CN114184714A - Method for distinguishing exosome sources - Google Patents
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- CN114184714A CN114184714A CN202210139448.6A CN202210139448A CN114184714A CN 114184714 A CN114184714 A CN 114184714A CN 202210139448 A CN202210139448 A CN 202210139448A CN 114184714 A CN114184714 A CN 114184714A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention discloses a method for distinguishing exosome sources, which is characterized in that exosomes are subjected to enzymolysis through trypsin to form a mixture containing different peptide fragments, the mixture is subjected to peptide map analysis, and the sources of the mixture can be known after the mixture is compared with a peptide map; the method using the source of the exosome of the region can also detect the medicine or cosmetic components containing the exosome. The invention has the beneficial effects that: the exosome with unknown source can be quickly and efficiently identified, the detection cost is low, the detection means is simple, and the detection sensitivity is high; the invention can be used for detecting the components of the medicine or the cosmetics, and accurately judges the components of the medicine or the cosmetics by detecting the sources of exosomes.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a method for distinguishing exosome sources.
Background
As a new field, the exosome industry needs a plurality of methods for identifying and characterizing exosomes, and currently, the identification means of exosomes mainly comprise an electron microscope, a Nanoparticle Tracking Analyzer (NTA), a western blot and the like, but the instruments are expensive, and the detection method and the operation process of the instruments are complex. These characterization techniques, while possible to characterize exosomes, are unable to distinguish exosomes of different origins.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for distinguishing the source of exosomes.
The technical scheme adopted by the invention is as follows: a method for distinguishing the source of exosome includes such steps as enzymolyzing exosome to obtain mixture containing different peptide segments, analyzing the peptide map of mixture, and comparing it with the map of peptide map to find its source.
Preferably, exosomes are enzymatically hydrolyzed by trypsin, papain, pepsin, chymotrypsin, carboxypeptidase, or proteinase K.
Preferably, the exosome sample is concentrated or diluted to make the protein concentration of the sample be 1mg/ml, and the samples are respectively and fully dialyzed by 1% ammonium bicarbonate solution for liquid exchange;
adding trypsin according to the proportion of 1:50 (mg/mg), and carrying out water bath enzymolysis at 37 ℃ for 16-24 h;
after termination of the reaction, the supernatant was centrifuged and used for peptide mapping.
Preferably, the reaction is stopped by adding 50% acetic acid solution in a ratio of 1: 10.
Preferably, the peptide map analysis is performed by chromatography.
Preferably, advanced Bio Peptide Map is used as a chromatographic column, and the column temperature is set to be 30 ℃;
taking 0.1% trifluoroacetic acid water solution as mobile phase A liquid, taking 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B liquid, the flow rate is 0.5ml per minute, gradient elution is carried out for 70 minutes, the A liquid is 100% -30%, the B liquid is 0-70%, and the detection wavelength is 214 nm.
Preferably, the exosomes from known sources are subjected to enzymolysis and then subjected to peptide map analysis to form a peptide map;
and then carrying out the same treatment on exosomes with unknown sources, and comparing the formed peptide map with the peptide map to obtain the sources of the exosomes.
Preferably, the characteristic peaks of the peptide map are compared to the peptide map.
Use of a method for distinguishing a source of exosomes for detecting an application of a pharmaceutical or cosmetic ingredient containing exosomes.
The invention has the advantages and positive effects that: the exosome with unknown source can be quickly and efficiently identified, the detection cost is low, the detection means is simple, and the detection sensitivity is high; the invention can be used for detecting the components of the medicine or the cosmetics, and accurately judges the components of the medicine or the cosmetics by detecting the sources of exosomes.
