CN110845569A - Method for extracting total protein from echinococcus granulosus - Google Patents
Method for extracting total protein from echinococcus granulosus Download PDFInfo
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- CN110845569A CN110845569A CN201911121096.6A CN201911121096A CN110845569A CN 110845569 A CN110845569 A CN 110845569A CN 201911121096 A CN201911121096 A CN 201911121096A CN 110845569 A CN110845569 A CN 110845569A
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- echinococcus granulosus
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- 201000003808 Cystic echinococcosis Diseases 0.000 title claims abstract description 68
- 241000244170 Echinococcus granulosus Species 0.000 title claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 31
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000006166 lysate Substances 0.000 claims abstract description 33
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000005119 centrifugation Methods 0.000 claims abstract description 14
- 230000009089 cytolysis Effects 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 claims abstract description 8
- 238000010814 radioimmunoprecipitation assay Methods 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 5
- 206010014096 Echinococciasis Diseases 0.000 description 8
- 208000009366 Echinococcosis Diseases 0.000 description 8
- 238000005336 cracking Methods 0.000 description 4
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000000751 protein extraction Methods 0.000 description 3
- 241000244160 Echinococcus Species 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 206010048282 zoonosis Diseases 0.000 description 2
- 241000282421 Canidae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting total protein from echinococcus granulosus, which comprises the following steps: (1) according to RIPA: preparing a lysate with PMSF being 100: 1; (2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2ml centrifuge tube, transferring the echinococcus granulosus into the centrifuge tube for centrifugation, discarding the supernatant after centrifugation, absorbing PBS after centrifugation, adding lysate into each group, placing the echinococcus granulosus into a refrigerator for lysis, slightly shaking the echinococcus granulosus in an oscillator, resetting the echinococcus granulosus in the refrigerator for lysis, shaking the echinococcus granulosus for 1 time every 5min, and crushing the echinococcus granulosus in a cell ultrasonic crusher; (3) centrifuging by a low-temperature high-speed centrifuge; (4) and (4) sucking the supernatant into another precooled marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction. The method can extract protein with higher concentration from the same quantity of echinococcus granulosus, and provides guarantee for further experiments.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for extracting total protein from echinococcus granulosus.
Background
Echinococcosis is a common zoonosis disease, which not only brings harm to public health but also affects economic development. Dogs and foxes are the final hosts of infection, and dogs ingest vesicles containing echinococcus protocoid larvae, and humans become infected by contact with dogs or by eating water sources and vegetables containing eggs. Echinococcus granulosus causes damage to single or multiple organs, including primarily the liver (70%), lungs (20%), and perhaps 10% of the parasitic organs, in any part of the body (such as the brain and muscle tissue). Echinococcosis is geographically widespread and mainly concentrated in parts of the continental europe, including the mediterranean region, russia, central asia, the midwest region of china, australia, the united states (particularly the south), and the northeast of africa.
Echinococcosis (also known as echinococcosis) is a zoonosis caused by the parasitism of echinococcus middle-taenii larvae (echinococcosis) in intermediate hosts such as humans, amphibians and rodents. Echinococcosis presents global distribution, the threatened population in only middle and sub-regions reaches 2.7 hundred million, nearly 300 million global patients are estimated, and 20 million new cases are estimated every year. The harm caused by the disease is serious, and the treatment cost of the echinococcosis and the economic loss of the animal husbandry are up to $ 30 hundred million each year. In China, echinococcosis is wide in epidemic range, and the number of threatened population and patients is huge.
