CN116286998A - Cell model for screening EGFR inhibitor and construction method and application thereof - Google Patents
Cell model for screening EGFR inhibitor and construction method and application thereof Download PDFInfo
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- CN116286998A CN116286998A CN202310546225.6A CN202310546225A CN116286998A CN 116286998 A CN116286998 A CN 116286998A CN 202310546225 A CN202310546225 A CN 202310546225A CN 116286998 A CN116286998 A CN 116286998A
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Abstract
The invention provides a cell model for screening EGFR inhibitors, and a construction method and application thereof. According to the reporter gene method based on the cell level, an EFGR receptor is expressed in HEK293 cell expression, and a STAT3 controlled luciferase expression element is transferred, when the added EGF binds EGFR, a STAT3 signal path is started, activated STAT3 can bind to a nucleotide sequence shown as SEQ ID NO. 2, so that the cell expresses luciferase, when a corresponding EGFR drug is added, the expression of the luciferase is weakened, and then the luciferase reacts with an additional luciferase substrate to generate chemiluminescence, and the chemiluminescence intensity of the luciferase can be quantitatively evaluated for biological activity of the EGFR drug. The invention has the advantages of simple and rapid operation, high sensitivity, good repeatability and suitability for high-throughput screening.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a cell model for screening EGFR inhibitors, and a construction method and application thereof.
Background
EGFR is an expression product of a proto-oncogene C-ErbB1, is a receptor for cell proliferation and signaling of an Epidermal Growth Factor (EGF), is one of members of the epidermal growth factor receptor (HER) family, belongs to a tyrosine kinase type receptor, and consists of three parts: extracellular ligand binding domain, single-stranded transmembrane domain, and intracellular tyrosine kinase domain. After the EGFR extracellular region is combined with the ligand, the EGFR extracellular region is changed into a dimer from a monomer, an intracellular kinase region is activated, three signal paths of STAT and PI3-K, RAS are mainly activated, and the cascade reaction of the three signal paths plays an important role in various physiological activities such as proliferation, differentiation, migration, apoptosis and the like of cells. Thus, excessive activation of the EGFR signaling pathway, such as non-small cell lung cancer, breast cancer, head and neck cancer, colon cancer, ovarian cancer, and the like, is detected in a variety of malignancies. Currently, molecular targeted drugs developed for EGFR are largely divided into two categories: (1) Small molecule Tyrosine Kinase Inhibitors (TKIs), such as gefitinib and erlotinib, inhibit EGFR intracellular domain tyrosine kinase activity; (2) Monoclonal antibodies (mabs), such as cetuximab and panitumumab, bind to the extracellular region of EGFR and block ligand-dependent activation of EGFR. The drugs block EGFR-mediated intracellular signaling pathways through different pathways, thereby inhibiting tumor growth, metastasis and angiogenesis, and promoting tumor cell apoptosis.
In the development process of EGFR drugs, it is very important to establish a cell biological activity evaluation method capable of reacting with a drug action Mechanism (MOA), and the drug activity is usually evaluated by adopting a method for inhibiting the growth of a tumor cell line of high-expression EGFR, however, the method has the defects of instability, insensitivity and unfavorable high-throughput screening, and development of a new system for EGFR antibody drug screening and/or small molecule inhibitor biological activity detection is needed.
Disclosure of Invention
The invention aims to provide a cell model for screening EGFR inhibitors, and a construction method and application thereof.
According to the invention, after ligand EGF is combined with receptor EGFR, EGFR receptor is dimerized, intracellular area is activated, cascade reaction of downstream signals is caused, a STAT3 signal response element synthesized by genes is connected to upstream of reporter luciferase by utilizing a STAT3 signal channel which can be activated, a reporter luciferase expression vector driven by the STAT3 response element is constructed, the expression vector is packaged into lentivirus, HEK293 cells are infected, and stable cell strain (named HEK293-STAT3/Luc cell strain) of STAT3 driven luciferase is obtained through signal channel activity screening. On the basis of the stable cell strain, a carrier expressing receptor EGFR is used for transfection, and the EGFR small molecule inhibitor and antibody activity detection stable cell strain (named HEK293-STAT 3/Luc-EGFR) is finally obtained through EGF activation screening.
