CN104237439B - A kind of method detecting protein disulfide and whether rupture - Google Patents
A kind of method detecting protein disulfide and whether rupture Download PDFInfo
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- CN104237439B CN104237439B CN201410526145.5A CN201410526145A CN104237439B CN 104237439 B CN104237439 B CN 104237439B CN 201410526145 A CN201410526145 A CN 201410526145A CN 104237439 B CN104237439 B CN 104237439B
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Abstract
A kind of method detecting protein disulfide and whether rupture, adopt the free sulfhydryl groups in iodoacetic acid and protein to react, unreacted iodoacetic acid is removed by ultrafiltration, use the disulfide bond in dithiothreitol (DTT) cut-out protein again, and then adopt iodoacetamide to react with the sulfydryl newly produced, use trypsase that proteolysis is become polypeptide, detect the mass-to-charge ratio information of polypeptide by high resolution mass spec, contrast with the disulfide bond information of native protein, determine whether disulfide bond ruptures.The present invention can be effective to determine that sulfydryl exists with form that is free or disulfide bond; What cause after iodoacetic acid and iodoacetamide and sulfydryl react is of poor quality within 1Da, therefore uses high resolution mass spec can conclude the difference of the two very accurately; Whether protein disulfide the accurate of fracture occurs judges to lay the foundation, for specific protein modification and actual production provide theoretical foundation as correlation theory research.
Description
Technical field
The present invention relates to a kind of method detecting protein disulfide and whether rupture.
Background technology
Disulfide bond is oxidized and form the covalent bond of-S-S-form by 2 sulfydryls.Disulfide bond divides two kinds, and namely in molecule and intermolecular disulfide bond, intramolecular disulfide bond is present in independent polypeptied chain and is used for the tertiary structure of firm protein, and intermolecular disulfide bond is present in and is used for the quaternary structure of stable protein between peptide chain.The fracture of disulfide bond can impel the spatial structure generation change to a certain degree of protein, and then affects biological nature and the reactivity of protein, and such as, the fracture of disulfide bond can accelerate ovalbumin generation glycosylation.At present, the mankind just constantly attempt changing protein structure by certain technology, thus transform its performance.Such as, can BSA Structure be changed by ultrasonic technology, and then accelerate the speed that glycosylation occurs for it.Is the change of protein structure relevant with disulfide bonds? the answer of this problem is conducive to the directional transformation instructing protein function character, therefore, find a kind of can accurately detect protein disulfide whether occur rupture method seem very necessary.
The present invention adopts the free sulfhydryl groups in iodoacetic acid and protein to react, unreacted iodoacetic acid is removed by ultrafiltration, the disulfide bond of dithiothreitol (DTT) cleave proteins matter is used to produce new sulfydryl again, the sulfydryl newly produced in iodoacetamide and protein is adopted to react, trypsase is used to become polypeptide by by the proteolysis after iodoacetamide effect, detect the mass-to-charge ratio information of polypeptide by high resolution mass spec, contrast with the disulfide bond information of native protein, determine whether disulfide bond ruptures.The present invention adopts that the free sulfhydryl groups of iodoacetic acid and protein reacts, iodoacetamide and reacting through the sulfydryl that dithiothreitol (DTT) cleave proteins matter disulfide bond obtains, and uses high resolution mass spec to detect, does not have document to carry out relevant report both at home and abroad.
Summary of the invention
The present invention aims to provide a kind of method detecting protein disulfide and whether rupture, adopt that the free sulfhydryl groups of iodoacetic acid and protein reacts, iodoacetamide and reacting through the sulfydryl that dithiothreitol (DTT) cleave proteins matter disulfide bond obtains, high resolution mass spec is used to detect, compare with the disulfide bond information of native protein, determine whether disulfide bond ruptures.
