CN114480406B - IL-1 signal path response element and application thereof - Google Patents
IL-1 signal path response element and application thereof Download PDFInfo
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
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Abstract
The invention discloses an IL-1 signal channel response element and application thereof, and the invention prepares an effector cell for stably expressing an IL-1 signal channel response element reporter gene, stimulates and activates the expression of the reporter gene by using IL-1 alpha and IL-1 beta, blocks the IL-1 signal channel by using IL-1Ra, and fits a four-parameter curve according to the measured signal value of the reporter gene to determine the biological activity of protein. The invention establishes a rapid, accurate and more sensitive detection method aiming at the biological activity measurement of IL-1 alpha, IL-1 beta and IL-1Ra medicaments, has short experimental period and simple operation, and also reduces the deviation caused by objective factors in the long-time incubation process of cells.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an IL-1 signal pathway response element and application thereof.
Background
Interleukin 1 (IL-1) is one of the earliest cytokines found in humans. It is a group of hormone-like peptide substances which have wide physiological actions such as maintaining the stability of internal environment, anti-infection, anti-tumor, promoting hematopoiesis and the like, and occupy important positions in cytokine networks. IL-1 family members consist of IL-1α, IL-1β and an interleukin-1 receptor antagonist (interlukin-1 receptor antagonist,IL-1 Ra). IL-1Ra is an antagonist common to IL-1 alpha and IL-1 beta, and its protein sequence is obviously similar to that of IL-1 alpha and IL-1 beta in receptor binding site, i.e. C-terminal region. IL-1Ra competitively inhibits IL-1 biological activity by binding to IL-1 receptor and has a dose-dependent relationship.
The U.S. Food and Drug Administration (FDA) approved the rh-IL-1Ra of Amgen, inc. for the treatment of refractory rheumatoid arthritis where conventional drugs were ineffective in 2001, 11. The biological activity of rh-IL-1Ra is embodied by inhibiting the activity of IL-1, is a naturally occurring cytokine competitive inhibitor discovered at present, and is a natural antagonistic mechanism formed by avoiding excessive inflammatory response of an organism.
Current methods for determining the biological activity of IL-1, IL-1Ra and IL-1 antibodies are primarily methods for inhibiting cell proliferation, which are primarily based on the fact that IL-1 killing of A375-S2 cells is dose dependent. The method is generally used for incubating for 72-120 h at 37 ℃, and has long experimental period and large variability.
Disclosure of Invention
The present invention aims to overcome the above-mentioned shortcomings of the prior art, and provides an IL-1 signal path response element and application thereof.
It is a first object of the present invention to provide an IL-1 signaling pathway response element.
A second object of the present invention is to provide a recombinant vector.
It is a third object of the present invention to provide a recombinant cell.
It is a fourth object of the invention to provide a method for detecting the biological activity of IL-1. Alpha. Or IL-1. Beta.
It is a fifth object of the present invention to provide a method of binding interleukin-1 receptor antagonist to the biological activity of IL-1α or IL-1β.
It is a sixth object of the present invention to provide the use of said IL-1 signaling pathway response element, said recombinant vector, and/or said recombinant cell for detecting a biological activity of an interleukin-1 receptor antagonist, IL-1 alpha or IL-1 beta.
In order to achieve the above object, the present invention is realized by the following means:
HepG2 cells belong to human liver cancer cells, the surfaces of which express IL-1R (interleukin-1 receptor), IL-1 binds to IL-1R, and activates IL-1 signaling pathway response elements through IRAK proteins; will contain a nucleotide sequence as set forth in SEQ ID NO: 1. the plasmid pGL-4.11-IL1RE of the IL-1 signaling pathway response element and luciferase reporter shown was placed into HepG2 cells,
IL-1 signaling pathway response elements are activated upon binding of IL-1 to IL-1R and initiate expression of a luciferase reporter linked to the response elements. IL-1Ra and IL-1 competitively bind to IL-1R and block an IL-1 signal path, so that the concentration of IL-1Ra is inversely proportional to the luciferase expression quantity of effector cells, a four-parameter curve is fitted according to the measured signal value of a reporter gene to determine the biological activity of an IL-1Ra antibody, and the corresponding titer is obtained by comparing half inhibition concentrations of the four-parameter curve of a sample and a reference sample.
