. Mu. Mol. L) -1 ) After incubation for 30 min in dark place, 3000 r min -1 Centrifugation (4 ℃) for 5 min to remove supernatant, washing 2 times with calcium-free D-Hanks buffer, removing residual Fluo-4AM, adding 500. Mu.L of calcium-free D-Hanks buffer for further incubation for 30 min, ensuring that Fluo-4AM is completely esterified in cells, and detecting Fluo-4AM labeling rate in a flow cytometer FITC channel.
3. Degree of inflammatory mediator release from cell supernatants
(1) Purpose of detection
The method is characterized in that inflammatory mediators released by degranulation after the cell is stimulated by the medicine are detected, and mast cells are the same as basophil degranulation release mediators, such as histamine, aminohexosidase, prostaglandin D2 (PGD 2), TNF-alpha and the like, representative mediators of histamine and aminohexosidase are selected, and the change of the content of cell supernatant substances after the medicine is added is detected by an ELISA method, so that the granule release degree can be reflected.
(2) ELISA kit Source
HIS ELISA Kit Histamine detection Kit from Elabscience; the Ratβ -Hex ELISA Kit Rat hexosaminidase detection Kit and the Mouse β -Hex ELISA Kit Mouse hexosaminidase detection Kit are all from Jiang Lai organisms.
(3) Method for detecting medium in cell supernatant
Taking cells in logarithmic phase in growth state, counting cells, and culturing in culture medium according to 1.5X10 5 ·mL -1 Is prepared by preparing a plurality of 96-well plates, inoculating 100. Mu.L of the suspension per well, and placing the inoculated plates in an incubator (37 ℃ C., 5% CO) 2 ) Culturing 24-72 h. Cell densities in the well plates were discarded as appropriate.
50. Mu.L of cell supernatant was taken per well 30 min after dosing, 3-5 multiplex wells were set per concentration, and the supernatant medium content was determined using histamine and beta-hexosaminidase ELISA kit, respectively, and according to the instructions for use. Further calculating the relative release rate of the medium: the lysate group was defined as "total enzyme group" (total amount released after cell lysis), release ratio = (drug group relative concentration/total enzyme group relative concentration) ×100%.
EXAMPLE 1 construction of stably transfected cell lines
1. Construction of an over-expression plasmid
1.1 Construction of r-MRGPRX2 overexpression plasmid
In this part of the experiment, the r-MRGPRX2 gene was inserted into the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector, the vector plasmid being provided by the Feng Hui organism.
NCBI accession number of r-MRGPRX2 gene sequence is: NM-001002280.1.
(1) Carrier map
The linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector map is shown in FIG. 1. The vector was cut completely with BamHI-EcoRI and 1% Agarose gel electrophoresis was used to recover large fragments, which were linearized vectors.
(2) Cleavage pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector
The miniplasmid pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro was digested with BamHI-EcoRI in a double digestion system as shown in Table 1:
TABLE 1
Note that: after reaction 5 h at 7 ℃, large fragments were recovered on a 1% agarose gel.
(3) Acquisition of r-MRGPRX2 Gene fragment
The R-MRGPRX2 gene sequence design primer (R-MRGPRX 2-F/R is a recombinant primer) is selected to construct a gene subcloning vector, and the sequence is shown in Table 2:
TABLE 2
Diluting the synthesized primer into working solution with the final concentration of 10 mu mol/L, and carrying out PCR amplification by using the diluted primer and the template. The system is shown in Table 3:
TABLE 3 Table 3
The materials are added into a thin-wall tube, evenly mixed and separated, then placed into a PCR instrument, and the final reaction procedure after adjustment is shown in Table 4:
TABLE 4 Table 4
After the completion of PCR, agarose gel electrophoresis was performed, and the target gene was recovered.
