CN113699169A - Luciferase reporter gene iGLuc, gene system and application thereof - Google Patents
Luciferase reporter gene iGLuc, gene system and application thereof Download PDFInfo
- Publication number
- CN113699169A CN113699169A CN202110862005.5A CN202110862005A CN113699169A CN 113699169 A CN113699169 A CN 113699169A CN 202110862005 A CN202110862005 A CN 202110862005A CN 113699169 A CN113699169 A CN 113699169A
- Authority
- CN
- China
- Prior art keywords
- igluc
- reporter gene
- gene
- luciferase
- beta
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of biological engineering. In particular to a luciferase reporter gene iGLuc, a gene system and application thereof. The nucleotide sequence of the reporter gene iGLuc is shown in SEQ ID NO. 1. The amino acid sequence of the reporter gene iGLuc is shown in SEQ ID NO. 2. The protein cleavage site between the N end and the C end of the IL-1 beta precursor in the reporter gene iGLuc provided by the invention can be replaced by the cleavage site of other protease, then the corresponding protease is adopted for cleavage, the cleavage efficiency of the protease can be reflected by the activity level of the luciferase, and the cleavage activity of the corresponding protease can also be reflected, and a luciferase reporter gene system for detecting the cleavage activity of the protease and the application thereof in detecting the cleavage activity of the protease are constructed according to the principle, and the application thereof in screening protease inhibitors or agonists in a quantitative manner.
Description
Technical Field
The invention belongs to the technical field of biological engineering. In particular to a luciferase reporter gene iGLuc, a gene system and application thereof.
Background
The reporter gene is a gene for coding a certain protein or enzyme which is easy to detect and does not generate characters, the coding sequence of the reporter gene is fused with other target genes to be expressed under certain conditions, and the expression of the target genes can be calibrated through signal detection of the reporter gene. The reporter gene has the characteristics of convenient detection, high sensitivity, high-throughput screening and the like, and is widely applied to various cell events such as gene promoter analysis, gene expression, drug screening and the like. With the progress of technology and detection methods, the application of luciferase is more and more extensive, and the reporter gene can be used for detecting the maturation of protein.
Interleukin 1 β (IL-1 β) is an important proinflammatory cytokine, and induces expression of cell adhesion molecules, attracts accumulation of neutrophils, activates immune cells, induces gene expression and secretion of inflammatory cytokines, and the like. The production of IL-1 β is tightly regulated by the body, which promotes the transcription and expression of the IL-1 β precursor when the body receives signals from injury-related molecular patterns (DAMPs) or pathogen-related molecular patterns (PAMPs); the IL-1 beta precursor is cut and matured by the activated caspase-1, and the mature IL-1 beta secretion cell plays an immunoregulation function.
Disclosure of Invention
The object of the present invention is to design a novel reporter gene system for detecting protease cleavage activity.
The invention also aims to provide a reporter gene strategy that a reporter gene system can construct any protease cleavage activity, wherein a porcine interleukin 1 beta gene is used as a framework, a P17 cleavage site in the interleukin 1 beta is replaced by any protease cleavage site, and a fusion gene is constructed by the protease cleavage site and a luciferase (GLuc) gene.
Another object of the present invention is to provide the use of the above reporter gene for detecting protease cleavage activity.
Another object of the present invention is to provide the use of the above reporter gene for screening protease inhibitors or agonists in a quantitative manner.
In order to achieve the purpose, the invention adopts the technical scheme that:
a luciferase reporter gene iGLuc is disclosed, and the nucleotide sequence of the reporter gene iGLuc is shown in SEQ ID NO. 1.
Furthermore, the amino acid sequence of the reporter gene iGLuc is shown in SEQ ID NO. 2.
The invention also provides a construction method of the luciferase reporter gene iGLuc.
A luciferase reporter gene iGLuc takes a porcine interleukin 1 beta gene as a framework, a sequence corresponding to a P17 cleavage site in interleukin 1 beta is replaced by an endonuclease site KpnI, and the fusion gene is constructed by the luciferase reporter gene iGLuc and a luciferase GLuc gene.