To achieve the object of the present invention, in a first aspect, the present invention provides a method for constructing a cell model for screening an EGFR inhibitor, comprising the steps of:
A. total gene synthesis of luciferase expression elements driven by STAT3 (signal transduction and transcription activator 3, geneID: 6774 in ncbi), construction of synthetic DNA sequences onto lentiviral expression vectors;
B. c, co-transfecting HEK293 cells with the recombinant lentiviral expression vector constructed in the step A and the lentiviral packaging auxiliary plasmid to obtain recombinant cells HEK293-STAT3/Luc;
C. constructing a eukaryotic expression vector for expressing human EGFR;
D. and C, introducing the recombinant expression vector constructed in the step C into HEK293-STAT3/Luc cells in the step B, and screening positive transformants to serve as a cell model for screening EGFR inhibitors.
The DNA sequence synthesized in the step A is shown as SEQ ID NO. 1.
Wherein STAT3 can be combined with a nucleotide sequence shown as SEQ ID NO. 2.
Preferably, the lentiviral expression vector of step A is pLenti-CMV-GFP-hygromycin (available from ACRObiosystems); the lentiviral packaging helper plasmids described in step B are PMD (available from ACROBiosystems) and PSD (available from ACROBiosystems); the starting vector for the eukaryotic expression vector described in step C was pCMV-GFP-puromycin (available from ACROBiosystems).
In a second aspect, the invention provides a cell line (HEK 293-STAT 3/Luc-EGFR-4) constructed according to the method.
In a third aspect, the invention provides the use of said cell line (HEK 293-STAT 3/Luc-EGFR-4) for screening EGFR inhibitors, i.e.for detecting biological activity of EGFR small molecule inhibitors and/or antibodies by means of a luciferase reporter gene.
In a fourth aspect, the invention provides a method of screening for an EGFR inhibitor, comprising the steps of:
(1) And (3) digesting the cell line by pancreatin, inoculating the cell line into a reaction pore plate, culturing for a period of time in a culture medium, adding samples to be detected with different concentrations into the pore, adding EGF protein with a certain concentration into the pore, simultaneously setting the EGF protein added, the EGF protein not added and the sample to be detected as positive control and the EGF protein not added and the sample to be detected as negative control, uniformly mixing, placing the mixture into an incubator for incubation for a period of time, adding luciferase substrates into each pore, uniformly mixing, lysing cells, detecting a luminescence signal value, and calculating the neutralization rate according to the following formula:
neutralization rate = (positive control luminescence signal value-to-be-measured sample luminescence signal value)/(positive control luminescence signal value-negative control luminescence signal value) ×100%
(2) Establishing an equation of the relation between the concentration and the neutralization rate of the sample to be detected, and calculating EC 50 Value and according to EC 50 The value determines the inhibitory activity of the test sample on EGFR.
Further, the method comprises: the cell lines were trypsinized with 3X 10 total accesses per well of 0.1mL in 96-well plates 4 Culturing cells of each cell in DMEM medium containing 10% serum for about 24h, adding samples to be tested with different concentrations at 40 μl/well into 96-well plate, simultaneously adding EGF protein with final concentration of 50 ng/mL at 40 μl/well, setting positive control containing EGF protein and no sample to be tested, negative control containing EGF protein and sample to be tested, mixing uniformly, and placing at 37deg.C and 5% CO 2 Incubator incubates about 18 h. Luciferase substrate was added at 80. Mu.L/well and mixed well, cells were lysed and luminescence signal values were detected.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention uses the reporter gene method based on the cell level for quantitative evaluation of EGFR small molecule inhibitor and antibody activity for the first time. Compared with the method for quantitatively evaluating the activity of the drug by adopting the method for inhibiting the growth of the tumor cell line with high expression of EGFR, the invention utilizes the downstream STAT3 signal path activated by EGFR, and based on the action Mechanism (MOI) of the target drug, the developed firefly reporter gene detection method has the advantages of simple and convenient operation, high speed, high sensitivity, good repeatability and suitability for high-throughput screening.