Drawings
FIG. 1 is a 293 cell exosome peptide map in example 1 of the present invention;
FIG. 2 is a repeated view of a peptide map of 293 cell exosomes in example 2 of the present invention;
FIG. 3 is a diagram of mesenchymal stem cell exosome peptides in example 1 of the present invention;
fig. 4 is a repeated view of a peptide map of mesenchymal stem cell exosomes in example 2 of the present invention;
FIG. 5 is a diagram of a milk exosome peptide according to example 1 of the present invention;
FIG. 6 is a repeat of a peptide map of milk exosomes of example 2 of the present invention;
FIG. 7 is peptide map of 293 cell exosomes, mesenchymal stem cell exosomes and milk exosomes in example 2 of the present invention;
FIG. 8 is a schematic diagram showing characteristic peaks of peptide patterns of 293 cell exosomes, mesenchymal stem cell exosomes and milk exosomes in example 2 of the present invention;
FIG. 9 is a peptide map of an unknown sample in example 3 of the present invention;
FIG. 10 is a diagram of a milk exosome peptide according to example 4 of the present invention;
fig. 11 is a diagram of mesenchymal stem cell exosome peptides in example 4 of the present invention;
FIG. 12 is a 293 cell exosome peptide map in example 4 of the present invention;
FIG. 13 is peptide map of 293 cell exosomes, mesenchymal stem cell exosomes and milk exosomes in example 3 of the present invention;
FIG. 14 is a peptide map of an unknown sample in example 5 of the present invention.
Detailed Description
Embodiments of the present invention will be described below with reference to the accompanying drawings.
The invention discloses a method for distinguishing exosome sources, according to the characteristics of exosomes, the exosomes from different sources do not contain completely identical proteins, after enzymolysis, exosome proteins form a mixture containing multiple peptide fragments, according to partial difference of the peptide fragments formed after enzymolysis of exosomes from different sources, peptide map analysis is carried out, finally, peptide map maps corresponding to different types of exosomes are obtained, and the exosome sources are identified and analyzed according to the peptide map maps. The specific analysis method comprises the steps of firstly carrying out enzymolysis on exosomes from known sources and then carrying out peptide map analysis to form a peptide map; and carrying out the same treatment on exosomes with unknown sources, comparing the formed peptide map with the peptide map, comparing the characteristic peaks of the peptide map and the peptide map, and obtaining the sources of the exosomes through comparison according to the characteristic peak conditions of the peptide map. In certain embodiments of the invention the exosomes may be enzymatically hydrolyzed using trypsin, papain, pepsin, chymotrypsin, carboxypeptidase or proteinase K.
Taking enzymolysis of exosomes by trypsin as an example; firstly, concentrating or diluting an exosome sample to ensure that the protein concentration of the sample is 1mg/ml, and respectively and fully dialyzing and replacing the solution by using 1% ammonium bicarbonate solution; adding trypsin according to the proportion of 1:50 (mg/mg), and carrying out water bath enzymolysis at 37 ℃ for 16-24 h; the reaction was stopped by adding 50% acetic acid solution at a ratio of 1:10, the supernatant was collected by centrifugation, and the resulting supernatant was subjected to peptide mapping by chromatography.
The method can judge the component sources by detecting the components in the finished product medicine or the finished product cosmetic and detecting the sources of the exosomes contained in the finished product medicine or the finished product cosmetic.
The protocol of the present invention will be described with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commercial specifications, and the instruments, reagents and consumables used in the examples are all available from commercial companies, if no specific description is provided, wherein the 293 cell used in some examples of the present invention is Expi 293.
Example 1:
1.1 exosomal enzymolysis:
respectively taking a milk exosome sample, a mesenchymal stem cell exosome sample and a 293 cell exosome sample, respectively concentrating the samples until the protein concentration is 1mg/ml, respectively and fully dialyzing the samples by using 1% ammonium bicarbonate solution, adding trypsin solution into the solution of a sample according to the ratio of 1:50 (mg/mg), carrying out enzymolysis for-16-24 h in water bath at 37 ℃, then adding 50% acetic acid solution according to the ratio of 1:10 to stop the reaction, turning the mixture upside down and mixing the mixture uniformly, centrifuging the mixture at 12,000g multiplied by 10min per minute, and taking out the supernatant for later use.