The invention therefore claims the following:
an IL-1 signaling pathway response element having a nucleotide sequence set forth in SEQ ID NO: 1.
A recombinant vector is a plasmid containing the IL-1 signal pathway response element.
Preferably, the plasmid further comprises a reporter gene, and the IL-1 signaling pathway response element is located upstream of the reporter gene.
More preferably, the reporter is a luciferase reporter.
Even more preferably, the reporter gene is a luciferase reporter gene.
A recombinant cell, which is a cell containing the recombinant vector.
More preferably, the cell is any one of CHO cells, 293 cells, hela cells, hepG2 cells, a549 cells and human liver cancer cells.
The invention further claims a method for detecting the biological activity of IL-1 alpha or IL-1 beta comprising mixing cells of the recombinant vector with a gradient concentration of IL-1 alpha or IL-1 beta, respectively, culturing, removing the supernatant, washing the cells, lysing the cells, performing a sufficient reaction, performing solid-liquid separation, collecting the supernatant, mixing with a luciferase substrate, performing a sufficient reaction, and detecting a chemiluminescent value, wherein the recombinant vector comprises a luciferase reporter gene thereon.
Preferably, the gradient concentration of IL-1α or IL-1β, IL-1α initial concentration of 250ng/mL,2 times serial dilution of 8 concentration gradient; IL-1β initial concentration was 125ng/mL, 2-fold serial dilutions were made for 8 concentration gradients.
Preferably, the incubation time is 4 to 22 hours.
More preferably, the incubation time is 18 hours.
Preferably, the concentration of the recombinant vector-containing cells is 1X 10 4 ～8×10 5 And/or holes.
More preferably, the concentration of cells containing the recombinant vector is 5X 10 4 Cell concentration of individual/well
The invention also contemplates the use of said IL-1 signaling pathway response element, said recombinant vector, and/or said recombinant cell for detecting a biological activity of an interleukin-1 receptor antagonist, IL-1 alpha or IL-1 beta.
The invention also claims a method for combining the biological activity of IL-1 alpha or IL-1 beta by using the interleukin-1 receptor antagonist, cells containing recombinant vectors, IL-1 alpha or IL-1 beta with known biological activity are respectively mixed with IL-1Ra with gradient concentration or interleukin-1 receptor antagonist with gradient concentration to be tested, cultured, cell supernatant removed, cells washed, cell lysed, solid-liquid separated, supernatant removed, mixed with luciferase substrate, fully reacted, and detected chemiluminescence values, wherein the recombinant vectors contain luciferase reporter genes.
Preferably, the IL-1α or IL-1β concentration is 10 pg/mL-2 μg/mL (for activating reporter gene expression)
More preferably, the IL-1α or IL-1β concentration is 30ng/mL.
Preferably, the gradient concentration of IL-1Ra, IL-1Ra starting concentration is 1pg/mL-1mg/mL.
More preferably, the gradient concentration of IL-1Ra, IL-1Ra initial concentration is 10pg/mL ~ 100 μg/mL.
The initial concentration of IL-1Ra is only sufficient to ensure that a plateau occurs.
Further preferably, the IL-1Ra initial concentration is 63ng/mL, which ensures that a plateau can occur.
Preferably, the incubation time is 4 to 22 hours.
More preferably, the incubation time is 18 hours.
Preferably, the concentration of the cells containing the recombinant vector of claim 3 is 1X 10 4 ～8×10 5 And/or holes.
More preferably, the concentration of cells containing the recombinant vector is 3X 10 4 Cell concentration of individual/well
Preferably, the adjacent concentration of the interleukin-1 receptor antagonist to be tested is 1:2-5 in gradient concentration.
More preferably, the gradient concentration of the interleukin-1 receptor antagonist to be tested is adjacent to a concentration of 1:4.