(4) Ligation of Gene fragments with vectors
The recovered and purified target fragment was ligated with the recovered and purified vector pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, the ligation product was designated as r-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as shown in FIG. 2. Because the primer contains a homologous arm sequence with the vector, the homologous recombinase is used for recombining the recovered fragment with the linearization vector, and the recombination process of recovering the target gene and the vector is completed.
(5) Ligation product transformation competent cells
mu.L ligation product was used to transform 100. Mu.L DH5a competent cells: mixing the product with competent cells, ice-bathing for 30 min, heat-shocking at 42 deg.C for 90 s, immediately placing on ice for 2 min, adding 500 μl of LB medium preheated to room temperature, shaking at 37 deg.C for 1 h at 180 rpm, centrifuging at 5000 rpm for 3 min, discarding 500 μl of culture supernatant, mixing the rest 100 μl with a pipettor, uniformly coating on LB plate containing 50 μg/mL ampicillin resistance, inverting, and culturing overnight in a 37 deg.C constant temperature incubator.
(5) PCR identification and sequencing
4 single colonies were picked and inoculated into LB medium containing 5 mL of 50. Mu.g/mL ampicillin resistance, and shaking cultured at a constant temperature of 37℃at 220 rpm for 5 h. And (3) carrying out PCR identification by using the cultured bacterial liquid, wherein the identified and picked bacterial colonies are correct, and carrying out r-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro sequencing, wherein the sequencing result is shown in figure 3.
1.2 Construction of m-MRGPRX2 overexpression plasmid
For the whole procedure, reference is made to 1.1, and the m-MRGPRX2 gene was inserted in the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector in this section of the experiment, the following is not repeated.
NCBI accession number of m-MRGPRX2 gene sequence is: NM-001034868.3.
(1) Acquisition of m-MRGPRX2 Gene fragment
As shown in Table 5, the m-MRGPRX2 gene sequence design primers (m-MRGPRX 2-F/R is a recombinant primer) were selected for constructing a gene subcloning vector.
TABLE 5
(2) Ligation of Gene fragments with vectors
The recovered and purified target fragment was ligated with the recovered and purified vector pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, the ligation product was designated as m-MRGPRX2-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as shown in FIG. 4. The plasmid sequencing method was the same as in example 1, and the sequencing results are shown in FIG. 5.
2. Plasmid transfection cell line procedure
The experiment in this section is to construct a MRGPRX2 gene over-expression stable transgenic cell line, and the over-expression plasmids carrying r-MRGPRX2 and m-MRGPRX2 genes are respectively transfected into RBL-2H3 cells and P815 cells and stably expressed.
RBL-2H3 cells and P815 cells were purchased from the national academy of sciences cell bank (Shanghai).
(1) Pre-experiment preparation of cells and determination of MOI (multiplicity of infection)
Information about RBL-2H3 cell line and P815 cell line was determined, including cell culture conditions, proliferation rate of cells, mycoplasma contamination. The literature was consulted to determine the MOI values of lentiviruses in RBL-2H3 and P815 cell lines and based on the data obtained by consulting, gradient experiments were designed to fudge the optimal MOI.
(2) Screening to determine the amount of Puro (Puromycin)
Referring to the lethal dose information of Puro screening of stably transformed cell lines in RBL-2H3 cell line and P815 cell line, and referring to the obtained data, 3 drug concentration gradients were determined (if there was no relevant information, the drug concentration gradient range was increased by an amount of up to 6).