Drawings
FIG. 1 shows the detection of IL-6 activation STAT3 activity controlling luciferase expression in a preferred embodiment of the invention. Wherein EC is 50 =3.594ng/mL。
FIG. 2 shows the activity screening assay of HEK293-STAT3/Luc-EGFR monoclonal cell lines in a preferred embodiment of the invention.
FIG. 3 is a flow assay of HEK293-STAT3/Luc-EGFR monoclonal cell lines in a preferred embodiment of the invention. Wherein, 1: HEK293-STAT3/Luc,2: HEK293-STAT3/Luc-EGFR-4.
FIG. 4 shows the EGF activating activity assay of HEK293-STAT3/Luc-EGFR-4 monoclonal cell line according to a preferred embodiment of the present invention. Where maximum induction fold = 56.
FIG. 5 shows the activity of EGFR antibody drug in accordance with the preferred embodiment of the present invention.
FIG. 6 shows the activity of EGFR small molecule inhibitors in accordance with a preferred embodiment of the present invention.
FIG. 7 shows stability detection of STAT 3-driven luciferase expression in accordance with the preferred embodiments of the invention.
FIG. 8 shows the stability test of EGFR expression level of cell membrane surface receptor in the preferred embodiment of the present invention. Wherein, 1: control, 2: p10,3: p15,4: p20.
Detailed Description
The invention is based on a cell level reporter gene method, which is characterized in that an EFGR receptor is expressed in HEK293 cell expression, and a STAT3 controlled Luciferase (Luciferase, genbank: BAF 48390.1) expression element is transferred, when the added EGF is combined with EGFR, a STAT3 signal path is started, activated STAT3 can be combined with a nucleotide sequence shown as SEQ ID NO. 2, so that the cells express the Luciferase, when a corresponding EGFR drug is added, the expression of the Luciferase is weakened, and then the Luciferase reacts with an external Luciferase substrate to generate chemiluminescence, and the chemiluminescence intensity of the Luciferase can quantitatively evaluate the biological activity of the EGFR drug.
The invention adopts the following technical scheme:
(1) Constructing a STAT3 reaction element driven luciferase expression vector;
(2) Constructing HEK293-STAT3/Luc stable cell lines;
(3) Constructing a human EGFR expression vector;
(4) Constructing HEK293-STAT3/Luc-EGFR stable cell strain;
(5) Detection of biological activity of EGFR small molecule inhibitors and antibodies.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 construction of STAT3-driven luciferase expression vector
1. Construction of pLenti-STAT3/Luc-hygromycin plasmid
Total gene synthesis of luciferase expression elements driven by STAT3, reference may be made to von Laue S, finidori J, maama M, shen XY, julice S, dobson PR, ross RJ. Stimulation of endogenous GH and interleukin-6 receptors selectively activates different Jaks and Stats, with a STAT5 specific synergistic effect of dexamethasone J endocrinol 2000 May 165 (2): 301-11.
The synthesized DNA fragment (SEQ ID NO: 1) was constructed on pLenti-CMV-GFP-hygromycin (available from ACRObiosystems) to give a recombinant plasmid designated pLenti-STAT3/Luc-hygromycin.
2. Lentiviral packaging of pLenti-STAT3/Luc-hygromycin plasmid
The pLenti-CMV-GFP-hygromycin expression plasmid was mixed with the lentiviral packaging helper plasmid PMD and PSD (helper plasmid supplied by Beijing Baipe Sitting Biotech Co., ltd.) in a certain ratio and co-transfected into 293T cells. After 72h of transfection, the culture supernatant containing the virus was collected and centrifuged at 3000 rpm at 4℃for 5min. Mixing supernatant with PEG8000/NaCl at volume ratio of 1:3, standing at 4deg.C overnight, and centrifuging at high speed for 45min. Discarding supernatant, and re-suspending and dissolving the precipitate with pre-cooled PBS to obtain lentivirus concentrate, and storing at-80deg.C.