Furthermore, the interleukin 1 beta gene skeleton is human interleukin 1 beta;
the nucleotide sequence of the reporter gene iGLuc-h constructed by taking the human interleukin 1 beta gene as a framework is shown as SEQ ID NO. 3, and the amino acid sequence of the reporter gene iGLuc-h is shown as SEQ ID NO. 4.
Furthermore, the interleukin 1 beta gene skeleton is murine interleukin 1 beta;
the murine interleukin 1 beta gene is a reporter gene iGLuc-m constructed by a framework, the nucleotide sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 5, and the amino acid sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 6.
Further, the endonuclease site also comprises a site into which a foreign gene can be inserted.
Furthermore, the construction strategy of the reporter gene iGLuc has different species commonalities and comprises the reporter gene constructed by adopting the same principle or similar principles.
The invention also provides a recombinant plasmid of the luciferase reporter gene iGLuc.
A recombinant plasmid containing the luciferase reporter gene iGLuc.
The invention also provides a preparation method of the luciferase reporter gene system for detecting the protease cleavage activity.
A luciferase reporter gene system for detecting protease cleavage activity is prepared by the following steps:
(1) inserting a nucleic acid sequence corresponding to the amino acid sequence of the protease cleavage site into a KpnI site in a reporter gene iGLuc to construct a recombinant plasmid;
(2) co-expressing the constructed recombinant plasmid and corresponding protease in cells, and detecting the activity of luciferase (GLuc) in cell supernatant;
the cell is a eukaryotic cell, and the expression vector is pCAGGS.
The invention also provides application of the luciferase reporter gene system.
The application of a luciferase reporter gene system in detecting protease activity and screening protease inhibitors or agonists in a quantitative manner.
According to the studies of the inventors, it was found that the N-terminus of the IL-1. beta. precursor has a function of inhibiting luciferase (GLuc) activity. The IL-1 beta precursor and Gluc are fused to construct a new luciferase reporter gene IL-1 beta-Gluc, when the IL-1 beta precursor is mature (not cut by caspase-1), luciferase is in a suppressed state, when the IL-1 beta precursor is cut and mature by caspase-1, the luciferase activity is obviously enhanced, and the level of the luciferase activity can reflect the level of the cut of the IL-1 beta precursor by caspase-1.
After the luciferase reporter gene iGLuc provided by the invention is expressed, only a low-level luciferase signal can be detected in the supernatant of the transfected iGLuc, and the luciferase reporter gene iGLuc is dose-dependent.
The luciferase reporter gene system provided by the invention has the advantages that the luciferase signal is gradually enhanced along with the increase of the transfection dosage, and the specificity is realized.
The luciferase reporter gene system provided by the invention has specificity because the luciferase signal is gradually weakened along with the increase of the dosage of the inhibitor.
The protein cleavage site between the N-terminal and the C-terminal of the IL-1 beta precursor in the reporter gene iGLuc provided by the invention can be replaced by the cleavage site of other proteases (including the cleavage site of viral proteases, the cleavage site of cellular proteases and the like), and then the corresponding proteases are adopted for cleavage, so that the cleavage efficiency of the proteases can be reflected by the activity level of the luciferases, and the cleavage activity of the corresponding proteases can also be reflected.
Drawings
FIG. 1 is a schematic diagram of the plasmid iGLuc provided by the present invention.
FIG. 2 shows the results of luciferase activity detection of the luciferase reporter genes iGLuc and Gluc provided by the present invention after HEK293T cells were transfected with dose gradients, respectively.
FIG. 3 is a schematic diagram of the recombinant plasmid nsp4-iGLuc provided by the invention.
FIG. 4 shows the luciferase activity detection results of the nsp4-iGLuc, iGLuc or Gluc co-transfected HEK293T cells respectively with nsp4 eukaryotic expression plasmids with dose gradients.
FIG. 5 is a schematic diagram of the recombinant plasmid S273R-iGLuc provided by the present invention.
FIG. 6 shows the results of luciferase activity detection of HEK293T cells co-transfected with S273R-iGLuc, iGLuc or Gluc and a dose-gradient S273R eukaryotic expression plasmid, respectively.
FIG. 7 is a schematic diagram of the recombinant plasmid Casp3-iGLuc provided by the present invention.