Day 1: the RBL-2H3 cell line and P815 cell line cells were plated in 6-well plates to a density of about 90% by day;
day 2: adding Puro to the cells at a set concentration;
day 4: changing liquid, and adding Puro with set concentration again;
day 7: observing to find out the hole with the lowest drug concentration at the cell lethality of 100%, wherein the drug concentration used in the hole is the Puro screening concentration;
(3) Screening and constructing stable-rotation cell strains
Cell plating: respectively inoculating RBL-2H3 cells into 6-hole plates to ensure that the density of the cells reaches about 70% in the next day;
viral infection: calculating the volume of the slow virus to be added according to the MOI value determined by a pre-experiment;
liquid replacement: according to the actual situation, the liquid is replaced, and for some cells with weak tolerance, the liquid is replaced in time; some cells with strong tolerance can be infected for 48-72 hours and then liquid exchange is carried out;
infection efficiency was observed: 72 h after infection, observing infection efficiency, which should not be lower than 40% at the lowest;
puro screening: the optimal acting time is 3-10 days, the common concentration range of Puro is 1-10 mug/mL, and virus infection can be carried out after the optimal screening concentration is determined through a pre-experiment;
infection: drug concentrations pre-experimentally determined after infection with 72 h (infection time depending on the specifics of the cells and infection efficiency) before addition to 6-well plates;
puro is added: puro drug concentrations pre-determined experimentally prior to addition to 6-well plates;
liquid replacement: according to the color of the culture medium and the growth condition of cells, the screening culture medium is replaced every 3-5 days, and when a large number of cells die, the Puro concentration can be halved to maintain screening;
and (3) observation: the state, growth condition, gene expression level and proportion of cells are observed every day until the proportion of fluorescent cells is more than 90% under a microscope.
Example 2 q-PCR test of transfection Effect
1. Method of
And (3) adding a fluorescent group into a PCR reaction system by using a q-PCR technology, monitoring the whole PCR process in real time by utilizing fluorescent signal accumulation, and finally quantitatively analyzing an unknown template by using a standard curve. The change of the amplification product amount of each cycle in the PCR amplification reaction can be detected in real time according to the change of the fluorescent signal, and the initial template can be quantitatively analyzed through the analysis of the Ct value and the standard curve.
(1) Primer design
As shown in Table 6, the primer pair information for gene 1 r-MRGPRX 2:
TABLE 6
As shown in table 7, the primer pair information for inner reference primer 1:
TABLE 7
As shown in Table 8, the primer pair information for gene 2 m-MRGPRX 2:
TABLE 8
As shown in table 9, the primer pair information for inner reference primer 2:
TABLE 9
(2) RNA extraction
1000. Mu.L of Trizol was added to a 1.5. 1.5 mL centrifuge tube containing the cell lines, and after thoroughly mixing, 200. Mu.L of chloroform was then added thereto, and after shaking mixing, the mixture was allowed to stand for 5 minutes and centrifuged at 12000 rpm at 4℃for 10 minutes. The 1.5 mL centrifuge tube was removed from the centrifuge and the upper colorless transparent aqueous phase was aspirated into another clean 1.5 mL centrifuge tube (the sample would be divided into three layers: lower organic phase layer, middle and upper aqueous phase layer, RNA in the upper aqueous phase). Adding equal volume of isopropanol, mixing, standing for 10min, and centrifuging at 12000 rpm and 4deg.C for 10min. After centrifugation, colloidal precipitation, which is the RNA to be extracted, can be seen on the wall or bottom of the centrifuge tube. The supernatant was carefully discarded and the RNA pellet was washed with 1mL of 75% ethanol. The supernatant was then removed as clean as possible by centrifugation at 7000 rpm at 4℃for 5 min.
And standing and drying at room temperature for about 5-10 min. 25. Mu.L of DEPC H was added to all centrifuge tubes 2 O, blow with gun head several times to dissolve RNA fully, store at-80 ℃.
And (3) RNA concentration detection: RNA concentration was detected using a nucleic acid protein detector.
(3) RNA concentration and purity determination:
as shown in table 10, the concentration of RNA extracted in the different cell lines:
table 10
(4) Reverse transcription PCR
The reverse transcription reaction solutions were prepared according to the compositions shown in Table 11:
TABLE 11
The following reactions were performed on a PCR instrument: 72℃for 5 min, and then quenched on ice.