Its concrete technology step is as follows:
1, protein free sulfhydryl groups and iodoacetic acid react
(1) preparation of protein solution: dissolve the protein solution obtaining 0.1-2.0mg/ml with the ammonium bicarbonate solution of 10-100mM;
(2) preparation of iodoacetic acid solution: with the iodoacetic acid solution of ultrapure water preparation 50-150mM;
(3) protein free sulfhydryl groups reacts with iodoacetic acid: mixed with 3-10 μ l iodoacetic acid solution by 10-30 μ l protein solution, and after mixing, lucifuge places 20-60min;
(4) iodoacetic acid having neither part nor lot in reaction is removed: adopt the ultra-filtration centrifuge tube of 1000-10000Da to remove the unnecessary iodoacetic acid having neither part nor lot in reaction.
2, dithiothreitol (DTT) cleave proteins matter disulfide bond
(1) dithiothreitol (DTT) solution preparation: with the dithiothreitol (DTT) solution of ultrapure water preparation 50-150mM;
(2) dithiothreitol (DTT) cleave proteins matter disulfide bond: add dithiothreitol (DTT) solution 2-10 μ l in protein solution, 50-100 DEG C of process 3-30min, is cooled to room temperature;
3, iodoacetamide and reacting through the dithiothreitol (DTT) sulfydryl that disulfide bond obtains that ruptures
(1) preparation of iodoacetamide solution: with the iodoacetamide solution of ultrapure water preparation 50-150mM;
(2) sulfydryl that iodoacetamide and the disulfide bond that ruptures through dithiothreitol (DTT) obtain reacts: in protein solution, add iodoacetamide solution 5-15 μ l, after mixing, lucifuge places 20-60min;
4, trypsin digestion protein
(1) tryptic preparation: with the trypsin solution of ultrapure water preparation 0.1-1.0mg/ml;
(2) trypsin digestion protein: be 100:1-100:1 according to protein and trypsase mass ratio g/g, mixes trypsase with protein solution, in the shaking table of 30-50 DEG C, places 8-24h;
(3) tryptic hydrolysis reaction is stopped: the trifluoroacetic acid 2-10 μ l adding 1-10% stops tryptic hydrolysis reaction, obtains final proteolysis potpourri.
5, high resolution mass spec detects
(1) loading: the protein hydrolysate of 2-15 μ g is injected into high performance liquid chromatograph, desalination 3-10min;
(2) high resolution mass spec detects: connect high performance liquid chromatograph and high resolution mass spec equipment, adopts spectrum 30-60min, the Information in Mass Spectra of record protein hydrolysate;
6, data analysis
(1) mass-spectrogram analysis: the Information in Mass Spectra arranging protein hydrolysate, iodoacetic acid and sulfydryl reaction can make peptide masses increase 57.09-58.02Da, and iodoacetamide and sulfydryl reaction can make peptide masses increase 57.01-57.03Da;
(2) in protein, sulfydryl dissociates situation analysis: the mass-to-charge ratio according to protein hydrolysate increases information, conclude that sulfydryl reacts with iodoacetic acid or reacts with iodoacetamide, if react with iodoacetic acid, illustrate that the sulfydryl in protein is free, if react with iodoacetamide, illustrate that the sulfydryl in protein combines, namely exist with the form of disulfide bond;
(3) determine whether the disulfide bond in protein ruptures: there is information according to the disulfide bond of native protein in database, the disulfide bond information obtained in contrast experiment, determine whether the disulfide bond in protein ruptures.
The art of this patent feature: adopt that the free sulfhydryl groups of iodoacetic acid and protein reacts, iodoacetamide and reacting through the sulfydryl that dithiothreitol (DTT) cleave proteins matter disulfide bond obtains, high resolution mass spec is used to detect, according to the disulfide bond information that mass-spectrogram provides, the disulfide bond information of contrast native protein, accurately judges whether the disulfide bond of protein ruptures.
beneficial effect of the present invention:
1, iodoacetic acid and iodoacetamide can react with free sulfhydryl groups, but reacting the latter two quality caused increases situation difference, can be effective to determine that sulfydryl exists with formation that is free or disulfide bond; What 2, iodoacetic acid and iodoacetamide and sulfydryl caused after reacting is of poor quality within 1Da, therefore, uses high resolution mass spec can conclude the difference of the two very accurately; What 3, whether protein disulfide occurred to rupture accurately judges to lay the foundation, for specific protein modification and actual production provide foundation as correlation theory research.