Echinococcus granulosus is currently an important research subject for researchers, and extraction of total protein in echinococcus granulosus is an essential experimental step for the experimenters, but the methods are different, and the technical scheme in the prior art includes: taking the echinococcus granulosus treated in the experiment, washing the echinococcus granulosus with PBS buffer solution for 3 times, discarding the supernatant, and performing the following steps according to the weight of 3mg of the original joint: adding lysate into protein extract at a ratio of 150 μ L, pulverizing with cell ultrasonic pulverizer, standing for lysis for 15min, centrifuging at 4 deg.C and 1000rp for 15min, standing for 20min, and collecting supernatant. This prior art has the following drawbacks: standing, cracking is not sufficient, and the concentration of extracted protein is low. Often the protein concentration extracted is so low that subsequent experiments are unsuccessful. Therefore, mastering the right protein extraction procedure can save a lot of time for the experimenter.
Disclosure of Invention
The invention aims to provide a method for extracting total protein from echinococcus granulosus. The method can fully crack echinococcus granulosus, improve protein extraction concentration, and provide guarantee for subsequent experiments.
The specific technical scheme is as follows:
a method for extracting total protein from echinococcus granulosus, comprising the steps of:
(1) preparing a lysate RIPA lysate to be placed at room temperature, taking PMSF out of a refrigerator at the temperature of-20 ℃, immediately placing on ice after complete dissolution, and performing RIPA: preparing a lysate with PMSF being 100: 1;
(2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2mL centrifuge tube, moving the echinococcus granulosus into the centrifuge tube, centrifuging the echinococcus granulosus for 3min at the temperature of 4 ℃ at 800rpm-1000rpm, discarding the supernatant after centrifugation, resuspending and washing the echinococcus granulosus for 3 times by 1 XPBS, absorbing each PBS group after centrifugation, adding 0.2-0.4mL of lysate, placing the PBS group in a refrigerator for lysis for 5min-10min at the temperature of 4 ℃, slightly shaking the PBS group in an oscillator after 5min-10min, resetting the PBS group in the refrigerator for lysis at the temperature of 4 ℃, shaking the PBS group for 1 time every 5min, after 4 times of shaking, crushing the echinococcus granulosus in a cell ultrasonic crusher, precooling the echinococcus granulosus, taking ice blocks for soaking, avoiding a variable;
(3) centrifuging with a low-temperature high-speed centrifuge at 4 deg.C and 13000rpm for 15 min;
(4) and (5) sucking the supernatant into another cold-marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction.
Further, in the step (2), the centrifugal speed of the centrifuge is 800rpm, 0.3mL of lysate is added to each group after centrifugation, the mixture is placed in a refrigerator at 4 ℃ for lysis for 5min, and after 5min, the mixture is slightly shaken by an oscillator and then reset in the refrigerator at 4 ℃ for lysis.
Further, in the step (2), the ultrasonic condition is as follows: amplitude transformer
Time of day1s, 2s apart.
Compared with the prior art, the invention has the beneficial effects that:
there is no normalization step for echinococcus granulosus protein extraction, which results in too low concentration of extracted protein according to the method for extracting protein from tissue and the method for extracting protein from cell due to the special structure of echinococcus granulosus. The method can extract protein with higher concentration from the same quantity of echinococcus granulosus, and provides guarantee for further experiments.
Drawings
FIG. 1 is a schematic flow diagram of the process for extracting total protein from echinococcus granulosus.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.
Example 1
Referring to fig. 1, a method for extracting total protein from echinococcus granulosus, comprising the steps of:
(1) preparing a lysate RIPA lysate, placing the lysate at room temperature, taking PMSF out of a refrigerator at the temperature of-20 ℃, immediately placing the PMSF on ice after completely dissolving (no ice crystal appears in a bottle after light is cured), and carrying out the steps of: preparing a lysate with PMSF being 100: 1;
(2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2ml centrifuge tube, transferring the echinococcus granulosus into the centrifuge tube, centrifuging the echinococcus granulosus completely to the bottom of the centrifuge tube at the temperature of 4 ℃ at 800rpm, centrifuging the echinococcus granulosus at the lowest speed of 800rpm for 3min, centrifuging the supernatant, re-suspending, washing and centrifuging the echinococcus granulosus for 3 times by 1 XPBS, absorbing each PBS group after centrifuging, adding 0.2 lysate, easily and insufficiently cracking the protein under the condition of less than 0.2ml, placing the echinococcus granulosus into a 4 ℃ refrigerator for cracking for 5min, slightly vibrating the echinococcus granulosus in an oscillator after 5min, resetting the echinococcus granulosus in the 4 ℃ refrigerator for cracking, vibrating the echinococcus granulosus for 1 time every 5min, vibrating the echinococcus granulosus for 4 times, crushing the echinococcus granulosus in an ultrasonic cell crusher, precooling the ultrasonic;
(3) centrifuging with a low-temperature high-speed centrifuge at 4 deg.C and 13000rpm for 15 min;
(4) and (4) sucking the supernatant into another precooled marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction.