EXAMPLE 2 construction of HEK293-STAT3/Luc cell lines
HEK293 cells were resuscitated 2 days in advance and plated in 6 well plates at 1X 10 according to a total 3 mL/well access 6 HEK293 cells were cultured in DMEM medium containing 10% serum for about 24 hours, the supernatant was discarded, 100mL of lentiviral concentrate was diluted with the same medium for 3mL, slowly added to a 6-well plate inoculated with HEK293 cells, and polybrene was added at a final concentration of 8. Mu.g/mL at 37℃with 5% CO 2 Culturing in an incubator for about 48 hours to obtain the recombinant cell HEK293-STAT3/Luc.
EXAMPLE 3 Activity detection of STAT3-driven luciferase expression
Pancreatin digestion of HEK293-STAT3/Luc cells obtained in example 2 at a total of 3X 10 in 0.1 mL/well Access in 96 well plates 4 HEK293-STAT3/Luc cells were cultured in DMEM medium containing 10% serum at about 24. 24 h. Human IL-6 (Cat. No. IL6-H4218, beijing Baisai Biotech Co., ltd.) protein (500 ng/mL, 125 ng/mL, 31.25 ng/mL, 7.813 ng/mL, 1.953 ng/mL, 0.488 ng/mL, 0.122 ng/mL, 0.031 ng/mL) was added to the 96-well cell culture plates at various concentrations, and 80. Mu.L/well was used as a negative control without IL-6 protein. Mixing, and placing at 37deg.C and 5% CO 2 Incubator incubates about 18 h. ONE-Glo ™ Luciferase reagent (Promega, cat. No. E6120) was added at 80. Mu.L/well and the cells were lysed by mixing. 100 mu L/Kong Zhuairu 96-well white bottom plate detection luminescence signal value (RLU) is taken, the activation times are calculated, and the activation times = sample RLUs/negative control RLUs, analyzing and drawing a dose-response curve by using GraphPad Prism software, calculating EC (electronic control) by taking the abscissa as the concentration of a sample and the ordinate as the activation multiple 50 Values. As a result, IL-6 was effective in activating STAT 3-driven luciferase expression and was concentration-dependent, as shown in FIG. 1.
EXAMPLE 4 construction of human EGFR expression vectors
The EGFR (Genbank, NP-005219) receptor from a total gene-synthesized human was constructed on pCMV-GFP-puromycin (available from Beijing Baiposis Biotechnology Co., ltd.) to give a recombinant plasmid designated pCMV-EGFR-puromycin.
EXAMPLE 5 construction of HEK293-STAT3/Luc-EGFR cell lines
1. Transfection
Pancreatin digestion of the HEK293-STAT3/Luc cell line from example 2, the total number of accesses in 6-well plates was 1X 10 at3 mL/well 6 HEK293-STAT3/Luc cells were cultured in DMEM medium containing 10% serum for about 24 hours, the supernatant was discarded, 5. Mu.g of pCMV-GFR-puromycin plasmid was transfected with PEI at 37℃with 5% CO 2 The incubator was incubated overnight for about 24 hours, and selection medium (DMEM+10% FBS+ mg/mL puromycin) was added and the selection medium was changed every 3-4 days for 3 weeks to obtain HEK293-STAT3/Luc-EGFR.
2. HEK293-STAT3/Luc-EGFR cell strain monoclonal sorting
The HEK293-STAT3/Luc-EGFR cell line obtained above was digested with pancreatin. Counting, diluting the cells to 10/mL with DMEM medium containing 10% serum, inoculating the cells into a 96-well plate according to 0.1 mL/well, culturing for about 3 weeks, observing under a microscope, selecting the cell holes with monoclonal growth, transferring the cell holes to a 24-well plate, and transferring the cell holes to a 6-well plate for expansion culture after 1 week.