Compared with the prior art, the invention has the following beneficial effects:
the invention prepares an effector cell for stably expressing an IL-1 signal channel response element reporter gene, stimulates and activates the expression of the reporter gene by using IL-1 alpha and IL-1 beta, blocks the IL-1 signal channel by using IL-1Ra, and fits a four-parameter curve according to the measured signal value of the reporter gene to determine the biological activity of the protein. The invention establishes a rapid, accurate and more sensitive detection method aiming at the biological activity measurement of IL-1 alpha, IL-1 beta and IL-1Ra medicaments, has short experimental period and simple operation, and also reduces the deviation caused by objective factors in the long-time incubation process of cells.
Drawings
FIG. 1 is a plasmid map of pGL4.11-IL1 RE.
Fig. 2 is a graph of signal activation at 3h, 6h, 10h, 24 h.
FIG. 3 is a graph showing the detection of rh-IL-1α activity.
FIG. 4 is a graph showing the detection of rh-IL-1β activity.
FIG. 5 shows the rh-IL-1RA binding rh-IL-1. Alpha. Activity.
FIG. 6 shows the rh-IL-1RA binding rh-IL-1. Beta. Activity.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
HepG2 cells are derived from ATCC; pGL4.11[ luc2P/Hygro ] plasmid was purchased from Promega;
IL-1 alpha, IL-1 beta and IL-1Ra for activity test are derived from Shanghai Jinuo Biotechnology Co., ltd, and luciferase reporter gene detection kit is purchased from Biyun Tian Co.
EXAMPLE 1 acquisition of HepG2 cell lines stably expressing the IL-1 Signal pathway response element reporter Gene
1. Experimental method
1. preparation of pGL4.11-IL1RE plasmid
Designing IL-1 signal path response element combination (IL-1 RE), and the sequence is shown in SEQ ID NO:1, synthesized in the Guangzhou department of Optimago.
SEQ ID NO：1：
GGTACCGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATA CAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGCTAGCCTCGAG GATATCAAGATCTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT。
IL-1RE and PGL4.11 synthesized by double cleavage with FastDiget restriction enzymes KpnI and HindIII, inc. Thermo Fisher Scientific, were Gel-electrophoresed, and then the linearized plasmid DNA fragments were recovered for use using the Gel Extraction kit, inc. of Omega.
The two linearized plasmid DNA fragments were ligated using T4 ligase from Thermo Fisher Scientific to construct pGL4.11-IL1RE plasmid (see FIG. 1). Plasmid was transfected into DH 5. Alpha. Competent cells by heat shock, and plasmid was extracted using the endotoxin-free plasmid extraction kit from the root of Tian after expansion culture.
2. Plasmid transfection
pGL4.11-IL1RE plasmid transfection was performed on HepG2 cells using an electroporation transfection system (invitrogen), and the next day after transfection, 300. Mu.g/mL hygromycin B was added for pressure screening, thereby killing cells into which no plasmid had been introduced. Then, the cells were expanded by limiting dilution to a maximum of 1 cell/well, and the cells were gradually expanded by adding a medium containing 300. Mu.g/mL hygromycin B, thereby obtaining HepG2 cells (HepG 2/pGL4.11-IL1RE cell lines) transfected with pGL4.11-IL1RE plasmid.
3. Screening of HepG2/pGL4.11-IL1RE cell lines
The HepG2/pGL4.11-IL1RE cell lines after the screening were isolated according to 3X 10 4 The wells/well were plated into 96-well plates, 50 μl per well; IL-1α was diluted in 2-fold series at an initial concentration of 500ng/mL, diluted 12 degrees, and added thereto. After 24h, the cell supernatant was removed, the cells were washed three times with PBS, lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the obtained lysate was added 100. Mu.L of a luciferase substrate (Biyun Tian, cat. RG 006). And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
Screening to obtain luciferase chemiluminescence value detection, and selecting a monoclonal HepG2/pGL4.11-IL1RE cell strain with high signal to noise ratio and good fitting degree after IL-1 stimulation for subsequent experiments.
