CN112481279A - Application of horseradish peroxidase gene as reporter gene - Google Patents
Application of horseradish peroxidase gene as reporter gene Download PDFInfo
- Publication number
- CN112481279A CN112481279A CN202011234692.8A CN202011234692A CN112481279A CN 112481279 A CN112481279 A CN 112481279A CN 202011234692 A CN202011234692 A CN 202011234692A CN 112481279 A CN112481279 A CN 112481279A
- Authority
- CN
- China
- Prior art keywords
- gene
- hrp
- reporter gene
- cells
- horseradish peroxidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an HRP reporter gene-based method for identifying activity of a cytokine. The method is simple and rapid in operation process, independent of specific cells, specific culture media and fluorescent microscopes and chemiluminescent instruments, can be popularized to more common biological laboratories, and has good practical value. Since changes caused by a small amount of cytokines can be reflected by using reporter cells, cost can be saved. Cell pathway changes are faster than in cell growth or toxicity experiments, so the use of HRP reporter gene can save valuable experimental time. The method can be used as a basis for preparing a kit for detecting cell active substances or transfection reagents.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of horseradish peroxidase gene as a reporter gene.
Background
Various chemical molecules, polypeptides, proteins and other bioactive molecules can activate or inhibit signal pathways by combining with corresponding receptors on the cell surface to complete biological functional effects. These biological activities include: promoting proliferation and differentiation of target cells, enhancing anti-infection and cell killing effects, promoting or inhibiting expression of other cytokines and membrane surface molecules, promoting inflammatory process, influencing cell metabolism, etc. These bioactive molecules have been widely used in research reagents and drug development. Therefore, how to establish an efficient and stable detection system for bioactive molecules is the key of scientific research and production.
Reporter genes (Reporter genes) encode proteins or enzymes that are conveniently detected and can be used to detect changes in a biological system of interest, thereby identifying and screening for desirable experimental results. According to different signal path characteristics, a reporter gene is added after a DNA response element of a specific signal path, and the expression of the reporter gene changes under the condition of signal stimulation. Common reporter gene systems include: luciferase gene (luc), chloramphenicol acetyltransferase gene (cat), green fluorescent protein gene (gfp), and the like. However, the above reporter gene system requires special reagents and fluorescence reading instruments, and is expensive, or cannot be observed by naked eyes, which is inconvenient for experiments. Therefore, the research and development of a new reporter gene detection method can not only make up for the defects of other detection methods, but also hopefully obtain a more convenient and reliable detection method, which has important significance for objectively evaluating the activity of bioactive molecules.
Horseradish peroxidase (HRP) exists in plant horseradish and is widely applied to the field of biochemistry mainly because of the capability of amplifying weak signals and enhancing the detectability of target molecules. The HRP product is not only widely used for a plurality of biochemical detection projects, but also widely used for immune (ELISA) kits.
Disclosure of Invention
The invention aims to provide an application of horseradish peroxidase gene as a reporter gene, provides a new idea for identifying activities of transfection reagents, chemical molecules, polypeptides, antibodies, proteins, antibodies and the like, and solves the defects of long time consumption, complex steps, high cost, low sensitivity and the like of the existing detection method.
In order to realize the purpose, the application of the horseradish peroxidase gene as a reporter gene provided by the invention comprises the following steps: firstly, setting a target to be identified, determining DNA of a corresponding original piece of a promoter corresponding to a signal path according to the target and the activated signal path, cloning the DNA of the original piece and a combined gene of HRP genes into an expression vector, then transfecting cells, treating the transfected cells by using the target with single or multiple concentrations, activating the signal path to express the HRP genes, and then adding a chromogenic substrate of HRP for qualitative or quantitative evaluation.
In the above protocol, the quantitative evaluation was carried out by treating cells transfected with a reporter gene with various concentrations of the target, detecting the color development value of the HRP chromogenic substrate, and calculating the EC50 value.
Alternatively, the chromogenic substrate for HRP is TMB.
In the above embodiment, the target to be identified is selected from one of transfection reagents, chemical molecules, polypeptides, antibodies, proteins, and antibodies.
Alternatively, the sequence of the HRP gene is shown in the gene bank GenBank under accession number ANS 10151.1.
The invention also provides an activity identification vector based on the HRP reporter gene, which comprises a preset HRP gene, and an editable promoter insertion site is arranged in front of the HRP gene.
