CN114480406A - IL-1 signal path response element and application thereof - Google Patents

IL-1 signal path response element and application thereof Download PDF

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CN114480406A
CN114480406A CN202111088353.8A CN202111088353A CN114480406A CN 114480406 A CN114480406 A CN 114480406A CN 202111088353 A CN202111088353 A CN 202111088353A CN 114480406 A CN114480406 A CN 114480406A
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CN114480406B (en
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周亚凤
申兆兴
黎勇
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Guangdong Cuidian Biotechnology Co ltd
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Abstract

The invention discloses an IL-1 signal path response element and application thereof, and the invention prepares an effector cell for stably expressing an IL-1 signal path response element reporter gene, stimulates and activates the expression of the reporter gene by IL-1 alpha and IL-1 beta, blocks the IL-1 signal path by 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 sensitive detection method aiming at the IL-1 alpha, IL-1 beta and IL-1Ra drug biological activity measurement, has short experimental period and simple operation, and reduces the deviation caused by objective factors in the process of incubating cells for a long time.

Description

IL-1 signal path response element and application thereof
Technical Field
The invention relates to the technical field of molecular biology, in particular to an IL-1 signal channel 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 effects, such as maintaining the stability of internal environment, resisting infection, resisting tumor, promoting hematopoiesis, and the like, and occupy important positions in a cytokine network. The IL-1 family member consists of IL-1 alpha, IL-1 beta and interleukin-1 receptor antagonist (IL-1 Ra). IL-1Ra is an antagonist common to IL-1 α and IL-1 β, and its protein sequence is significantly similar to IL-1 α and IL-1 β in the receptor binding site, i.e., the C-terminal region. IL-1Ra inhibits IL-1 biological activity by competing with IL-1 receptor binding and is dose-dependent.
The United states Food and Drug Administration (FDA) approved rh-IL-1Ra from Amgen on the market at 11 months 2001 to treat intractable rheumatoid arthritis where conventional drugs are ineffective. 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 organisms.
The current methods for measuring the biological activity of IL-1, IL-1Ra and IL-1 antibodies are mainly methods for inhibiting cell proliferation, and the methods are mainly based on that the killing of A375-S2 cells by IL-1 shows dose dependence. The method generally incubates for 72-120 h at 37 ℃, and has long experimental period and large variability.
Disclosure of Invention
It is an object of the present invention to overcome the above-mentioned deficiencies of the prior art and to provide an IL-1 signal path response element and an application thereof.
It is a first object of the present invention to provide an IL-1 signal path responsive element.
The second objective of the invention is to provide a recombinant vector.
The third objective of the invention is to provide a recombinant cell.
It is a fourth object of the present invention to provide a method for detecting the biological activity of IL-1 α or IL-1 β.
It is a fifth object of the invention to provide a method of binding the biological activity of an interleukin-1 receptor antagonist to IL-1 alpha or IL-1 beta.
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 in the detection of a biological activity having an interleukin-1 receptor antagonist, IL-1 α or IL-1 β.
In order to achieve the purpose, the invention is realized by the following scheme:
HepG2 cell belongs to human liver cancer cell, its surface expression has IL-1R (interleukin-1 receptor), IL-1 combines with IL-1R, through IRAK protein activation IL-1 signal path response element; and (2) adding a nucleotide sequence shown as SEQ ID NO: the plasmid pGL-4.11-IL1RE of the IL-1 signal path response element and the luciferase reporter gene shown in 1 is put into HepG2 cells,
since IL-1 binds to IL-1R, it activates the IL-1 signaling pathway response element and initiates expression of the luciferase reporter gene linked to the response element. IL-1Ra and IL-1 competitively bind to IL-1R to block IL-1 signal path, therefore IL-1Ra concentration is inversely proportional to luciferase expression quantity of effector cells, biological activity of IL-1Ra antibody is determined by fitting a four-parameter curve according to measured reporter gene signal value, and corresponding potency is obtained by comparing half inhibition concentration of the four-parameter curve of sample and reference.
The invention therefore claims the following:
an IL-1 signaling pathway response element having a nucleotide sequence set forth in SEQ ID NO: 1 is shown.