The reverse transcription reaction solution (20. Mu.L in total) shown in Table 12 was added to the PCR tube:
table 12
The reverse transcription reaction was performed on a PCR instrument under the following conditions: 60min at 42℃and 10min at 72 ℃.
The cDNA was immediately subjected to the experiment or stored at 4 ℃.
(5) Real-time fluorescent quantitative PCR reaction
The configuration of the reaction system is shown in Table 13:
TABLE 13
The reaction condition settings are shown in table 14:
TABLE 14
The amplification procedure is shown in table 15:
TABLE 15
The melting procedure is shown in table 16:
table 16
2. Results
(1) MRGPRX2-RBL-2H3 cell contrast RBL-2H3 cell q-PCR detection result
The comparison of the detection results of the r-MRGPRX2 gene expression level detected by q-PCR is shown in FIG. 6, and the obvious difference (P < 0.001) of the target gene expression level in the transfected cell strain compared with that of the original cell is shown, which proves that the r-MRGPRX2 over-expression stable transgenic cell strain is basically established successfully, namely MRGPRX2-RBL-2H3, and the r-MRGPRX2 over-expression stable transgenic cell strain is submitted to China Center for Type Culture Collection (CCTCC) for preservation at 2023 and 5 months and 31 days, wherein the preservation number is CCTCC NO: C2023151.
(2) MRGPRX2-P815 cell to P815 cell q-PCR detection result
The comparison of the detection results of the m-MRGPRX2 gene expression level detected by q-PCR is shown in FIG. 7, and the obvious difference (P < 0.001) of the target gene expression level in transfected cell lines compared with that of original cells proves that the m-MRGPRX2 over-expression stable transgenic cell line is basically established successfully, is abbreviated as MRGPRX2-P815, and is submitted to China Center for Type Culture Collection (CCTCC) for preservation in 2023, 5 and 31 days, with the preservation number of CCTCC NO: C2023150.
Effect example 1 detection of atracurium besilate allergy-free reaction
1. Concentration of drug action
The drug effect concentrations selected are shown in table 17:
table 17 cell and drug for comparative detection of degranulation of cells
2. Cell Annexin V positive rate comparison detection result after administration of atracurium besylate
As can be seen from the data in Table 18, the variation trend of the positive rate of Annexin V after the administration of MRGPRX2-RBL-2H3 cells is the same as that of RBL-2H3 cells, namely, compared with a blank cell group, the positive drug C48/80 and the positive drug of atracurium MRGPRX2-RBL-2H3 cells have obvious difference (P < 0.05 or P < 0.01) in the variation degree of the positive rate of Annexin V after the administration of the same drug action concentration, and the exocytosis effect is enhanced and the cell degranulation phenomenon is enhanced after the administration. As shown in FIG. 8, the contrast results of the positive rate of Annexin V after administration of MRGPRX2-RBL-2H3 cells and RBL-2H3 cells show that the positive rate of Annexin V of MRGPRX2-RBL-2H3 cells in the blank cell group, the positive drug C48/80 and the atracurium drug group is higher than that of RBL-2H3 cells, and the change degree is significantly different (P < 0.05 or P < 0.01).
The positive rate of Annexin V changes in P815 and MRGPRX2-P815 cells after drug action are shown in Table 18 and FIG. 9, and the rule of the results is similar to that of MRGPRX2-RBL-2H3 cells.
The result proves that the MRGPRX2-RBL-2H3 and MRGPRX2-P815 constructed based on two over-expressed stable transfer cell strains can reflect the anaphylactic risk by detecting the positive rate of Annexin-V, namely the exocytosis condition of cells, the exocytosis effect of the over-expressed stable transfer cell strain is increased compared with that of the original cell strain after the atracurium is dosed, the index proves that the MRGPRX2 stable transfer cell strain is more suitable for in vitro detection of the drug, and the MRGPRX2 is probably a drug target generated by the atracurium anaphylactic reaction.