The principle of the invention is as follows: since HRP is not present in mammalian cells and insect cells, the activity of the substance can be rapidly and simply identified by transfecting cells with HRP gene instead of luciferase gene (luc) or other reporter gene, and then inducing the cells with the molecule to be tested. Through the substrate color development of adding HRP, not only can see the experimental trend with naked eyes, but also can read OD450 value through the enzyme-labeling instrument, more accurately obtain the activity of the substance to be detected. The method does not depend on a fluorescence microscope and a chemiluminescence instrument, and has the advantages of sensitivity, simplicity, low cost, high flux, wide application range and the like. The method can be expanded to the detection and identification of transfection reagents, chemical molecules, polypeptides, antibodies, proteins, antibodies and other biological reagents and active substances.
The HRP gene is a substitute of a GFP gene or a Luciferase Reporter gene, and the application of the HRP gene as a Reporter gene can quickly, simply and conveniently identify various biochemical substances. Compared with luciferase gene or green fluorescent protein, the fluorescent protein does not need to depend on a fluorescent microscope and a chemiluminescent instrument. The invention has low requirement on instruments, can be suitable for common laboratories, and can observe approximate signal difference by naked eyes in a short time, so that the experimental scheme can be adjusted according to the signal. Since the half-maximal effect concentration of the cytokine can be accurately obtained, the sensitivity of HRP as a reporter gene is extremely high.
The invention has the beneficial effects that: the method has simple and quick operation process, does not depend on specific cells, special culture medium and fluorescent microscope and chemiluminescent instruments, can be popularized to more common biological laboratories, and has good practical value. Since changes caused by a small amount of cytokines can be reflected by using reporter cells, cost can be saved. Cell pathway changes are faster than in cell growth or toxicity experiments, so the use of HRP reporter gene can save valuable experimental time. The method can be used as a basis for preparing a kit for detecting cell active substances or transfection reagents.
Drawings
FIG. 1 is a schematic structural diagram of a vector expressing an HRP reporter gene. Including basic elements such as upstream promoter and HRP reporter gene.
FIG. 2 is an ED50 value for testing Human TNF α activity using the method of the present invention.
FIG. 3 is an ED50 value for Human TNF α activity measured by biological assays.
FIG. 4 is an ED50 value for the determination of Mouse TNF α activity by the method of the present invention.
FIG. 5 is an ED50 value for IL-1 β activity measured by the method of the present invention
FIG. 6 shows the efficiency of transfection of mammalian cells with various transfection reagents tested by the method of the present invention.
FIG. 7 shows the EC50 values of transfection reagents for detection of HEK293 cells in mammalian cells by the method of the present invention; a, B, C of the three transfection reagents respectively correspond to HighGene, Expi293 and PEI.
FIG. 8 shows the efficiency of transfection of insect cell Sf9 with three reagents tested by the method of the present invention.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments. The reagents or products used in the following examples, which are not indicated, are commercially available and are not intended to limit the scope of the present invention.
The following example is a verification of the process of the invention.
The specific implementation method of the HRP reporter gene method is as follows:
1. selecting a signal path: selecting a signal pathway based on the detection of the identified biochemical.
HEK293 is the most commonly used cell. Next, a promoter and an HRP gene sequence (GenBank: ANS10151.1) were genetically synthesized.
2. Preparing a recombinant plasmid: the synthesized HRP gene fragment is cloned to a multiple cloning site of pcDNA3.1 or a similar expression vector, then the original promoter of the vector is replaced by the synthesized specific promoter sequence, and the plasmid is transformed, expanded and extracted for later use.
3. Transfection: HEK293 cells were prepared (other cells could be selected and tried as required for the experiment).
The transfection was carried out according to the instructions of the HighGene (cat. ABClonal RM 09014; ABClonal Science Inc.) transfection reagent, with the following methods, appropriately adjusted according to the experimental conditions:
1) on the first day, a proper amount of cells are inoculated in a six-well cell culture plate (different cell inoculation densities are different, conditions need to be groped), so that the cell density can reach 70% -90% in transfection on the second day (the cell density needs to be controlled well, and the density has great influence on the transfection efficiency and the cell state after transfection);
2) the next day, adding 4 μ g of plasmid into a centrifugal tube containing 200 μ L of serum-free DMEM basal medium, blowing, beating and mixing uniformly, then adding 8 μ L of HighGene transfection reagent, blowing, beating and mixing uniformly (after adding HighGene, soft blowing is recommended);
3) dripping 200 μ L of plasmid/HighGene transfection reagent compound into 6-well cell culture plate hole, and gently shaking the cell culture plate to mix them uniformly;
4) after 4-6 hours of cell transfection, half of the fresh complete medium was replaced.