FIG. 8 shows the results of luciferase activity assays of the HEK293T cells co-transfected with Caspase 3-iGLuc, iGLuc or Gluc and dose-gradient Caspase-3 eukaryotic expression plasmids, respectively.
FIG. 9 shows the results of luciferase activity assays for Caspase 3-iGLuc reporter genes inhibiting Caspase-3 activity with different doses of specific inhibitors provided by the present invention.
FIG. 10 is a schematic diagram of plasmids iGLu-h and iGLu-m provided by the present invention.
FIG. 11 shows the results of luciferase activity detection of the S273R-iGLuc, S273R-iGLuc-h and S273R-iGLuc-m co-transfected HEK293T cells with the S273R eukaryotic expression plasmid.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.
A luciferase reporter gene iGLuc is disclosed, and the nucleotide sequence of the reporter gene iGLuc is shown in SEQ ID NO. 1.
Furthermore, the amino acid sequence of the reporter gene iGLuc is shown in SEQ ID NO. 2.
The invention also provides a construction method of the luciferase reporter gene iGLuc.
A luciferase reporter gene iGLuc takes an interleukin 1 beta gene as a framework, a sequence corresponding to a P17 cleavage site in the interleukin 1 beta is replaced by an endonuclease site KpnI, and the fusion gene is constructed by the luciferase reporter gene iGLuc and the luciferase GLuc gene.
Furthermore, the interleukin 1 beta gene skeleton is human interleukin 1 beta;
the nucleotide sequence of the reporter gene iGLuc-h constructed by taking the human interleukin 1 beta gene as a framework is shown as SEQ ID NO. 3, and the amino acid sequence of the reporter gene iGLuc-h is shown as SEQ ID NO. 4.
Furthermore, the interleukin 1 beta gene skeleton is murine interleukin 1 beta;
the murine interleukin 1 beta gene is a reporter gene iGLuc-m constructed by a framework, the nucleotide sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 5, and the amino acid sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 6.
Further, the endonuclease site also comprises a site into which a foreign gene can be inserted.
Furthermore, the construction strategy of the reporter gene iGLuc has different species commonalities and comprises the reporter gene constructed by adopting the same principle or similar principles.
The invention also provides a recombinant plasmid of the luciferase reporter gene iGLuc.
A recombinant plasmid containing the luciferase reporter gene iGLuc.
The invention also provides a preparation method of the luciferase reporter gene system for detecting the protease cleavage activity.
A luciferase reporter gene system for detecting protease cleavage activity is prepared by the following steps:
(1) inserting a nucleic acid sequence corresponding to the amino acid sequence of the protease cleavage site into a KpnI site in a reporter gene iGLuc to construct a recombinant plasmid;
(2) co-expressing the constructed recombinant plasmid and corresponding protease in cells, and detecting the activity of luciferase (GLuc) in cell supernatant;
the cell is a eukaryotic cell, and the expression vector is pCAGGS.
The invention also provides application of the luciferase reporter gene system.
The application of a luciferase reporter gene system in detecting protease activity and screening protease inhibitors or agonists in a quantitative manner.
The protein cleavage site between the N-terminal and the C-terminal of the IL-1 beta precursor in the reporter gene iGLuc provided by the invention can be replaced by the cleavage site of other proteases (including the cleavage site of viral proteases, the cleavage site of cellular proteases and the like), and then the corresponding proteases are adopted for cleavage, so that the cleavage efficiency of the proteases can be reflected by the activity level of the luciferases, and the cleavage activity of the corresponding proteases can also be reflected.
Example 1
Construction of luciferase reporter Gene iGLuc
According to the nucleotide sequence shown as SEQ ID NO:1, an iGLuc gene is synthesized, then the gene is cloned into a eukaryotic expression vector pCAGGS, and the plasmid is named as iGLuc (shown in figure 1); a control plasmid named GLuc is constructed by synthesizing GLuc gene and then cloning the gene into a eukaryotic expression vector pCAGGS. And transfecting HEK293T cells with iGLuc and Gluc respectively according to a dose gradient, collecting supernatant after 24 hours, and detecting the activity of luciferase by using a Gluc luciferase detection kit of Thermo company. The results are shown in FIG. 2, where only low levels of luciferase signal could be detected in the supernatants transfected with iGLuc, while high levels of luciferase signal could be detected in the supernatants transfected with GLuc-Flag and were dose dependent.