2. Experimental results
The experimental results are shown in Table 1, and the results show that the monoclonal HepG2/pGL4.11-IL1RE cell strain 1 has better reactivity (Table 1) under the stimulation of IL-1 alpha, the signal value and the signal to noise ratio are high, and the monoclonal HepG2/pGL4.11-IL1RE cell strain 1 is selected as an experimental cell.
Table 1:
example 2 Effect of incubation time and cell Density on signal to noise ratio
1. Experimental method
HepG2/pGL4.11-IL1RE cells were grown at 1X 10 4 3X 10 per well 4 Per well, 6×10 4 Individual/well, 8×10 4 The individual/well was added to 96 Kong Baiban; IL-1α initial concentration 500ng/mL, 2-fold serial dilution 12 concentration gradients; 37 ℃,5% CO 2 Culturing. Treatments were performed after culturing for 3 hours, 6 hours, 10 hours and 24 hours, respectively.
After 18h incubation, the cell supernatant was removed, the cells were washed three times with PBS and lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the resulting lysates, 100. Mu.L of luciferase substrate was added. And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
And comparing the influence of incubation time and cell density on the signal-to-noise ratio according to the four-parameter curve fitted by the measured chemiluminescence values and the signal-to-noise ratio.
2. Experimental results
The signal activation curves under the conditions of 3h, 6h, 10h and 24h are shown in FIG. 2. The values of the plateau and signal to noise ratio of the different cell densities on the stimulation signal path can be seen in the following tables 3h (Table 2), 6h (Table 3), 10h (Table 4). It can be seen that both the lower plateau and the upper plateau are gradually increasing with increasing cells, but the signal to noise ratio is gradually decreasing due to the higher cell background value. Select 3×10 4 IL-1 alpha stimulation is carried out on each hole, the stimulation time is 18-24 hours, a good fitting curve can be obtained, and the signal to noise ratio is maximum.
Table 2:
| parameters (parameters) | 1×10 4 Individual/holes | 3×10 4 Individual/holes | 6×10 4 Individual/holes | 8×10 4 Individual/holes |
| Bottom | 1221 | 1851 | 3341 | 5231 |
| Top | 1982 | 3272 | 4651 | 7321 |
| S/B | 1.623 | 1.768 | 1.392 | 1.400 |
Table 3:
| parameters (parameters) | 1×10 4 Individual/holes | 3×10 4 Individual/holes | 6×10 4 Individual/holes | 8×10 4 Individual/holes |
| Bottom | 1331 | 2061 | 3761 | 5872 |
| Top | 2981 | 5821 | 6801 | 8414 |
| S/B | 2.239669421 | 2.824357108 | 1.808295666 | 1.432901907 |
Table 4:
| parameters (parameters) | 1×10 4 Individual/holes | 3×10 4 Individual/holes | 6×10 4 Individual/holes | 8×10 4 Individual/holes |
| Bottom | 1531 | 2761 | 4651 | 7321 |
| Top | 3671 | 7082 | 9421 | 15010 |
| S/B | 2.397779229 | 2.565012677 | 2.025585896 | 2.050266357 |
Example 2 an assay for detecting L-1 alpha Activity
1. Experimental method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was used at 3X 10 4 The individual/well was added to 96 Kong Baiban; the self-produced IL-1 alpha of the company is diluted to the initial concentration of 250ng/mL, and 8 concentration gradients are serially diluted by 2 times; IL-1α at different concentrations was added to monoclonal HepG2/pGL4.11-IL1RE cell line 1, respectively, at 37℃with 5% CO 2 Culturing.
After 18h incubation, the cell supernatant was removed, the cells were washed three times with PBS and lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the resulting lysates, 100. Mu.L of luciferase substrate was added. And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
The biological activity of IL-1α was calculated from a four-parameter curve fitted to the measured chemiluminescent values.