3. HEK293-STAT3/Luc-EGFR monoclonal cell strain functional activity screening
Pancreatin digestion of HEK293-STAT3/Luc-EGFR monoclonal cell lines in 96 well plates at a total of 3X 10 at 0.1 mL/well 4 HEK293-STAT3/Luc-EGFR cells were cultured in DMEM medium containing 10% serum for about 24 h. Human EGF (Beijing Baiposi Biotechnology Co., ltd., cat.N.) was added to 96-well cell culture plates at various concentrationsEGF-H52H3 protein (100 ng/mL), 80. Mu.L/well, no proteome added as negative control. Mixing, and placing at 37deg.C and 5% CO 2 Incubator incubates about 18 h. ONE-Glo ™ Luciferase reagent was added at 80. Mu.L/well and the cells were lysed by mixing. The activation fold was calculated by taking 100 μl/Kong Zhuairu 96 well white bottom plate detection luminescence signal value (RLU), and the results are shown in fig. 2, where cell clones 1, 3, 4, 5, 6, and 7 all have better activation fold, and the activation fold of cell clone No. 4 (HEK 293-STAT 3/Luc-EGFR-4) is highest, reaching 44.1 fold.
4. Flow detection of HEK293-STAT3/Luc-EGFR monoclonal cell strain
Pancreatin digested monoclonal cell line HEK293-STAT3/Luc-EGFR-4 at a ratio of 5X 10 5 The cells/tube were placed in a flow tube, wherein HEK293-STAT3/Luc cells were set as negative cells, PE anti-human EGFR (Biolegend, cat. No. 352904) was added at 5uL/Test, mixed incubation was performed at 4℃for 20 min, washed with PBS wash, centrifuged to remove unbound antibody, and 300 uL PBS was used to resuspend cells for detection on a flow cytometer. As shown in FIG. 3, the EGFR positive rate of the HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain is 99.39%, and the uniformity is good.
EXAMPLE 6 detection of Activity of HEK293-STAT3/Luc-EGFR-4 monoclonal cell line activation
The HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain screened by pancreatin digestion was inoculated in a 96-well plate according to a total of 3X 10 at 0.1 mL/well 4 HEK293-STAT3/Luc-EGFR-4 cells were cultured in DMEM medium containing 10% serum for about 24 h. Human EGF protein (800 ng/mL, 400 ng/mL, 200 ng/mL, 100ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL) was added to 96-well cell culture plates at various concentrations, 80. Mu.L/well, and no proteome was added as a negative control. Mixing, and placing at 37deg.C and 5% CO 2 Incubator incubates about 18 h. ONE-Glo ™ Luciferase reagent was added at 80. Mu.L/well and the cells were lysed by mixing. Taking 100 mu L/Kong Zhuairu 96-well white bottom plate detection luminescence signal value (RLU), calculating an activation multiple, wherein the activation multiple=sample RLUs/negative control RLUs, analyzing and drawing a dose-response curve by using GraphPad Prism software, and the abscissa is the concentration of the sampleDegree, ordinate is activation multiple. As shown in FIG. 4, EGF can effectively activate STAT3 to drive expression of luciferase by binding to EGFR receptor on cell surface, and has concentration dependence, and the highest activation multiple can reach 56 times.
EXAMPLE 7 detection of antibody pharmaceutical Activity
The HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain screened by pancreatin digestion was inoculated in a 96-well plate according to a total of 3X 10 at 0.1 mL/well 4 HEK293-STAT3/Luc-EGFR-4 cells were cultured in DMEM medium containing 10% serum for about 24 hours, at 40. Mu.L/well in 96-well cell culture plates, cetuximab (available from Beijing Baisai Biotechnology Co., ltd.) antibodies (50 ug/mL, 12.5 ug/mL, 3.13. Mu.g/mL, 0.78. Mu.g/mL, 0.20. Mu.g/mL, 0.05. Mu.g/mL, 0.01. Mu.g/mL and 0.003. Mu.g/mL) were added at different concentrations, EGF protein was added at a final concentration of 50 ng/mL, the non-added antibody set was set as a positive control, the non-added protein and antibody set were negative control, and the mixture was homogenized at 37℃and 5% CO 2 Incubator incubates about 18 h. ONE-Glo ™ Luciferase reagent was added at 80. Mu.L/well and the cells were lysed by mixing. The neutralization rate was calculated by taking 100. Mu.L/Kong Zhuairu 96-well white bottom plate detection luminescence signal value (RLU).