A recombinant vector which is a plasmid containing the IL-1 signal path 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 gene is a luciferase reporter gene.
Even more preferably, the reporter gene is the luciferase reporter gene.
A recombinant cell is a cell containing the recombinant vector.
More preferably, the cell is any one of a CHO cell, 293 cell, Hela cell, HepG2 cell, a549 cell and human hepatoma cell.
The invention further claims a method for detecting the biological activity of IL-1 alpha or IL-1 beta, which comprises the steps of mixing the cells of the recombinant vector with IL-1 alpha or IL-1 beta with gradient concentration respectively, culturing, removing cell supernatant, washing the cells, cracking the cells, fully reacting, carrying out solid-liquid separation, taking supernatant, mixing with luciferase substrate, fully reacting, and detecting chemiluminescence values, wherein the recombinant vector contains luciferase reporter genes.
Preferably, the gradient concentration of IL-1 alpha or IL-1 beta, IL-1 alpha initial concentration is 250ng/mL, 2 times serial dilution 8 concentration gradient; IL-1. beta. initial concentration of 125ng/mL, 2-fold serial dilution of 8 concentration gradients.
Preferably, the culture time is 4-22 h.
More preferably, the incubation time is 18 h.
Preferably, the concentration of the cells containing the recombinant vector is 1X 104～8×105Per well.
More preferably, the concentration of cells containing the recombinant vector is 5X 104Cell concentration per well
The invention also claims the IL-1 signal path response element, the recombinant vector and/or the application of the recombinant cell in detecting the biological activity of interleukin-1 receptor antagonist, IL-1 alpha or IL-1 beta.
The invention also claims a method for combining IL-1 alpha or IL-1 beta biological activity by the interleukin-1 receptor antagonist, which comprises the steps of mixing the cells containing the recombinant vector, IL-1 alpha or IL-1 beta with known biological activity and IL-1Ra with gradient concentration or interleukin-1 receptor antagonist to be detected with gradient concentration respectively, culturing, removing cell supernatant, washing cells, cracking the cells, carrying out solid-liquid separation, taking the supernatant, mixing the supernatant with luciferase substrate, fully reacting, and detecting chemiluminescence value, wherein the recombinant vector contains luciferase reporter gene.
Preferably, the IL-1 alpha or IL-1 beta concentration is between 10pg/mL and 2. mu.g/mL (for activating reporter gene expression)
More preferably, the IL-1 α or IL-1 β concentration is 30 ng/mL.
Preferably, the gradient concentration of IL-1Ra, IL-1Ra starting concentration is from 1pg/mL to 1 mg/mL.
More preferably, the gradient concentration of IL-1Ra, IL-1Ra starting concentration is 10pg/mL to 100. mu.g/mL.
The initial concentration of IL-1Ra is only sufficient to ensure that there is a plateau.
Further preferably, the initial concentration of IL-1Ra is 63ng/mL, which ensures that a plateau phase occurs.
Preferably, the culture time is 4-22 h.
More preferably, the incubation time is 18 h.
Preferably, the concentration of the cells containing the recombinant vector of claim 3 is 1X 104～8×105Per well.
More preferably, the concentration of cells containing the recombinant vector is 3X 104Cell concentration per well
Preferably, the adjacent concentration of the interleukin-1 receptor antagonist to be detected in the gradient concentration is 1: 2-5.
More preferably, the adjacent concentration of the gradient concentration of interleukin-1 receptor antagonist to be tested is 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 IL-1 alpha and IL-1 beta, blocks the IL-1 signal channel by 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 sensitive detection method aiming at the IL-1 alpha, IL-1 beta and IL-1Ra drug biological activity measurement, has short experimental period and simple operation, and reduces the deviation caused by objective factors in the process of incubating cells for a long time.
Drawings
FIG. 1 is a diagram of the plasmid pGL4.11-IL1 RE.
Fig. 2 is a signal activation graph under the conditions of 3h, 6h, 10h and 24 h.
FIG. 3 is a graph showing the detection of rh-IL-1. alpha. activity.