2. Experimental results
The results are shown in Table 5 below, and a four parameter curve fitting fit was performed. The fitting results are shown in fig. 3, and the median effective amount of IL-1α, ed50=1.50 ng/mL, is calculated from the fitting curve. Half of the effective dose is low, the dose-to-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the detection of the biological activity of IL-1 alpha.
Table 5:
| RUC | 15388 | 15350 | 14434 | 12380 | 9972 | 7074 | 6116 | 5826 |
| rh-IL-1α(ng/mL) | 250 | 62.5 | 15.625 | 3.906 | 0.977 | 0.244 | 0.061 | 0.0153 |
example 3A method for detecting IL-1. Beta. Activity
1. Experimental method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was used at 3X 10 4 The individual/well was added to 96 Kong Baiban; the initial concentration of the company is 125ng/mL IL-1 beta, and 8 concentration gradients are serially diluted by 2 times; IL-1β at different concentrations was added to monoclonal HepG2/pGL4.11-IL1RE cell line 1, 37℃and 5% CO, respectively 2 Culturing.
After 18h incubation, the cell supernatant was removed, the cells were washed three times with PBS and lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the resulting lysates, 100. Mu.L of luciferase substrate was added. And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
The biological activity of IL-1β was calculated from a four-parameter curve fitted to the measured chemiluminescent values.
2. Experimental results
The results are shown in Table 6 below, 8 points (see Table 6 below) including 2 upper plateau points and 2 lower plateau points were selected for four parameter curve fitting. The fitting results are shown in fig. 4, and the median effective amount of IL-1β, ed50=1.30 ng/mL, is calculated from the fitting curve. The half effective amount is low, the amount-to-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the detection of the biological activity of IL-1 beta.
Table 6:
| RUC | 13976 | 13796 | 11598 | 10474 | 9176 | 7204 | 6580 | 5976 |
| rh-IL-1β(ng/mL) | 125 | 31.25 | 7.813 | 1.953 | 0.488 | 0.122 | 0.031 | 0.008 |
EXAMPLE 4 method of IL-1Ra binding to IL-1 alpha Activity
1. Experimental method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was used at 3X 10 4 Add to white 9 per well6 in a well plate; IL-1α, which was detected as active in example 2, was added to a white 96-well plate to a final concentration of 30ng/mL; then, the initial concentration of IL-1Ra produced by the company is 1.969ng/mL, 8 concentration gradients are diluted in 4-fold series, and the obtained mixture is respectively added into a white 96-well plate with the monoclonal HepG2/pGL4.11-IL1RE cell strain 1 and IL-1 alpha obtained in the example 1, and the temperature is 37 ℃ and the concentration is 5% CO 2 Culturing.
After 18h incubation, the cell supernatant was removed, the cells were washed three times with PBS and lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the resulting lysates, 100. Mu.L of luciferase substrate was added. And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
2. Experimental results
The results are shown in Table 7 below, 8 points including 2 upper plateau points and 2 lower plateau points were selected for four parameter curve fitting. The fitting results are shown in FIG. 5, and the biological activity ED of IL-1Ra binding IL-1 alpha is calculated 50 =0.0184 ng/mL. The half effective amount is low, the amount-to-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the biological activity detection of IL-1 Ra.
Table 7:
| RUC | 5275 | 5525 | 6450 | 8795 | 12210 | 14480 | 15105 | 14980 |
| rh-IL-1RA(ng/mL) | 1.969 | 0.493 | 0.123 | 0.0308 | 0.008 | 0.002 | 0.0005 | 0.0001 |
EXAMPLE 5 method of IL-1Ra binding to IL-1 beta Activity
1. Experimental method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was used at 3X 10 4 Add individual/wells to a white 96-well plate; IL-1β, which was detected as active in example 2, was added to a white 96-well plate to a final concentration of 30ng/mL; then, the initial concentration of IL-1Ra produced by the company is 7.875ng/mL, 8 concentration gradients are diluted in 4-fold series, and the obtained mixture is respectively added into a white 96-well plate with the monoclonal HepG2/pGL4.11-IL1RE cell strain 1 and IL-1 alpha obtained in the example 1, and the temperature is 37 ℃ and the concentration is 5% CO 2 Culturing.