Example 2
Luciferase reporter gene system for detecting nsp4 cleavage activity
The protease nsp4 of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cleaves self-proteins, maturing viral-encoded polyprotein, a process that is important for viral replication. A nucleic acid sequence (tccaatcatgaggggaag) corresponding to the amino acid Sequence (SNHEGK) of the nsp4 protease cleavage site was inserted into the KpnI site of the reporter gene iGLuc to construct a recombinant plasmid nsp4-iGLuc (shown in FIG. 3). The nsp4-iGLuc, iGLuc or Gluc and nsp4 eukaryotic expression plasmids with a dose gradient are co-transfected into HEK293T cells respectively, and after 24 hours, the supernatant is collected and the luciferase activity is detected by adopting a Gluc luciferase detection kit of Thermo company. The results are shown in FIG. 4, and the reporter gene nsp4-iGLuc containing the nsp4 cleavage site has specificity due to the gradually increased luciferase signal with the increase of the nsp4 transfection dose.
Example 3
Luciferase reporter gene system for detecting S273R cleavage activity
Protease S273R of African Swine Fever Virus (ASFV) cleaves self-proteins, maturing virus-encoded polyprotein, a physiological process necessary for viral replication. A nucleic acid sequence (AAGGTAGGGGGGAATGAA) corresponding to the amino acid sequence (KVGGNE) of the protease cleavage site of S273R was inserted into the KpnI site of the reporter gene iGLuc to construct a recombinant plasmid S273R-iGLuc (shown in FIG. 5). S273R-iGLuc, iGLuc or Gluc and a dose-gradient S273R eukaryotic expression plasmid are co-transfected into HEK293T cells respectively, and after 24 hours, the supernatant is collected, and the luciferase activity is detected by adopting a Gluc luciferase detection kit of Thermo company. The results are shown in FIG. 6, and the reporter gene S273R-iGLuc containing the S273R cleavage site has specificity with increasing luciferase signal with increasing transfection dose of S273R.
Example 4
Luciferase reporter gene system for detecting Caspase-3 cleavage activity and application thereof
Caspase-3 is an important protease expressed by cells and plays an important role in apoptosis. A nucleic acid sequence (GACATGCCAGATGCTGCG) corresponding to the protease cleavage site amino acid sequence (DMPDAA) of Caspase-3 was inserted into KpnI site of reporter gene iGLuc to construct recombinant plasmid Casp3-iGLuc (shown in FIG. 7). The method comprises the steps of co-transfecting Casp3-iGLuc, iGLuc or Gluc and Caspase-3 eukaryotic expression plasmids with dose gradient HEK293T cells respectively, collecting supernatant after 24 hours, and detecting luciferase activity by adopting a Gluc luciferase detection kit of Thermo company. The results are shown in FIG. 8, and the reporter gene Casp3-iGLuc containing Caspase-3 cleavage site has gradually increased luciferase signal and specificity with the increase of Caspase-3 transfection dose. Further, the specific inhibitor with different doses is used for inhibiting Caspase-3 activity, and the result is shown in FIG. 9, the luciferase signal of the Casp3-iGLuc reporter gene is gradually weakened along with the increase of the dose of the inhibitor, and the specificity is realized.
Example 5
Reporter gene detection S273R cleavage activity constructed by interleukin 1 beta gene skeletons from different species
Synthesizing human-derived and murine interleukin 1 beta gene skeleton reporter genes respectively according to nucleotide sequences SEQ ID NO:3 and SEQ ID NO:5, cloning the genes into a eukaryotic expression vector pCAGGS, and naming plasmids as iGLu-h and iGLu-m (shown in figure 10); a nucleic acid sequence (AAGGTAGGGGGGAATGAA) corresponding to the amino acid sequence (KVGGNE) of the protease cleavage site of S273R was inserted into KpnI sites in reporter genes iGLuc-h and iGLuc-m to construct recombinant plasmids S273R-iGLuc-h and S273R-iGLuc-m (shown in FIG. 10). S273R-iGLuc, S273R-iGLuc-h and S273R-iGLuc-m and S273R eukaryotic expression plasmid are co-transfected into HEK293T cells respectively, and after 24 hours, supernatant is collected, and the activity of luciferase is detected by adopting a GLuc luciferase detection kit of Thermo company. The results are shown in FIG. 11, and the reporter genes S273R-iGLuc, S273R-iGLuc-h and S273R-iGLuc-m containing the S273R cleavage site co-transfect luciferase with the S273R eukaryotic expression plasmid have obviously enhanced signals and specificity.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are included in the protective scope of the present invention.