Neutralization% = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%
Analyzing and drawing a dose-response curve by using GraphPad Prism software, calculating EC (ethylene glycol) by taking the concentration of a sample as an abscissa and the neutralization rate as an ordinate 50 Values. As shown in FIG. 5, the Cetuximab antibody can effectively inhibit the activation activity of EGF and is concentration dependent, EC 50 The value is 1.793 mug/mL, and the screened HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain can be used for activity detection of EGFR antibody drugs.
Example 8 detection of Small molecule drug Activity
The HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain screened by pancreatin digestion was inoculated in a 96-well plate according to a total of 3X 10 at 0.1 mL/well 4 HEK293-STAT3/Luc-EGFR-4 cells were cultured in DMEM medium containing 10% serum for about 24 hours, and added at 40. Mu.L/well to a 96-well cell culture plateAdding EGFR small molecule inhibitor Erlotinib (Beyotide, SC0168-10 mM) at different concentrations, diluting with concentration of 1 μM, 0.2 μM, 0.04 μM, 0.008 μM, 0.0016 μM, 0.00032 μM, 0.000064 μM and 0.000013 μM, adding EGF protein at final concentration of 50 ng/mL at 40 μL/hole, setting protein adding, small molecule inhibitor adding as positive control, protein adding and small molecule inhibitor group as negative control, mixing, and standing at 37deg.C and 5% CO 2 Incubator incubates about 18 h. ONE-Glo ™ Luciferase reagent was added at 80. Mu.L/well and the cells were lysed by mixing. The neutralization rate was calculated by taking 100. Mu.L/Kong Zhuairu 96-well white bottom plate detection luminescence signal value (RLU).
Neutralization% = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%
Analyzing and drawing a dose-response curve by using GraphPad Prism software, calculating EC (ethylene glycol) by taking the concentration of a sample as an abscissa and the neutralization rate as an ordinate 50 Values. As shown in FIG. 6, the Erlotinib small molecule inhibitor can effectively inhibit EGFR activation activity and has concentration dependence, EC 50 The value is 0.01 mu M, and the screened HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain can be used for activity detection of EGFR small molecule inhibitors.
Example 9 stability test of HEK293-STAT3/Luc-EGFR-4 monoclonal cell line
1. Stability detection of STAT 3-driven luciferase expression
Resuscitate successive growing HEK293-STAT3/Luc-EGFR-4 monoclonal cell lines at 3X 10 in 96 well plates according to a total of 0.1 mL/well access 4 HEK293-STAT3/Luc-EGFR-4 cells were cultured in DMEM medium containing 10% serum for about 24 h. Human EGF protein (800 ng/mL, 400 ng/mL, 200 ng/mL, 100ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL) was added to 96-well cell culture plates at various concentrations, 80. Mu.L/well, and no proteome was added as a negative control. After mixing, the mixture was incubated at 37℃in a 5% CO2 incubator at about 18 h. ONE-Glo ™ Luciferase reagent was added at 80. Mu.L/well and the cells were lysed by mixing. Taking 100 mu L/Kong Zhuairu 96-well white bottom plate detection luminescence signal value (RLU), calculating an activation multiple, wherein the activation multiple=sampleSample RLUs/negative control RLUs, analyzing and drawing a dose-response curve by using GraphPad Prism software, wherein the abscissa is the concentration of the sample, the ordinate is the activation multiple, and calculating EC 50 Values. As shown in FIG. 7 and Table 1, the activation activities of HEK293-STAT3/Luc-EGFR-4 monoclonal cell lines of different generations exhibited better stability, CV% was less than 20%, and the EC50 of the EGF protein measured exhibited better stability, CV% was less than 20%.