After 18h incubation, the cell supernatant was removed, the cells were washed three times with PBS and lysed with 110. Mu.L of lysate (Biyun day), and shaken in a microplate shaker at 200rpm/min for 5min. 100. Mu.L of lysate was aspirated. To the resulting lysates, 100. Mu.L of luciferase substrate was added. And (3) carrying out signal detection after oscillating for 5min at 200rpm/min in a microplate oscillator at room temperature, and detecting a chemiluminescence value.
2. Experimental results
The results are shown in Table 8 below, 8 points (see Table 8 below) including 2 upper plateau points and 2 lower plateau points were selected for four parameter curve fitting. The fitting results are shown in FIG. 6, and the biological activity of IL-1Ra binding IL-1 beta is calculated as ED 50 = 0.09219ng/mL. The half effective amount is low, the amount-to-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the biological activity detection of IL-1 Ra.
Table 8:
| RUC | 5525 | 6105 | 7765 | 11125 | 12940 | 15540 | 15020 | 16975 |
| rh-IL-1RA(ng/mL) | 7.875 | 1.969 | 0.4929 | 0.123 | 0.0301 | 0.0077 | 0.0019 | 0.0005 |
it should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of protection of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Sequence listing
<110> Guangdong Cuiythe biotechnology Co., ltd
<120> an IL-1 signal pathway response element and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 218
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggtaccggag gaaaaactgt ttcatacaga aggcgtggag gaaaaactgt ttcatacaga 60
aggcgtggag gaaaaactgt ttcatacaga aggcgtggag gaaaaactgt ttcatacaga 120
aggcgtggag gaaaaactgt ttcatacaga aggcgtgcta gcctcgagga tatcaagatc 180
tagagggtat ataatggaag ctcgacttcc agaagctt 218
Claims (9)
1. Use of an IL-1 signaling pathway response element for detecting the biological activity of an interleukin-1 receptor antagonist, IL-1 a or IL-1 β, wherein the nucleotide sequence of said IL-1 signaling pathway response element is as set forth in SEQ ID NO:1, said use being for the purpose of non-disease therapeutic diagnosis.
2. Use of a recombinant vector for detecting the biological activity of an interleukin-1 receptor antagonist, IL-1 alpha or IL-1 beta, wherein the recombinant vector is a plasmid comprising an IL-1 signal pathway response element, wherein the IL-1 signal pathway response element has a nucleotide sequence as set forth in SEQ ID NO:1, said use being for the purpose of non-disease therapeutic diagnosis.
3. The use according to claim 2, wherein the plasmid further comprises a reporter gene.
4. Use of a recombinant cell for detecting the biological activity of an interleukin-1 receptor antagonist, IL-1 alpha or IL-1 beta, wherein the recombinant cell is a cell comprising a recombinant vector, the recombinant vector is a plasmid comprising an IL-1 signaling pathway response element, and the nucleotide sequence of the IL-1 signaling pathway response element is as set forth in SEQ ID NO:1, said use being for the purpose of non-disease therapeutic diagnosis.
5. The use according to claim 4, wherein the cell is any one of CHO cell, 293 cell, hela cell, hepG2 cell, a549 cell and human hepatoma cell.
6. A method for detecting the biological activity of IL-1 a or IL-1 β, characterized in that a cell containing a recombinant vector containing a luciferase reporter gene thereon is mixed with a gradient concentration of IL-1 a or IL-1 β, cultured, cell supernatant removed, washed, lysed, fully reacted, solid-liquid separated, supernatant taken, mixed with a luciferase substrate, fully reacted, and a chemiluminescent value detected, respectively;
the recombinant vector is a plasmid containing an IL-1 signal path response element, and the nucleotide sequence of the IL-1 signal path response element is shown as SEQ ID NO:1 is shown in the specification;
the method is for the purpose of non-disease therapeutic diagnosis.