4. Cell treatment: after 1 day of transfection, cells were harvested for cell counting, and 96-well plates were seeded at 0.5X 105 cells/well at 100. mu.L/well and cultured overnight to allow cells to adhere.
5. Protein treatment: and (2) carrying out gradient dilution on the protein to be detected by using a culture medium of cells used for transfection, wherein the maximum concentration is 1 mu g/mL, the 10-time or 5-time gradient dilution is carried out, the protein to be detected is diluted into 9 gradients, a protein-free control is arranged, each gradient is provided with 2-3 multiple holes, and the diluted protein is added according to 100 mu L/hole.
6. Detection by a microplate reader: after 4h of protein treatment [ specific time to be according to different protein exploration ], 100. mu.L/well of TMB color development solution is added, or centrifugation is carried out, supernatant is carefully absorbed and discarded, color development is carried out while observation is carried out, color development is carried out for about 15min, 50. mu.L/well stop solution is added, and OD450 is detected by a microplate reader (OD 630 is used as reference wavelength).
7. And (3) data analysis: the data were processed into pictures using Origin 8 to obtain EC50 values (EC50, half maximal effect concentration means the concentration that causes 50% of the maximal effect and is also the dose that causes 50% of the individual to be effective).
It should be noted that the above specific operation steps are an embodiment of the method of the present invention, and those skilled in the art can substitute other similar or identical operations for the steps therein on the basis of understanding the present invention, so that the above specific operation steps are not intended to limit the scope of the present invention.
Example 1
TNF- α activates the NF-. kappa.B and MAPK signaling pathways by binding to receptors. Both of these signaling pathways are present in HEK293 cells. The DNA sequence of the NF-. kappa.B binding site was cloned as a promoter on the vector of FIG. 2.
The method of the invention is used for detecting the activity of Human TNF alpha (ABClonal RP00993_9692082805), and the obtained ED50 is as follows: 23pg/mL (FIG. 2); biological assays (cytotoxicity experiments) gave ED50 values: 20pg/mL (FIG. 3). The detection result obtained by the HRP reporter gene method is almost completely consistent with the cytotoxicity experimental detection method, which fully illustrates the feasibility of the HRP reporter gene method and the reliability of the result. However, cytotoxicity experiments require special experimental materials and are not easily available in general laboratories. The advantages of the HRP reporter gene approach can be seen.
Example 2
The signal path and the promoter have certain conservation. We used recombinant mouse TNF α to verify the conservation of the DNA sequence of the NF-. kappa.B binding site on the human cell line HEK 293. According to the above experimental scheme, the activity of Mouse TNFa (ABClonal RP01071_9693041602) was detected by HRP reporter gene method, and the ED50 was: 0.23ng/mL (FIG. 4). This suggests that the cross-species cytokine can also be used on the same vector, which would reduce the effort to construct the plasmid.
Example 3
When the activity of some cytokines is not obvious by biological detection and immunological detection, the HRP reporter gene method can be used.
For example, IL-1 β (ABClonal RP00002_967201031) activates the downstream NF-. kappa.B pathway by binding to the IL-1R1 receptor. According to the above experimental example 1, the method of using HRP reporter gene, ED50:5.9pg/mL was obtained (FIG. 5). But not by assays using cell proliferation.
Example 4
The invention also allows the determination of the transfection efficiency of transfection reagents in different cells. It is possible to measure which transfection reagent is appropriate for which cell by the exact OD value. And can accurately measure the EC50 value and figure out the amount of transfection reagent or plasmid needed to be the most appropriate.
1) The following example is to explore which transfection reagent will best transfect on which mammalian cells.
Subcloning the synthesized HRP gene fragment into pcDNA3.1 (or the multiple cloning site of similar mammal expression vector); and transforming and amplifying the plasmid for extraction.
We used the following transfection reagents, HighGene, respectively; expi 293; PEI.
(Expi293:Thermofisher,A14524 ExpiFectamine 293Transfection Kit)
The transfection was compared to 3 cells HEK293, Hela, U2 OS.
Following the above protocol, the supernatant was discarded after 3 days of transfection and TMB substrate was added to the cells to conclude that
For HEK293 cells, the HighGene transfection efficiency was close to that of the Expi293 transfection reagent.
The highGene is obviously superior to other transfection reagents for Hela and U2OS cells.
The results are shown in FIG. 6, where the BK group is Blank control of Blank with no transfection reagent.
2) Since transfection reagents are different in price, there is a need for an economic advantage in measuring the efficiency of various kinds of transfection. We next explored a comparison of the optimal transfection doses of the above transfection reagents (EC50 values) with fixed plasmid amounts and cell types.