TABLE 18 Annexin V Positive Rate of cells after administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2 cells and untransfected cellsCompared with the traditional Chinese medicine, the # is P < 0.05, and the # is P < 0.01; results toN=3.
3. Comparison and detection result of calcium ion level change of cells after administration of atracurium besilate
As can be seen from the data in Table 19, the variation trend of the Fluo-4AM labeling rate of MRGPRX2-RBL-2H3 cells after administration is the same as that of RBL-2H3 cells, namely, compared with a blank cell group, the variation degree of the Fluo-4AM labeling rate of MRGPRX2-RBL-2H3 cells after administration of atracurium has a significant difference (P < 0.05 or P < 0.01) under the same drug action concentration, and the calcium ion level is presumed to be increased after administration, so that the cell degranulation phenomenon is enhanced. As can be seen from FIG. 10, the comparison of the Fluo-4AM labeling rate of MRGPRX2-RBL-2H3 cells with that of RBL-2H3 cells shows that the Fluo-4AM labeling rate of MRGPRX2-RBL-2H3 cells is higher than that of RBL-2H3 cells in the blank cell group, the positive drug C48/80 and the atracurium drug group, and the variation degree is significantly different (P < 0.05 or P < 0.01).
The changes in Fluo-4AM labeling rate of P815 and MRGPRX2-P815 cells after drug action are shown in Table 19 and FIG. 11, and the rule of the results is similar to that of MRGPRX2-RBL-2H3 cells.
The result proves that the cell degranulation degree can be shown by detecting the marking rate of Fluo-4AM, namely the cell calcium ion level condition, on the basis of two over-expression stable transfer cell strains MRGPRX2-RBL-2H3 and MRGPRX2-P815, so that the anaphylactic risk is reflected, the calcium ion level of the over-expression stable transfer cell strains after the administration of atracurium is increased compared with that of the original cell strains, the index proves that the MRGPRX2 stable transfer cell strains are more suitable for in vitro detection of the drug, and MRGPRX2 is possibly a drug target generated by the atracurium anaphylactic reaction.
TABLE 19 Fluo-4AM labelling Rate of cells after administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor analysis of variance/independent sample T-test, representing RBL-2H3 cells and blank controlGroup comparison, P < 0.05, P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2 cells with P < 0.05, # with P < 0.01 compared to untransfected cells; results toN=3.
4. Extent of release of inflammatory mediators from cell supernatants following administration of atracurium besylate
As can be seen from the data in Table 20 and Table 22, the trend of changes in histamine and beta-hexosaminidase after administration of MRGPRX2-RBL-2H3 cells is the same as that of RBL-2H3 cells, i.e., compared with that of blank cell groups, the change degree of inflammatory mediators in cell supernatants of MRGPRX2-RBL-2H3 cells after administration of atracurium under the same drug action concentration is significantly different (P < 0.05 or P < 0.01), and it is presumed that the mediators released by cells after administration are increased and the level of degranulation of cells is increased. From FIGS. 12 and 14, it is clear that the results of comparing the inflammatory mediator level changes of MRGPRX2-RBL-2H3 cells with those of RBL-2H3 cells after administration show that there is a significant difference (P < 0.05 or P < 0.01) between the inflammatory mediator changes of MRGPRX2-RBL-2H3 cells in the blank cell group, the positive drug C48/80, and the atracurium drug group.
The changes in histamine and beta-hexosaminidase release rates of P815 and MRGPRX2-P815 cells after drug action are shown in Table 21, table 23, FIG. 13 and FIG. 15, and the result rule is similar to that of MRGPRX2-RBL-2H3 cells.
The results prove that the two over-expression stable transfer cell strains MRGPRX2-RBL-2H3 and MRGPRX2-P815 constructed by the invention can display the degree of cell degranulation by detecting the release rate of histamine and beta-aminohexosidase, namely the release level of inflammatory medium in cell supernatant, so as to reflect the anaphylactic risk, the release degree of the over-expression stable transfer cell strain medium after the administration of atracurium is increased compared with that of the original cell strain, and the index proves that the MRGPRX2 stable transfer cell strain is more suitable for in vitro detection of the drug, and MRGPRX2 is probably a drug target generated by atracurium anaphylactic reaction.