FIG. 4 is a graph of rh-IL-1 β activity assay.
FIG. 5 shows the binding of rh-IL-1RA to rh-IL-1 α activity.
FIG. 6 shows the binding of rh-IL-1RA to rh-IL-1 β activity.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
HepG2 cells were 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 from Shanghai Jingnuo Biotech, Inc., and luciferase reporter gene detection kit is purchased from Biyuntian.
EXAMPLE 1 acquisition of HepG2 cell line stably expressing IL-1 Signal pathway response element reporter Gene
First, experiment method
1. preparation of pGL4.11-IL1RE plasmid
Designing an IL-1 signal channel response element combination (IL-1RE), wherein the sequence is shown as SEQ ID NO: 1, synthesized by Onghama corporation, Guangzhou.
SEQ ID NO：1：
GGTACCGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATA CAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTT CATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGCTAGCCTCGAG GATATCAAGATCTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT。
IL-1RE and PGL4.11 synthesized by double-enzymatic cleavage using Fastdigest restriction enzymes KpnI and Hind III from Thermo Fisher Scientific were used, Gel-electrophoresed, and then the linearized plasmid DNA fragment was recovered by using Gel Extraction kit from Omega for use.
The two linearized plasmid DNA fragments were ligated using T4 ligase from Thermo Fisher Scientific to construct pGL4.11-IL1RE plasmid (see FIG. 1). The plasmid was transfected into DH 5. alpha. competent cells by heat shock, and plasmids were extracted using Tiangen endotoxin-free plasmid extraction kit after extensive culture.
2. Plasmid transfection
HepG2 cells were transfected with pGL4.11-IL1RE plasmid using electroporation transfection system (invitrogen), and the next day after transfection 300. mu.g/mL hygromycin B was added for pressure selection to kill cells into which no plasmid was introduced. Then, the cells were diluted to 1 cell/well by limiting dilution, and the cells were amplified by adding a medium containing 300. mu.g/mL Hygrmycin B, and gradually cultured in scale-up manner to obtain HepG2 cells (HepG2/pGL4.11-IL1RE cell line) transfected with pGL4.11-IL1RE plasmid.
3. Screening of HepG2/pGL4.11-IL1RE cell line
The screened HepG2/pGL4.11-IL1RE cell line is 3X 104Laying the seeds into a 96-well plate with each well being 50 mu L; IL-1. alpha. was diluted in 2-fold series at an initial concentration of 500ng/mL for 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 lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the obtained lysate, 100. mu.L of luciferase substrate (Biyuntian, cat # RG006) was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
Luciferase chemiluminescence values are obtained through screening for detection, and a monoclonal HepG2/pGL4.11-IL1RE cell strain which is high in signal-to-noise ratio and good in fitting degree after IL-1 stimulation is selected for subsequent experiments.
Second, experimental results
The experimental results are shown in Table 1, and the results show that the monoclonal HepG2/pGL4.11-IL1RE cell line 1 has better reactivity under the stimulation of IL-1 alpha (Table 1), the signal value and the signal-to-noise ratio are high, and the monoclonal HepG2/pGL4.11-IL1RE cell line 1 is selected as the experimental cell.
Table 1:
Figure BDA0003266370570000051
example 2 Effect of incubation time and cell Density on the Signal to noise ratio number
First, experiment method
HepG2/pGL4.11-IL1RE cells at 1X 104One/hole, 3X 1046 x 10 pieces/hole48 x 10 pieces/hole4Add one/well to a 96-well whiteboard; IL-1 alpha initial concentration 500ng/mL, 2 times serial dilution of 12 concentration gradient; 37 ℃ and 5% CO2And (5) culturing. The treatment was carried out after 3h, 6h, 10h and 24h of cultivation, respectively.
After 18h incubation, cell supernatants were removed, cells were washed three times with PBS, lysed with 110 μ L of lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the resulting lysates, 100 μ L luciferase substrate was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
The effect of incubation time and cell density on the signal-to-noise ratio number was compared according to a four-parameter curve fitted to the measured chemiluminescence values and the signal-to-noise ratio.