Mammalian HEK293 cells were transfected with the three transfection reagents HighGene, Expi293 and PEI according to the protocol described above. The pCMV-HRP plasmids were all: 1 μ g, and transfection in 24-well plates. The concentration of the transfection reagent is added in the following order: 0; 0.0625; 0.125; 0.25; 0.5; 1; 2; 4; 8; the volume of the solution is 16 mu L,
transferring the cells to a 96-well plate after two days of transfection, arranging three multiple wells for each cell, adding TMB for color development after the cells adhere to the wall, reacting for about 15min, adding a stop solution to stop the reaction, and calculating the EC50 of each transfection reagent.
The results are shown in A, B, C of FIG. 7.
The HighGene EC50 is: 1.23 mu L;
expi293 EC50 is: 4.35 μ L;
PEI EC50 was: 3.68 μ L.
The conclusion is that the cost performance of the HighGene is far better than that of Expi293
Example 5
Also, the invention can be used in other cells that do not express HRP, such as insect cells SF 9.
The synthesized HRP gene fragment was cloned into the insect intermediate vector pFAST (or into the multiple cloning site of a similar expression vector). Promoter sequences are shown as appendix sequences. Then we prepared Bacmid of HRP according to standard method, after extracting Bacmid.
1) We used the following transfection reagents, HighGene; expi 293; PEI transfects insect cells SF 9.
Following the protocol described above, the supernatant was discarded after 7 days of transfection and the cells were added with TMB substrate, which allowed the following conclusions to be reached:
transfected with SF9 cells, HighGene was optimized among 3 transfection reagents, as shown in FIG. 8, where the BK group was Blank control with Blank no transfection reagent.
The promoter sequences provided in the present invention are only examples and should not be used to limit the present invention.
Claims (6)
1. An application of horseradish peroxidase gene as a reporter gene comprises the following steps:
firstly, setting a target to be identified, determining DNA of a corresponding original piece of a promoter corresponding to a signal path according to the target and the activated signal path, cloning the DNA of the original piece and a combined gene of HRP genes into an expression vector, then transfecting cells, treating the transfected cells by using the target with single or multiple concentrations, activating the signal path to express the HRP genes, and then adding a chromogenic substrate of HRP for qualitative or quantitative evaluation.
2. The use of the horseradish peroxidase gene as a reporter gene according to claim 1, characterized in that: the quantitative evaluation is carried out by treating cells transfected with the reporter gene with various concentrations of the target, detecting the color development value of the HRP color development substrate, and calculating the EC50 value.
3. The use of the horseradish peroxidase gene as a reporter gene according to claim 1, characterized in that: the chromogenic substrate of the HRP is TMB.
4. The use of the horseradish peroxidase gene as a reporter gene according to claim 1, characterized in that: the target to be identified is selected from one of transfection reagents, chemical molecules, polypeptides, antibodies, proteins and antibodies.
5. The use of the horseradish peroxidase gene as a reporter gene according to claim 1, characterized in that: the sequence of the HRP gene is shown as the number ANS10151.1 in the GenBank of the gene database.
6. An activity identification vector based on an HRP reporter gene is characterized in that: the vector comprises a preset HRP gene, and an editable promoter insertion site is arranged in front of the HRP gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011234692.8A CN112481279A (en) | 2020-11-07 | 2020-11-07 | Application of horseradish peroxidase gene as reporter gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011234692.8A CN112481279A (en) | 2020-11-07 | 2020-11-07 | Application of horseradish peroxidase gene as reporter gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112481279A true CN112481279A (en) | 2021-03-12 |
Family
ID=74928818
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011234692.8A Pending CN112481279A (en) | 2020-11-07 | 2020-11-07 | Application of horseradish peroxidase gene as reporter gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112481279A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114032252A (en) * | 2021-11-01 | 2022-02-11 | 武汉爱博泰克生物科技有限公司 | Expression method and application of recombinant human MMP-7 protein |
| CN114045291A (en) * | 2021-11-01 | 2022-02-15 | 武汉爱博泰克生物科技有限公司 | Recombinant human Sema4C protein, expression vector, host cell and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173495A (en) * | 2013-02-25 | 2013-06-26 | 中国疾病预防控制中心病毒病预防控制所 | Novel rabies virus fake virus system as well as preparation and application thereof |