TABLE 20 Histamine release from cells after administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2-RBL-2H3 cells, P < 0.05, # is P < 0.01 compared to RBL-3H3 cells; results toN=3.
Table 21 histamine release from cells following administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing P815 cells as P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01 compared with the blank control group; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to P815 cells; results toN=3.
TABLE 22 beta-hexosaminidase Release from cells following administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2-RBL-2H3 cells, P < 0.05, # is P < 0.01 compared to RBL-2H3 cells; results toN=3.
Table 23 release rate of beta-hexosaminidase from cells after administration of atracurium besylate
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing P815 cells as P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01 compared with the blank control group; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to P815 cells; results toN=3.
Effect example 3 rocuronium bromide based allergy detection
1. Concentration of drug action
The drug effect concentrations selected are shown in table 24 below.
TABLE 24 cells and drugs for comparative detection of degranulation of cells
2. Comparison and detection result of positive rate of cell Annexin V after rocuronium bromide administration
As can be seen from the data in table 25 and fig. 16-17, the change of rocuronium bromide after administration is similar to the detection result of atracurium besilate, the two over-expressed stable transgenic cell strains MRGPRX2-RBL-2H3 and MRGPRX2-P815 constructed by the invention can reflect the anaphylactoid risk by detecting the positive rate of Annexin V, namely the condition of cell exocytosis, the exocytosis effect of the over-expressed stable transgenic cell strain is increased compared with that of the original cell strain after rocuronium bromide administration, and the index proves that the MRGPRX2 stable transgenic cell strain is more suitable for being used as a drug target generated by the allergic reaction of the rocuronium bromide.
Table 25 Annexin V positive rate of cells after rocuronium bromide administration
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2 cells with P < 0.05, # with P < 0.01 compared to untransfected cells; results toN=3.
3. Comparison and detection result of calcium ion level change of cells after rocuronium bromide administration
As can be seen from the data in Table 26 and FIGS. 18-19, the change of rocuronium bromide after administration is similar to the detection result of atracurium besilate, the two overexpression stable transgenic cell lines MRGPRX2-RBL-2H3 and MRGPRX2-P815 constructed by the invention can show the degree of cell degranulation by detecting the Fluo-4AM mark rate, namely the condition of cell calcium ion level, so as to reflect the anaphylactoid risk, the calcium ion level of the overexpression stable transgenic cell line after atracurium administration is increased compared with that of the original cell line, and the index proves that the MRGPRX2 stable transgenic cell line is more suitable for being used as a drug target generated by atracurium anaphylactic reaction.
TABLE 26 Fluo-4AM labelling Rate of cells after rocuronium bromide administration
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2 cells with P < 0.05, # with P < 0.01 compared to untransfected cells; results toN=3.
4. Degree of inflammatory mediator release from cell supernatants following rocuronium bromide administration
As can be seen from the data in tables 27-30 and FIGS. 20-23, the changes after rocuronium bromide administration are similar to the results of the example atracurium besylate assay, and the rules of the results are similar to MRGPRX2-RBL-2H3 cells. The results prove that the two over-expression stable transfer cell strains MRGPRX2-RBL-2H3 and MRGPRX2-P815 constructed by the invention can display the degree of cell degranulation by detecting the release rate of histamine and beta-aminohexosidase, namely the release level of inflammatory medium in cell supernatant, so as to reflect the anaphylactic risk, the release degree of the over-expression stable transfer cell strain medium after the administration of atracurium is increased compared with that of the original cell strain, and the index proves that the MRGPRX2 stable transfer cell strain is more suitable for in vitro detection of the drug, and MRGPRX2 is probably a drug target generated by atracurium anaphylactic reaction.