In the step (2), ultrasonic conditions are as follows: amplitude transformer
Time is 1s and interval is 2 s.
Example 2
Referring to fig. 1, a method for extracting total protein from echinococcus granulosus, comprising the steps of:
(1) preparing a lysate RIPA lysate, placing the lysate at room temperature, taking PMSF out of a refrigerator at the temperature of-20 ℃, immediately placing the PMSF on ice after completely dissolving (no ice crystal appears in a bottle after light is cured), and carrying out the steps of: preparing a lysate with PMSF being 100: 1;
(2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2mL centrifuge tube, moving the echinococcus granulosus into the centrifuge tube, centrifuging the echinococcus granulosus for 3min at 900rpm at 4 ℃, discarding supernatant after centrifugation, resuspending and washing the echinococcus granulosus for 3 times by 1 XPBS, absorbing PBS each group after centrifugation, adding 0,3mL of lysate, placing the lysate in a refrigerator at 4 ℃ for lysis for 8min, slightly shaking the lysate in an oscillator after 8min, resetting the lysate in the refrigerator at 4 ℃, shaking the lysate for lysis at 4 ℃, shaking the lysate for 1 time every 5min, after shaking for 4 times, crushing the cell in an ultrasonic crusher, precooling the cell in advance by the ultrasonic crusher, taking ice blocks for soaking, and avoiding an amplitude transformer from touching the tube wall of the centrifuge tube, and placing the centrifuge;
(3) centrifuging with a low-temperature high-speed centrifuge at 4 deg.C and 13000rpm for 15 min;
(4) and (5) sucking the supernatant into another cold-marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction.
In the step (2), ultrasonic conditions are as follows: amplitude transformer
Time is 1s and interval is 2 s.
Example 3
Referring to fig. 1, a method for extracting total protein from echinococcus granulosus, comprising the steps of:
(1) preparing a lysate RIPA lysate, placing the lysate at room temperature, taking PMSF out of a refrigerator at the temperature of-20 ℃, immediately placing the PMSF on ice after completely dissolving (no ice crystal appears in a bottle after light is cured), and carrying out the steps of: preparing a lysate with PMSF being 100: 1;
(2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2mL centrifuge tube, transferring the echinococcus granulosus into the centrifuge tube, centrifuging at 81000rpm at 4 ℃ and below 1000rpm to reduce the damage of the centrifugation to the echinococcus granulosus, centrifuging for 3min by using a centrifuge, discarding supernatant after centrifugation, resuspending and washing by 1 XPBS for 3 times, absorbing PBS each group after centrifugation, adding 0.4mL of lysate, generally reducing the protein concentration if the protein concentration is higher than 0.4mL, placing the echinococcus granulosus in a refrigerator at 4 ℃ for lysis for 10min, slightly shaking by using an oscillator after 10min, resetting the echinococcus granulosus in the refrigerator at 4 ℃ for lysis, shaking for 1 time every 5min, after shaking for 4 times, crushing by using a cell ultrasonic crusher, precooling the cell ultrasonic crusher, taking ice blocks for soaking, taking care to avoid an amplitude transformer rod from touching the tube wall of the centrifuge tube, and placing the centrifuge tube into;
(3) centrifuging with a low-temperature high-speed centrifuge at 4 deg.C and 13000rpm for 15 min;
(4) and (5) sucking the supernatant into another cold-marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction.