1.2 peptide mapping analysis:
chromatographic conditions are as follows: the instrument uses Agilent 1260 liquid chromatogram, the chromatographic column selects advanced Bio Peptide Map, the column temperature is set to 30 ℃, and the standby sample is placed in a sample manager at 4 ℃; using 0.1% trifluoroacetic acid water solution as mobile phase A solution and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B solution, the flow rate is 0.5ml per minute, gradient elution is carried out for 70 minutes (the A solution is from 100% to 30%, the B solution is from 0% to 70%), and the detection wavelength is 214 nm.
Respectively obtaining a 293 cell exosome peptide map as shown in figure 1, a mesenchymal stem cell exosome peptide map as shown in figure 3 and a cow milk exosome peptide map as shown in figure 5.
Example 2:
according to the experimental process, milk exosome samples, mesenchymal stem cell exosome samples and 293 cell exosome samples are taken for repeated experiments, and obtained maps are made into repeated maps, as shown in fig. 2, fig. 4 and fig. 6 respectively.
FIG. 7 is a comparison result of peptide maps of 293 cell exosomes, mesenchymal stem cell exosomes and milk exosomes, and it can be seen that the peptide maps of exosomes from different sources have obvious difference; as shown in FIG. 8, the peptide pattern characteristic peaks of the 293 cell exosome, the mesenchymal stem cell exosome and the milk exosome are shown, and the positions of the characteristic peaks of exosomes from different sources are also different.
Example 3:
an unknown sample was subjected to enzymatic hydrolysis and peptide mapping analysis in the same order as in example 1, and the results are shown in FIG. 9. As can be seen by comparison, 48min in FIG. 9 has a peak identical to that of the peptide map of the known milk exosome (FIG. 5), and other positions have no characteristic peak, so that the unknown sample is known as the milk exosome.
Example 4:
4.1 enzymatic hydrolysis of exosomes:
respectively taking a milk exosome sample, a mesenchymal stem cell exosome sample and a 293 cell exosome sample, respectively concentrating the samples until the protein concentration is 1mg/ml, adding proteinase K (the working concentration of the proteinase K is 50 mu g/ml-100 mu g/ml) into a solution of a test article, carrying out enzymolysis for 1h in water bath at 37 ℃, reversing the solution up and down, mixing the solution uniformly, centrifuging the solution at 12,000g multiplied by 10min per minute, and taking out supernatant for later use.
4.2 peptide mapping analysis:
chromatographic conditions are as follows: the instrument uses Agilent 1260 liquid chromatogram, the chromatographic column selects advanced Bio Peptide Map, the column temperature is set to 30 ℃, and the standby sample is placed in a sample manager at 4 ℃; using 0.1% trifluoroacetic acid water solution as mobile phase A solution and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B solution, the flow rate is 0.5ml per minute, gradient elution is carried out for 70 minutes (the A solution is from 100% to 30%, the B solution is from 0% to 70%), and the detection wavelength is 214 nm.
A peptide map of the milk exosomes as shown in fig. 10, a peptide map of the mesenchymal stem cell exosomes as shown in fig. 11 and a peptide map of the 293 cell exosomes as shown in fig. 12 were obtained, respectively.
Fig. 13 is a comparison result of peptide maps of 293 cell exosomes, mesenchymal stem cell exosomes and milk exosomes, and it can be seen that the peptide maps of exosomes from different sources have obvious differences.