Table 27 histamine release from cells following rocuronium bromide dosing
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing RBL-2H3 cells as compared to the blank group with P < 0.05 and P < 0.01; a represents MRGPRX2-RBL-2H3 cells, a is P < 0.05, aa is P < 0.01 compared with a blank control group; # represents MRGPRX2-RBL-2H3 cells, P < 0.05, # is P < 0.01 compared to RBL-3H3 cells; results toN=3.
Table 28 histamine release from cells following rocuronium bromide administration
Note that: the statistical method comprises the following steps: single factor analysis of variance/independent sample TtestIn the experiment, P815 cells were compared with the blank group, P < 0.05, P < 0.01; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01 compared with the blank control group; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to P815 cells; results toN=3.
Table 29 beta-hexosaminidase Release from cells after rocuronium bromide administration
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing P815 cells as P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01 compared with the blank control group; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to P815 cells; results toN=3.
TABLE 30 beta-hexosaminidase Release from cells after rocuronium bromide administration
Note that: the statistical method comprises the following steps: single factor anova/independent sample T test, representing P815 cells as P < 0.05 and P < 0.01 compared to the blank; a represents MRGPRX2-P815 cells, a is P < 0.05, aa is P < 0.01 compared with the blank control group; # represents MRGPRX2-P815 cells with P < 0.05 and # P < 0.01 compared to P815 cells; results toN=3.
In summary, the following technical effects are obtained by the stable cell line of embodiment 1 of the present invention:
(1) Through q-PCR detection, the MRGPRX2 gene expression level in the stable transgenic cell strain is improved by at least 60 times compared with that of a starting cell before transfection;
(2) Taking the Fluo-4AM labeling rate, the Annexin V positive rate, the histamine and the beta-aminohexosaminidase release rate as detection indexes, wherein the indexes can cover the whole process of anaphylactic reaction after the medicine stimulates the membrane receptor; the results show that under the stimulation effect of the same drug, the stable transfected cell strain provided by the invention has more obvious indexes than the original cells before transfection;
(3) The drug C48/80 is taken as a representative positive drug, two kinds of anesthetic agents of muscle relaxants are selected as examples, and the atracurium besylate and the rocuronium bromide are combined with the detection results of the indexes, so that the stable transgenic cell line can be practically applied to screening of allergy-like risk drugs, in particular to screening of muscle relaxants.

Claims (7)

1. The application of the stable transgenic cell strain over-expressing MRGPRX2 in screening of allergy-like risk medicaments is characterized in that the stable transgenic cell strain is a stable transgenic cell strain with a preservation number of CCTCC NO: C2023150.
2. The application of the stable transgenic cell strain over-expressing MRGPRX2 in preparing the anaphylactic reaction-like detection reagent is characterized in that the stable transgenic cell strain is a stable transgenic cell strain with a preservation number of CCTCC NO: C2023150.
3. The use according to claim 1 or 2, wherein the method of constructing the stably transfected cell line comprises the steps of transfecting the starting cells with an overexpression plasmid of MRGPRX2 and selecting.
4. The use according to claim 3, wherein the backbone plasmid of the over-expression plasmid is pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro.
5. The application according to claim 1 or 2, wherein the application comprises: and (3) contacting the drug to be detected with the stable transgenic cell strain to obtain a detection index result.
6. The use of claim 5, wherein the assay indicator is selected from the group consisting of intracellular calcium ion level change, degree of exocytosis, and rate of release of inflammatory mediators released by degranulation of cells.
7. The use of claim 6, wherein the change in intracellular calcium ion levels is detected by a calcium ion fluorescent probe; the exocytosis degree of the cells is detected by an Annexin V positive rate; the release rate of inflammatory mediators released by degranulation of the cells was measured by histamine and beta-hexosaminidase release rates.
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