It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A luciferase reporter gene iGLuc, characterized in that: the nucleotide sequence of the reporter gene iGLuc is shown in SEQ ID NO. 1.
2. The luciferase reporter gene iGLuc as claimed in claim 1, wherein: the amino acid sequence of the reporter gene iGLuc is shown in SEQ ID NO. 2.
3. The luciferase reporter gene iGLuc as claimed in claim 1, wherein: the reporter gene iGLuc is a fusion gene constructed by taking a porcine interleukin 1 beta gene as a framework, replacing a sequence corresponding to a P17 cleavage site in the interleukin 1 beta with an endonuclease site KpnI and a luciferase GLuc gene.
4. The luciferase reporter gene iGLuc according to claim 3, wherein: the interleukin 1 beta gene skeleton is human interleukin 1 beta;
the nucleotide sequence of the reporter gene iGLuc-h constructed by taking the human interleukin 1 beta gene as a framework is shown as SEQ ID NO. 3, and the amino acid sequence of the reporter gene iGLuc-h is shown as SEQ ID NO. 4.
5. The luciferase reporter gene iGLuc according to claim 3, wherein: the interleukin 1 beta gene skeleton is murine interleukin 1 beta;
the murine interleukin 1 beta gene is a reporter gene iGLuc-m constructed by a framework, the nucleotide sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 5, and the amino acid sequence of the reporter gene iGLuc-m is shown as SEQ ID NO. 6.
6. The luciferase reporter gene iGLuc according to claim 3, wherein: the endonuclease site also includes a site into which a foreign gene can be inserted.
7. The luciferase reporter gene iGLuc as claimed in claim 1, wherein: the reporter gene iGLuc construction strategy has different species commonalities and comprises reporter genes constructed by adopting the same principle or similar principles.
8. A recombinant plasmid containing the luciferase reporter gene iGLuc according to any one of claims 1 to 5.
9. A luciferase reporter system for detecting protease cleavage activity, comprising: the luciferase reporter gene system is prepared by the following steps:
(1) inserting a nucleic acid sequence corresponding to the amino acid sequence of the protease cleavage site into a KpnI site in a reporter gene iGLuc to construct a recombinant plasmid;
(2) co-expressing the constructed recombinant plasmid and corresponding protease in cells, and detecting the activity of luciferase (GLuc) in cell supernatant;
the cell is a eukaryotic cell, and the expression vector is pCAGGS.
10. The application of a luciferase reporter gene system in detecting protease activity and screening protease inhibitors or agonists in a quantitative manner.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110862005.5A CN113699169A (en) | 2021-07-29 | 2021-07-29 | Luciferase reporter gene iGLuc, gene system and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110862005.5A CN113699169A (en) | 2021-07-29 | 2021-07-29 | Luciferase reporter gene iGLuc, gene system and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113699169A true CN113699169A (en) | 2021-11-26 |
Family
ID=78650838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110862005.5A Pending CN113699169A (en) | 2021-07-29 | 2021-07-29 | Luciferase reporter gene iGLuc, gene system and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113699169A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114592004A (en) * | 2022-04-02 | 2022-06-07 | 南方医科大学珠江医院 | Recombinant expression vector system and application thereof in constructing stem cell directed induction report cell |
| CN117106100A (en) * | 2023-10-20 | 2023-11-24 | 杭州百裕生物科技有限公司 | Screening method of PRRSV protease inhibitor and inhibition effect evaluation method |
| CN117106101A (en) * | 2023-10-20 | 2023-11-24 | 杭州百裕生物科技有限公司 | Plasmid and ASFV protease inhibitor screening and drug effect evaluation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463343A (en) * | 2008-12-30 | 2009-06-24 | 同济大学 | Cell model for screening antiphlogistic medicament and method for screening medicament by using the same |
| 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 |
-
2021