Example 5:
an unknown sample was subjected to enzymatic hydrolysis and peptide mapping in this order according to the procedure of example 4, and the results are shown in FIG. 14. As can be seen by comparison, in FIG. 14, there are 4 peaks between 10 and 20min which are the same as the peaks of the peptide map of the known mesenchymal stem cell exosomes (FIG. 11), and there are no characteristic peaks at other positions, so that the unknown sample is known to be the mesenchymal stem cell exosomes.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (9)
1. A method of differentiating the source of exosomes, characterized by: carrying out enzymolysis on exosome to form a mixture containing different peptide fragments, carrying out peptide map analysis on the mixture, and comparing with a peptide map to know the source of the mixture.
2. A method of differentiating the origin of exosomes according to claim 1, characterized in that: exosomes are enzymatically hydrolyzed by trypsin, papain, pepsin, chymotrypsin, carboxypeptidase, or proteinase K.
3. A method of differentiating the origin of exosomes according to claim 2, characterized in that: concentrating or diluting the exosome sample to make the protein concentration of the sample be 0.1-10 mgmg/ml;
adding trypsin according to the proportion of 1:10-200 (mg/mg), and carrying out water bath enzymolysis at 37 ℃ for 16-24 h;
after termination of the reaction, the supernatant was centrifuged and used for peptide mapping.
4. A method of distinguishing a source of exosomes according to claim 3, characterised in that: the reaction was stopped by adding 50% acetic acid solution at a ratio of 1: 10.
5. A method of differentiating the origin of exosomes according to any of claims 1-4, characterized in that: peptide mapping analysis was performed by chromatography.
6. A method of differentiating the origin of exosomes according to claim 5, characterized in that: adopting a C18 chromatographic column, and setting the column temperature to be 30 ℃;
taking 0.1% trifluoroacetic acid water solution as mobile phase A liquid, taking 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B liquid, the flow rate is 0.5ml per minute, gradient elution is carried out for 70 minutes, the A liquid is 100% -30%, the B liquid is 0-70%, and the detection wavelength is 214 nm.
7. A method of differentiating the origin of exosomes according to claim 5, characterized in that: carrying out enzymolysis on exosomes from known sources and then carrying out peptide map analysis to form a peptide map;
and then carrying out the same treatment on exosomes with unknown sources, and comparing the formed peptide map with the peptide map to obtain the sources of the exosomes.
8. A method of differentiating the origin of exosomes according to claim 7, characterized in that: the characteristic peaks of the peptide pattern and the peptide pattern were compared.
9. Use of a method for differentiating the source of exosomes as claimed in any of claims 1 to 8 for the detection of pharmaceutical or cosmetic ingredients containing exosomes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117866890A (en) * | 2024-03-12 | 2024-04-12 | 山东翰康医学科技集团有限公司 | Preparation method for extracting and purifying adipose-derived mesenchymal stem cell exosomes |
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| CN108623657A (en) * | 2018-05-11 | 2018-10-09 | 苏州大学 | Polypeptide, recombinant DNA molecules, recombinant vector, excretion body and its application |
| CN110596403A (en) * | 2019-09-17 | 2019-12-20 | 中山大学附属第一医院 | Specific protein marker for ovarian cancer diagnosis and preparation method thereof |
| US20210263042A1 (en) * | 2018-11-09 | 2021-08-26 | Nx Prenatal Inc. | Tandem-paired column chemistry for high-throughput proteomic exosome analysis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108623657A (en) * | 2018-05-11 | 2018-10-09 | 苏州大学 | Polypeptide, recombinant DNA molecules, recombinant vector, excretion body and its application |
| US20210263042A1 (en) * | 2018-11-09 | 2021-08-26 | Nx Prenatal Inc. | Tandem-paired column chemistry for high-throughput proteomic exosome analysis |
| CN110596403A (en) * | 2019-09-17 | 2019-12-20 | 中山大学附属第一医院 | Specific protein marker for ovarian cancer diagnosis and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117866890A (en) * | 2024-03-12 | 2024-04-12 | 山东翰康医学科技集团有限公司 | Preparation method for extracting and purifying adipose-derived mesenchymal stem cell exosomes |
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