7. A method for binding the biological activity of an interleukin-1 receptor antagonist to IL-1 alpha or IL-1 beta, characterized in that a cell containing a recombinant vector, IL-1 alpha or IL-1 beta of known biological activity, is mixed with a gradient concentration of IL-1Ra or a gradient concentration of a test interleukin-1 receptor antagonist, respectively, cultured, decellularized supernatant, washed cells, lysed cells, subjected to solid-liquid separation, the supernatant is taken, mixed with a luciferase substrate, fully reacted, and the chemiluminescent value is detected, wherein the recombinant vector contains a luciferase reporter gene;
the recombinant vector is a plasmid containing an IL-1 signal path response element, and the nucleotide sequence of the IL-1 signal path response element is shown as SEQ ID NO:1 is shown in the specification;
the method is for the purpose of non-disease therapeutic diagnosis.
8. The method according to claim 6 or 7, wherein the incubation time is 4 to 22 hours.
9. The method according to claim 6 or 7, wherein the concentration of the cells containing the recombinant vector of claim 3 is 1X 10 4 ～8×10 5 And/or holes.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348376A (en) * | 1999-03-05 | 2002-05-08 | 新生制药公司 | Method for validating/invalidating target(s) and pathways |
| CN103221422A (en) * | 2010-07-29 | 2013-07-24 | 十一生物治疗股份有限公司 | Chimeric IL-1 receptor type I agonists and antagonists |
| CN107586826A (en) * | 2017-09-28 | 2018-01-16 | 中国食品药品检定研究院 | Transgenic cell measuring method for activity determines IL 5 or IL 5R Alpha antibodies activity |
| CN107760760A (en) * | 2017-09-28 | 2018-03-06 | 中国食品药品检定研究院 | A kind of method of the receptor antibody pharmaceutical biology activity of quick measure IL 6/IL 6 |
| CN110045131A (en) * | 2019-06-14 | 2019-07-23 | 迈威(上海)生物科技有限公司 | Method for measuring the biological activity of people's IL-33/ST2 pathway inhibitor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140294873A1 (en) * | 2011-11-08 | 2014-10-02 | Children Medical Center Corporation | Small molecule screen for inhibitors of nfat: ap-1: dna interactions |
-
2021
- 2021-09-16 CN CN202111088353.8A patent/CN114480406B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348376A (en) * | 1999-03-05 | 2002-05-08 | 新生制药公司 | Method for validating/invalidating target(s) and pathways |
| CN103221422A (en) * | 2010-07-29 | 2013-07-24 | 十一生物治疗股份有限公司 | Chimeric IL-1 receptor type I agonists and antagonists |
| CN107586826A (en) * | 2017-09-28 | 2018-01-16 | 中国食品药品检定研究院 | Transgenic cell measuring method for activity determines IL 5 or IL 5R Alpha antibodies activity |
| CN107760760A (en) * | 2017-09-28 | 2018-03-06 | 中国食品药品检定研究院 | A kind of method of the receptor antibody pharmaceutical biology activity of quick measure IL 6/IL 6 |
| CN110045131A (en) * | 2019-06-14 | 2019-07-23 | 迈威(上海)生物科技有限公司 | Method for measuring the biological activity of people's IL-33/ST2 pathway inhibitor |
Non-Patent Citations (4)
| Title |
|---|
| Expression vector pYL1, complere sequence;Liu Y等;GenBank;MH594278.1 * |
| NFAT and NF-κB factors——the distant relatives;Edgar Serfling等;IJBCB;第36卷(第7期);第1167页右栏最后一段 * |
| Optimisation and use of humanised RBL NF-AT-GFP and NF-AT-DsRed reporter cell lines suitable for high-throughput scale detection of allergic sensitisation in array format and identification of the ECM-integrin interaction as critical factor;Xiaowei Wang等;Mol Biotechnol;第56卷(第2期);全文 * |
| Synthetic construct clone FAT-CBR red-shifted luciferase (CBR) gene, complete cds;Szyska M等;GenBank;MF462285 * |
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