- 2021-07-29 CN CN202110862005.5A patent/CN113699169A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463343A (en) * | 2008-12-30 | 2009-06-24 | 同济大学 | Cell model for screening antiphlogistic medicament and method for screening medicament by using the same |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| JIE SONG: "Screening of PRRSV- and ASFV-encoded proteins involved in the inflammatory response using a porcine iGLuc reporter", JOURNAL OF VIROLOGICAL METHODS, pages 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114592004A (en) * | 2022-04-02 | 2022-06-07 | 南方医科大学珠江医院 | Recombinant expression vector system and application thereof in constructing stem cell directed induction report cell |
| CN114592004B (en) * | 2022-04-02 | 2023-04-25 | 南方医科大学珠江医院 | Recombinant expression vector system and application thereof in construction of stem cell directional induction report cells |
| CN117106100A (en) * | 2023-10-20 | 2023-11-24 | 杭州百裕生物科技有限公司 | Screening method of PRRSV protease inhibitor and inhibition effect evaluation method |
| CN117106101A (en) * | 2023-10-20 | 2023-11-24 | 杭州百裕生物科技有限公司 | Plasmid and ASFV protease inhibitor screening and drug effect evaluation method |
| CN117106100B (en) * | 2023-10-20 | 2024-02-06 | 杭州百裕生物科技有限公司 | Screening method of PRRSV protease inhibitor and inhibition effect evaluation method |
| CN117106101B (en) * | 2023-10-20 | 2024-06-07 | 合肥百裕生物科技有限公司 | Plasmid and ASFV protease inhibitor screening and drug effect evaluation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113699169A (en) | Luciferase reporter gene iGLuc, gene system and application thereof | |
| Schelle et al. | Selective replication of coronavirus genomes that express nucleocapsid protein | |
| EP0117059B1 (en) | Methods for producing human tpa and expression vectors therefor | |
| Tseng et al. | Modification of small hepatitis delta virus antigen by SUMO protein | |
| CN107075486A (en) | Express the mammalian cell of cytomegalovirus antigen | |
| CA2003248A1 (en) | Insect signal peptide mediated secretion of recombinant proteins | |
| Laux et al. | Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase‐1 knockout | |
| Probst et al. | Processing of proteinase precursors and their effect on hepatitis A virus particle formation | |
| Meents et al. | p27Kip1‐mediated controlled proliferation technology increases constitutive sICAM production in CHO‐DUKX adapted for growth in suspension and serum‐free media | |
| Breiner et al. | Cellular receptor traffic is essential for productive duck hepatitis B virus infection | |
| Zhao et al. | Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions | |
| Zhu et al. | Circ-udg derived from cyprinid herpesvirus 2 promotes viral replication | |
| JP3456992B2 (en) | Expression induction method using mutant DHFR gene | |
| JP6960409B2 (en) | promoter | |
| Senanayake et al. | Translation from the 5′ untranslated region (UTR) of mRNA 1 is repressed, but that from the 5′ UTR of mRNA 7 is stimulated in coronavirus-infected cells | |
| CA2012632C (en) | Process for the expression of a recombinant gene | |
| CN116083521A (en) | Fluorescence polarization method for screening novel coronavirus papain-like small molecule inhibitor and screening model | |
| JPH0740942B2 (en) | Expression of tPA in mammalian cells | |
| RU2385936C1 (en) | HGFopt GENE OF HEPATOCYTE GROWTH FACTOR | |
| US6866998B1 (en) | Cellular assays for identifying signaling peptides | |
| Noguchi et al. | Minimum essential region of CCG1/TAF II 250 required for complementing the temperature-sensitive cell cycle mutants, tsBN462 and ts13 cells, of hamster BHK21 cells | |
| Jalanko et al. | Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector | |
| CN110732019B (en) | Application of CG10032 protein and/or CG10032 gene | |
| CN113637675B (en) | Production method, nucleotide sequence, expression vector and expression system of human serum albumin | |
| CN117327681B (en) | Chaperone plasmid for promoting correct folding of mammalian cell expression protein and application thereof |
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 |