In the step (2), ultrasonic conditions are as follows: amplitude transformer
Time is 1s and interval is 2 s.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (3)
1. A method for extracting total protein from echinococcus granulosus, comprising the steps of:
(1) preparing a lysate RIPA lysate to be placed at room temperature, taking PMSF out of a refrigerator at the temperature of-20 ℃, immediately placing on ice after complete dissolution, and performing RIPA: preparing a lysate with PMSF being 100: 1;
(2) taking the echinococcus granulosus out of the incubator, placing the echinococcus granulosus on ice, taking a precooled 2mL centrifuge tube, moving the echinococcus granulosus into the centrifuge tube, centrifuging the echinococcus granulosus for 3min at the temperature of 4 ℃ at 800rpm-1000rpm, discarding the supernatant after centrifugation, resuspending and washing the echinococcus granulosus for 3 times by 1 XPBS, absorbing each PBS group after centrifugation, adding 0.2-0.4mL of lysate, placing the PBS group in a refrigerator for lysis for 5min-10min at the temperature of 4 ℃, slightly shaking the PBS group in an oscillator after 5min-10min, resetting the PBS group in the refrigerator for lysis at the temperature of 4 ℃, shaking the PBS group for 1 time every 5min, after 4 times of shaking, crushing the echinococcus granulosus in a cell ultrasonic crusher, precooling the echinococcus granulosus, taking ice blocks for soaking, avoiding a variable;
(3) centrifuging with a low-temperature high-speed centrifuge at 4 deg.C and 13000rpm for 15 min;
(4) and (4) sucking the supernatant into another precooled marked 2mL centrifugal tube to obtain the total cell protein obtained by extraction.
2. The method for extracting total protein from echinococcus granulosus according to claim 1, wherein in step (2), the centrifuge speed is 800rpm, 0.3mL of lysate is added to each group after centrifugation, the mixture is placed in a refrigerator at 4 ℃ for lysis for 5min, and the refrigerator is reset to 4 ℃ after 5min of gentle shaking by a shaker.
3. The method for extracting total protein from echinococcus granulosus according to claim 1, wherein in step (2), the ultrasound conditions are: amplitude transformer
Time is 1s and interval is 2 s.
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| CN201911121096.6A CN110845569A (en) | 2019-11-15 | 2019-11-15 | Method for extracting total protein from echinococcus granulosus |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992001051A1 (en) * | 1990-07-12 | 1992-01-23 | Institut Pasteur | Immunogenic peptide sequence from echinococcus granulosus, dna sequence coding for said peptide sequence, and diagnostic and therapeutic uses thereof |
| CN101948521A (en) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof |
| CN109613252A (en) * | 2018-10-22 | 2019-04-12 | 新疆医科大学第附属医院 | Echinococcus granulosus high immunity yolk antibody and test strips and preparation method and application |
-
2019
- 2019-11-15 CN CN201911121096.6A patent/CN110845569A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992001051A1 (en) * | 1990-07-12 | 1992-01-23 | Institut Pasteur | Immunogenic peptide sequence from echinococcus granulosus, dna sequence coding for said peptide sequence, and diagnostic and therapeutic uses thereof |
| CN101948521A (en) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof |
| CN109613252A (en) * | 2018-10-22 | 2019-04-12 | 新疆医科大学第附属医院 | Echinococcus granulosus high immunity yolk antibody and test strips and preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 张耀刚等: "细粒棘球蚴和多房泡球蚴在人体内蛋白质表达谱初步分析", 《中国人兽共患病学报》 * |
| 曹得萍等: "细粒棘球绦虫转酮醇酶克隆表达及免疫性分析", 《中国血吸虫病防治杂志》 * |
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Application publication date: 20200228 |