| CN103627779A (en) * | 2013-12-02 | 2014-03-12 | 山东大学 | Screening system of anti-tumor drugs based on TRAIL receptor signal channel |
| CN106008720A (en) * | 2016-05-25 | 2016-10-12 | 深圳市国创纳米抗体技术有限公司 | Fusion protein of horseradish peroxidase and antibody fragment and application |
| CN109837249A (en) * | 2017-11-28 | 2019-06-04 | 南京传奇生物科技有限公司 | For evaluating the active recombinant cell of anti-tnf-alpha pharmaceutical biology and its application |
| CN110029146A (en) * | 2018-12-20 | 2019-07-19 | 中国食品药品检定研究院 | The biological activity of reporter gene method measurement recombinant human horny cell growth factor-2 |
| CN111534529A (en) * | 2020-05-09 | 2020-08-14 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Reporter gene cell strain and construction method and application thereof |
-
2020
- 2020-11-07 CN CN202011234692.8A patent/CN112481279A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173495A (en) * | 2013-02-25 | 2013-06-26 | 中国疾病预防控制中心病毒病预防控制所 | Novel rabies virus fake virus system as well as preparation and application thereof |
| CN103627779A (en) * | 2013-12-02 | 2014-03-12 | 山东大学 | Screening system of anti-tumor drugs based on TRAIL receptor signal channel |
| CN106008720A (en) * | 2016-05-25 | 2016-10-12 | 深圳市国创纳米抗体技术有限公司 | Fusion protein of horseradish peroxidase and antibody fragment and application |
| CN109837249A (en) * | 2017-11-28 | 2019-06-04 | 南京传奇生物科技有限公司 | For evaluating the active recombinant cell of anti-tnf-alpha pharmaceutical biology and its application |
| CN110029146A (en) * | 2018-12-20 | 2019-07-19 | 中国食品药品检定研究院 | The biological activity of reporter gene method measurement recombinant human horny cell growth factor-2 |
| CN111534529A (en) * | 2020-05-09 | 2020-08-14 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Reporter gene cell strain and construction method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114032252A (en) * | 2021-11-01 | 2022-02-11 | 武汉爱博泰克生物科技有限公司 | Expression method and application of recombinant human MMP-7 protein |
| CN114045291A (en) * | 2021-11-01 | 2022-02-15 | 武汉爱博泰克生物科技有限公司 | Recombinant human Sema4C protein, expression vector, host cell and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Quality control of cell-based high-throughput drug screening | |
| Rodriguez et al. | Wound-healing assay | |
| Rodier | Detection of the senescence-associated secretory phenotype (SASP) | |
| CN112481279A (en) | Application of horseradish peroxidase gene as reporter gene | |
| CN106574292A (en) | Method for identifying desired cell type using miRNA expression as indicator | |
| CN112538461B (en) | GLP1R reporter gene stable transgenic cell strain, construction method and application | |
| WO2003027634A2 (en) | Identification of modulatory molecules using inducible promoters | |
| CN110231490A (en) | Probe and application thereof | |
| US20030170744A1 (en) | Multiplexed analysis of cellular responses using endogenous reporter genes | |
| US7960101B2 (en) | Method for using division arrested cells in screening assays | |
| CN107384869A (en) | A kind of monoclonal cell strain and its application in IL 6R inhibitor relative biological activities are determined | |
| CN111579796A (en) | High-throughput integrated phosphorylation proteomics detection method | |
| Ruan et al. | An engineered (CAGA) 12-EGFP cell-based biosensor for high-content and accurate detection of active TGF-β | |
| US10544472B2 (en) | Multiplexing transcription factor reporter protein assay process and system | |
| WO2003006947A2 (en) | Cell-screening assay and composition | |
| WO1990000619A1 (en) | Analysis of cell modifying substances | |
| CN112195218A (en) | Luciferase reporter gene-based biological activity detection reagent | |
| CN117821392B (en) | Recombinant cell for screening RXR alpha effector, construction method and application | |
| US20040137488A1 (en) | Methods for identifying the activity of gene products | |
| CN110004179B (en) | Universal detection method and detection kit for cannabinoid active substances | |
| CN113337571B (en) | Rapid and ultrahigh-sensitivity TNF alpha biological activity detection method | |
| CN110133241B (en) | Novel method for measuring biological activity of recombinant human soluble gp130-Fc fusion protein | |
| JP2002335998A (en) | Assay based on vegf-responding cell for measuring biological activity of vegf | |
| CN112280741A (en) | Application of FRT cell strain in preparation of preparation or kit for screening Piezo1 regulator | |
| KR100844581B1 (en) | Reporter adenovirus having serum responsive elementSRE promoter for high throughout screening system for G protein-coupled receptorGPCR modulators |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210312 |
|
| RJ01 | Rejection of invention patent application after publication |