Accompanying drawing explanation
Fig. 1 is process chart of the present invention.
Embodiment
Embodiment one
1, protein free sulfhydryl groups and iodoacetic acid react
(1) preparation of protein solution: dissolve the protein solution obtaining 1.0mg/ml with the ammonium bicarbonate solution of 50mM;
(2) preparation of iodoacetic acid solution: with the iodoacetic acid solution of ultrapure water preparation 100mM;
(3) protein free sulfhydryl groups reacts with iodoacetic acid: mixed with 3 μ l iodoacetic acid solution by 10 μ l protein solutions, and after mixing, lucifuge places 30min;
(4) iodoacetic acid having neither part nor lot in reaction is removed: adopt the ultra-filtration centrifuge tube of 3000Da to remove the unnecessary iodoacetic acid having neither part nor lot in reaction.
2, dithiothreitol (DTT) cleave proteins matter disulfide bond
(1) dithiothreitol (DTT) solution preparation: with the dithiothreitol (DTT) solution of ultrapure water preparation 100mM;
(2) dithiothreitol (DTT) cleave proteins matter disulfide bond: add dithiothreitol (DTT) solution 2 μ l in protein solution, 95 DEG C of process 5min, are cooled to room temperature;
3, iodoacetamide and reacting through the dithiothreitol (DTT) sulfydryl that disulfide bond obtains that ruptures
(1) preparation of iodoacetamide solution: with the iodoacetamide solution of ultrapure water preparation 100mM;
(2) sulfydryl that iodoacetamide and the disulfide bond that ruptures through dithiothreitol (DTT) obtain reacts: in protein solution, add iodoacetamide solution 5 μ l, after mixing, lucifuge places 20min;
4, trypsin digestion protein
(1) tryptic preparation: with the trypsin solution of ultrapure water preparation 0.1mg/ml;
(2) trypsin digestion protein: according to protein and trypsase mass ratio 50:1(g/g), trypsase is mixed with protein solution, in the shaking table of 37 DEG C, places 12h;
(3) tryptic hydrolysis reaction is stopped: the trifluoroacetic acid 2 μ l adding 10% stops tryptic hydrolysis reaction, obtains final proteolysis potpourri.
5, high resolution mass spec detects
(1) loading: the protein hydrolysate of 3 μ g is injected into high performance liquid chromatograph, desalination 5min;
(2) high resolution mass spec detects: connect high performance liquid chromatograph and high resolution mass spec equipment, adopts spectrum 60min, the Information in Mass Spectra of record protein hydrolysate;
6, data analysis
(1) mass-spectrogram analysis: the Information in Mass Spectra arranging protein hydrolysate, iodoacetic acid and sulfydryl react and make peptide masses increase 58.0055Da, and iodoacetamide and sulfydryl reaction can make peptide masses increase 57.0215Da;
(2) in protein, sulfydryl dissociates situation analysis: the mass-to-charge ratio according to protein hydrolysate increases information, conclude that sulfydryl reacts with iodoacetic acid or reacts with iodoacetamide, if react with iodoacetic acid, illustrate that the sulfydryl in protein is free, if react with iodoacetamide, illustrate that the sulfydryl in protein combines, namely exist with the form of disulfide bond;
(3) determine whether the disulfide bond in protein ruptures: there is information according to the disulfide bond of native protein in database, the disulfide bond information obtained in contrast experiment, but it is exist with disulfide formation that experimental result is shown as display in free sulfhydryl groups native protein information, illustrates that the disulfide bond of protein there occurs fracture.
Embodiment two
1, protein free sulfhydryl groups and iodoacetic acid react
(1) preparation of protein solution: dissolve the protein solution obtaining 1.0mg/ml with the ammonium bicarbonate solution of 100mM;
(2) preparation of iodoacetic acid solution: with the iodoacetic acid solution of ultrapure water preparation 100mM;
(3) protein free sulfhydryl groups reacts with iodoacetic acid: mixed with 3 μ l iodoacetic acid solution by 20 μ l protein solutions, and after mixing, lucifuge places 30min;
(4) iodoacetic acid having neither part nor lot in reaction is removed: adopt the ultra-filtration centrifuge tube of 2000Da to remove the unnecessary iodoacetic acid having neither part nor lot in reaction.