TABLE 1
2. Stability detection of EGFR expression level of cell membrane surface receptor
Continuous growth of HEK293-STAT3/Luc-EGFR-4 monoclonal cell lines of different generations by pancreatin digestion was performed at a rate of 5X 10 5 The cells/tube was placed in a flow tube, wherein HEK293-STAT3/Luc cells were set as negative cells, PE anti-human EGFR was added at 5. Mu.L/tube, mixed incubation was performed for 20 min at 4℃and washed with PBS wash, unbound antibody was removed by centrifugation, 300. Mu.L PBS resuspended cells, and detection was performed on-machine by flow cytometry. As shown in FIG. 8, HEK293-STAT3/Luc-EGFR-4 monoclonal cell strain surface receptor EGFR expression levels of different generations show better stability.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. A method of constructing a cell model for screening for EGFR inhibitors, comprising the steps of:
A. total gene synthesis of a luciferase expression element driven by STAT3, and constructing a synthesized DNA sequence on a lentiviral expression vector;
B. c, co-transfecting HEK293 cells with the recombinant lentiviral expression vector constructed in the step A and the lentiviral packaging auxiliary plasmid to obtain recombinant cells HEK293-STAT3/Luc;
C. constructing a eukaryotic expression vector for expressing human EGFR;
D. and C, introducing the recombinant expression vector constructed in the step C into HEK293-STAT3/Luc cells in the step B, and screening positive transformants to serve as a cell model for screening EGFR inhibitors.
2. The method according to claim 1, wherein the DNA sequence synthesized in step A is shown in SEQ ID NO. 1.
3. The method of claim 1 or 2, wherein the lentiviral expression vector of step a is pLenti-CMV-GFP-hygromycin;
b, the lentivirus packaging auxiliary plasmids are PMD and PSD;
the starting vector of the eukaryotic expression vector in the step C is pCMV-GFP-puromycin.
4. A cell line constructed according to the method of any one of claims 1-3.
5. Use of the cell line of claim 4 for screening for EGFR inhibitors.
A method of screening for an egfr inhibitor, comprising the steps of:
(1) Pancreatin digestion of the cell line of claim 4, inoculating into a reaction well plate, culturing in a culture medium for a period of time, adding samples to be tested with different concentrations into the well, adding EGF protein with a certain concentration into the well, setting the EGF protein added, the EGF protein not added and the sample to be tested as positive control and the EGF protein not added and the sample to be tested as negative control, mixing uniformly, placing into an incubator, incubating for a period of time, adding luciferase substrates into each well, mixing uniformly, lysing cells, detecting luminous signal values, and calculating the neutralization rate according to the following formula:
neutralization rate = (positive control luminescence signal value-to-be-measured sample luminescence signal value)/(positive control luminescence signal value-negative control luminescence signal value) ×100%
(2) Establishing an equation of the relation between the concentration and the neutralization rate of the sample to be detected, and calculating EC 50 Value and according to EC 50 The value determines the inhibitory activity of the test sample on EGFR.
7. The method according to claim 6, characterized in that the method comprises: the cell lines were trypsinized with 3X 10 total accesses per well of 0.1mL in 96-well plates 4 Culturing 24h cells in DMEM medium containing 10% serum, adding samples to be tested with different concentrations at 40 μl/well into 96-well plate, adding EGF protein with final concentration of 50 ng/mL at 40 μl/well, setting positive control containing EGF protein and no sample to be tested, negative control containing EGF protein and sample to be tested, mixing, and standing at 37deg.C and 5% CO 2 Incubator incubation 18 h; luciferase substrate was added at 80. Mu.L/well and mixed well, cells were lysed and luminescence signal values were detected.
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