Second, experimental results
The signal activation curves under the conditions of 3h, 6h, 10h and 24h are shown in FIG. 2. In the following tables 3h (table 2), 6h (table 3) and 10h (table 4) it can be seen that different cell densities have plateau values and signal-to-noise values above and below the stimulation signal pathway. From this, it can be seen that the lower plateau and the upper plateau are gradually increased with the increase of cells, but the signal-to-noise ratio is gradually decreased due to the higher background value of the cells. Selecting at 3 × 104And (3) carrying out IL-1 alpha stimulation on each hole, wherein a good fitting curve can be obtained within 18-24 h of stimulation time, and the signal-to-noise ratio is maximum.
Table 2:
parameter(s) 1×104One/hole 3×104One/hole 6×104One/hole 8×104One/hole
Bottom 1221 1851 3341 5231
Top 1982 3272 4651 7321
S/B 1.623 1.768 1.392 1.400
Table 3:
parameter(s) 1×104One/hole 3×104One/hole 6×104One/hole 8×104One/hole
Bottom 1331 2061 3761 5872
Top 2981 5821 6801 8414
S/B 2.239669421 2.824357108 1.808295666 1.432901907
Table 4:
parameter(s) 1×104One/hole 3×104One/hole 6×104One/hole 8×104One/hole
Bottom 1531 2761 4651 7321
Top 3671 7082 9421 15010
S/B 2.397779229 2.565012677 2.025585896 2.050266357
Example 2 assay for L-1. alpha. Activity
First, experiment method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was cultured at 3X 104Add one/well to a 96-well whiteboard; diluting the self-produced IL-1 alpha of the company to the initial concentration of 250ng/mL, and carrying out 2-time serial dilution on the obtained product for 8 concentration gradients; IL-1. alpha. was added to the monoclonal HepG2/pGL4.11-IL1RE cell line 1 at 37 ℃ with 5% CO2And (4) culturing.
After 18h incubation, cell supernatants were removed, cells were washed three times with PBS, lysed with 110 μ L of lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the resulting lysates, 100 μ L luciferase substrate was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
The biological activity of IL-1 α was calculated from a four parameter curve fitted to the measured chemiluminescence values.
Second, experimental results
The results are shown in table 5 below, where a four parameter curve fit was performed. The results of the fit are shown in fig. 3, and the half effective amount ED50 of IL-1 α is calculated as 1.50ng/mL from the fitted curve. The half effective dose is lower, the dose-effect relation between the biological activity and the protein is obvious, and the method can be well applied to the detection of the biological activity of the 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
First, experiment method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was cultured at 3X 104Add one/well to a 96-well whiteboard; the initial concentration of the company is 125ng/mL IL-1 beta, and 8 concentration gradients are serially diluted by 2 times; IL-1. beta. was added to monoclonal HepG2/pGL4.11-IL1RE cell line 1 at different concentrations, 5% CO at 37 ℃2And (4) culturing.
After 18h incubation, cell supernatants were removed, cells were washed three times with PBS, lysed with 110 μ L of lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the resulting lysates, 100 μ L luciferase substrate was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
The biological activity of IL-1. beta. was calculated from a four parameter curve fitted to the measured chemiluminescence values.
Second, experimental results
The results are shown in table 6 below, where 8 points (see table 6 below) including 2 upper plateau points and 2 lower plateau points were selected for four-parameter curve fitting. The results of the fit are shown in fig. 4, and the half-effective amount ED50 of IL-1 β was calculated as 1.30ng/mL from the fitted curve. Half of the effective dose is lower, the dose-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 the 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 methods of IL-1Ra binding to IL-1 α Activity
First, experiment method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was cultured at 3X 104Add one/well to white 96-well plate; IL-1. alpha. detected as active in example 2 was added to a white 96-well plate to a final concentration of 30 ng/mL; thereafter, the company's own IL-1Ra initial concentration was 1.969ng/mL, and 8 concentration gradients were serially diluted 4 times, and added to white 96-well plates containing the monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 and IL-1. alpha. at 37 ℃ with 5% CO2And (5) culturing.