2, dithiothreitol (DTT) cleave proteins matter disulfide bond
(1) dithiothreitol (DTT) solution preparation: with the dithiothreitol (DTT) solution of ultrapure water preparation 100mM;
(2) dithiothreitol (DTT) cleave proteins matter disulfide bond: add dithiothreitol (DTT) solution 2 μ l in protein solution, 95 DEG C of process 10min, are cooled to room temperature;
3, iodoacetamide and reacting through the dithiothreitol (DTT) sulfydryl that disulfide bond obtains that ruptures
(1) preparation of iodoacetamide solution: with the iodoacetamide solution of ultrapure water preparation 100mM;
(2) sulfydryl that iodoacetamide and the disulfide bond that ruptures through dithiothreitol (DTT) obtain reacts: in protein solution, add iodoacetamide solution 5 μ l, after mixing, lucifuge places 40min;
4, trypsin digestion protein
(1) tryptic preparation: with the trypsin solution of ultrapure water preparation 0.1mg/ml;
(2) trypsin digestion protein: according to protein and trypsase mass ratio 20:1(g/g), trypsase is mixed with protein solution, in the shaking table of 37 DEG C, places 18h;
(3) tryptic hydrolysis reaction is stopped: the trifluoroacetic acid 2 μ l adding 5% stops tryptic hydrolysis reaction, obtains final proteolysis potpourri.
5, high resolution mass spec detects
(1) loading: the protein hydrolysate of 5 μ g is injected into high performance liquid chromatograph, desalination 5min;
(2) high resolution mass spec detects: connect high performance liquid chromatograph and high resolution mass spec equipment, adopts spectrum 45min, the Information in Mass Spectra of record protein hydrolysate;
6, data analysis
(1) mass-spectrogram analysis: the Information in Mass Spectra arranging protein hydrolysate, iodoacetic acid and sulfydryl react and make peptide masses increase 58.0045Da, and iodoacetamide and sulfydryl reaction can make peptide masses increase 57.0225Da;
(2) in protein, sulfydryl dissociates situation analysis: the mass-to-charge ratio according to protein hydrolysate increases information, conclude that sulfydryl reacts with iodoacetic acid or reacts with iodoacetamide, if react with iodoacetic acid, illustrate that the sulfydryl in protein is free, if react with iodoacetamide, illustrate that the sulfydryl in protein combines, namely exist with the form of disulfide bond;
(3) determine whether the disulfide bond in protein ruptures: there is information according to the disulfide bond of native protein in database, the disulfide bond information obtained in contrast experiment, but it is exist with disulfide formation that experimental result is shown as display in free sulfhydryl groups native protein information, illustrates that the disulfide bond of protein there occurs fracture.
Claims (6)
1. detect the method whether protein disulfide ruptures, it is characterized in that:
A, protein free sulfhydryl groups and iodoacetic acid react;
B, dithiothreitol (DTT) cleave proteins matter disulfide bond;
C, iodoacetamide and react through the dithiothreitol (DTT) sulfydryl that disulfide bond obtains that ruptures;
D, trypsin digestion protein obtain protein hydrolysate;
E, high resolution mass spec detect;
F, data analysis;
Described data analysis comprises the steps:
(1) arrange the Information in Mass Spectra of protein hydrolysate, iodoacetic acid and sulfydryl reaction can make peptide masses increase 57.09-58.02Da, and iodoacetamide and sulfydryl reaction can make peptide masses increase 57.01-57.03Da;
(2) information is increased according to the mass-to-charge ratio of protein hydrolysate, conclude that sulfydryl reacts with iodoacetic acid or reacts with iodoacetamide, if react with iodoacetic acid, illustrate that the sulfydryl in protein is free, if react with iodoacetamide, illustrate that the sulfydryl in protein combines, namely exist with the form of disulfide bond;
(3) there is information according to the disulfide bond of native protein in database, the disulfide bond information obtained in contrast experiment, determine whether the disulfide bond in protein ruptures.
2. a kind of method detecting protein disulfide and whether rupture according to claim 1, is characterized in that:
Described protein free sulfhydryl groups and iodoacetic acid react and comprise the steps:
(1) dissolve with the ammonium bicarbonate solution of 10-100mM the protein solution obtaining 0.1-2.0mg/ml;
(2) with the iodoacetic acid solution of ultrapure water preparation 50-150mM;
(3) mixed with 3-10 μ l iodoacetic acid solution by 10-30 μ l protein solution, after mixing, lucifuge places 20-60min;
(4) ultra-filtration centrifuge tube of 1000-10000Da is adopted to remove the unnecessary iodoacetic acid having neither part nor lot in reaction.
3. a kind of method detecting protein disulfide and whether rupture according to claim 1, is characterized in that:
Described dithiothreitol (DTT) cleave proteins matter disulfide bond comprises the steps:
(1) with the dithiothreitol (DTT) solution of ultrapure water preparation 50-150mM;
(2) in protein solution, add dithiothreitol (DTT) solution 2-10 μ l, 50-100 DEG C of process 3-30min, is cooled to room temperature.
4. a kind of method detecting protein disulfide and whether rupture according to claim 1, is characterized in that:
The sulfydryl that described iodoacetamide and the disulfide bond that ruptures through dithiothreitol (DTT) obtain reacts and comprises the steps:
(1) with the iodoacetamide solution of ultrapure water preparation 50-150mM;
(2) in protein solution, add iodoacetamide solution 5-15 μ l, after mixing, lucifuge places 20-60min.
5. a kind of method detecting protein disulfide and whether rupture according to claim 1, is characterized in that:
Described trypsin digestion protein comprises the steps:
(1) with the trypsin solution of ultrapure water preparation 0.1-1.0mg/ml;
(2) be 100:1 according to protein and trypsase mass ratio g/g, trypsase mixed with protein solution, in the shaking table of 30-50 DEG C, places 8-24h;
(3) the trifluoroacetic acid 2-10 μ l adding 1-10% stops tryptic hydrolysis reaction, obtains final proteolysis potpourri.
6. a kind of method detecting protein disulfide and whether rupture according to claim 1, is characterized in that:
Described high resolution mass spec detects and comprises the steps:
(1) protein hydrolysate of 2-15 μ g is injected into high performance liquid chromatograph, desalination 3-10min;
(2) connect high performance liquid chromatograph and high resolution mass spec equipment, adopt spectrum 30-60min, the Information in Mass Spectra of record protein hydrolysate.
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| CN106854231B (en) * | 2015-12-09 | 2020-08-11 | 中国科学院大连化学物理研究所 | Method for enriching sulfhydrylation polypeptide |
| CN108333281A (en) * | 2018-02-08 | 2018-07-27 | 吉林大学 | A method of measuring recombinant human granulocyte colony stimulating factor disulfide bond composition |
| CN109839297B (en) * | 2018-07-31 | 2021-06-08 | 深圳市安帝宝科技有限公司 | Method for rapidly preparing glycated albumin standard substance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1688886A (en) * | 2002-08-13 | 2005-10-26 | 独立行政法人农业.生物系特定产业技术研究机构 | Method of detecting allergen protein |
| CN101893634A (en) * | 2009-05-20 | 2010-11-24 | 中国科学院生物物理研究所 | Specific detection method of protein or polypeptide cysteine sulfydryl modification and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1688886A (en) * | 2002-08-13 | 2005-10-26 | 独立行政法人农业.生物系特定产业技术研究机构 | Method of detecting allergen protein |
| CN101893634A (en) * | 2009-05-20 | 2010-11-24 | 中国科学院生物物理研究所 | Specific detection method of protein or polypeptide cysteine sulfydryl modification and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry;Ten-Yang Yen等;《JOURNAL OF MASS SPECTROMETRY》;20001231;第35卷;全文 * |
| Determination of Glycosylation Sites and Disulfide Bond Structures Using LC/ESI-MS/MS Analysis;TEN-YANG YEN 等;《METHODS IN ENZYMOLOGY》;20061231;第415卷;103-113 * |
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