After 18h incubation, cell supernatants were removed, cells were washed three times with PBS, lysed with 110 μ L of lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the resulting lysates, 100 μ L luciferase substrate was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
Second, experimental results
The results are shown in table 7 below, where 8 points including 2 upper plateau points and 2 lower plateau points were selected for four-parameter curve fitting. The results of the fit are shown in FIG. 5, and the biological activity ED of IL-1Ra binding to IL-1 α is calculated500.0184 ng/mL. Half of the effective amount is low, the dose-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the biological activity detection of the 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 methods of IL-1Ra binding to IL-1 β Activity
First, experiment method
The monoclonal HepG2/pGL4.11-IL1RE cell line 1 obtained in example 1 was cultured at 3X 104Add one/well to white 96-well plate; IL-1. beta. detected as active in example 2 was added to a white 96-well plate to a final concentration of 30 ng/mL; thereafter, the company's own IL-1Ra initial concentration was 7.875ng/mL, and 8 concentration gradients were serially diluted 4-fold and added to white 96-well plates containing the monoclonal HepG2/pGL4.11-IL1RE cell line 1 and IL-1. alpha. obtained in example 1, respectively, at 37 ℃ and 5% CO2And (5) culturing.
After 18h incubation, cell supernatants were removed, cells were washed three times with PBS, lysed with 110 μ L of lysis buffer (Bycy day), and shaken for 5min at 200rpm/min in a microplate shaker. Pipette 100. mu.L of lysate. To the resulting lysates, 100 μ L luciferase substrate was added. And oscillating at 200rpm/min for 5min in a microplate oscillator at room temperature, and then carrying out signal detection to detect a chemiluminescence value.
Second, experimental results
The results are shown in table 8 below, where 8 points (see table 8 below) including 2 upper plateau points and 2 lower plateau points were selected for four-parameter curve fitting. The fit results are shown in FIG. 6, and the biological activity of IL-1Ra binding to IL-1 β is calculated as ED500.09219 ng/mL. Half of the effective amount is low, the dose-effect relationship between the biological activity and the protein is obvious, and the method can be well applied to the biological activity detection of the 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 only used for illustrating the technical solutions of the present invention, and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and ideas, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Guangdong Cui point Biotech Co., Ltd
<120> IL-1 signal path response element and application 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 (10)

1. An IL-1 signaling pathway response element characterized by a nucleotide sequence as set forth in SEQ ID NO: 1 is shown.
2. A recombinant vector comprising the IL-1 signaling pathway response element of claim 1.
3. The recombinant vector according to claim 2, wherein the plasmid further comprises a reporter gene.
4. A recombinant cell comprising the recombinant vector according to claim 2.
5. The recombinant cell 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 alpha or IL-1 beta, which comprises mixing the cells containing the recombinant vector of claim 3 with a gradient concentration of IL-1 alpha or IL-1 beta, respectively, culturing, removing cell supernatant, washing the cells, lysing the cells, allowing the cells to react well, performing solid-liquid separation, collecting supernatant, mixing with a luciferase substrate, allowing the cells to react well, and detecting the chemiluminescence value, wherein the recombinant vector contains a luciferase reporter gene.
7. A method for binding interleukin-1 receptor antagonist to the biological activity of IL-1 alpha or IL-1 beta, which comprises mixing the cells of the recombinant vector of claim 3, IL-1 alpha or IL-1 beta of known biological activity, with IL-1Ra of gradient concentration or interleukin-1 receptor antagonist to be detected of gradient concentration, culturing, removing cell supernatant, washing the cells, lysing the cells, performing solid-liquid separation, collecting supernatant, mixing with luciferase substrate, reacting well, and detecting chemiluminescence value, wherein the recombinant vector contains luciferase reporter gene.
8. The method according to claim 6 or 7, wherein the cultivation 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 104～8×105One for each well.
10. Use of the IL-1 signaling pathway response element of claim 1, the recombinant vector of claim 2, and/or the recombinant cell of claim 4 to detect a biological activity having an interleukin-1 receptor antagonist, IL-1 α, or IL-1 β.
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