CN113122439A - Device for extracting protein and nucleic acid by breaking wall of fungus and method for extracting protein and nucleic acid by applying device - Google Patents
Device for extracting protein and nucleic acid by breaking wall of fungus and method for extracting protein and nucleic acid by applying device Download PDFInfo
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- CN113122439A CN113122439A CN202110500265.8A CN202110500265A CN113122439A CN 113122439 A CN113122439 A CN 113122439A CN 202110500265 A CN202110500265 A CN 202110500265A CN 113122439 A CN113122439 A CN 113122439A
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 46
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 44
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 44
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 44
- 241000233866 Fungi Species 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000000227 grinding Methods 0.000 claims abstract description 42
- 239000000725 suspension Substances 0.000 claims description 10
- 210000002421 cell wall Anatomy 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 7
- 238000001819 mass spectrum Methods 0.000 abstract description 7
- 230000001580 bacterial effect Effects 0.000 description 9
- 230000002538 fungal effect Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 241000222290 Cladosporium Species 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000010297 mechanical methods and process Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000005498 polishing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241001668502 Cladophialophora carrionii Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- 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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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a device for extracting protein and nucleic acid by breaking walls of fungi and a method for extracting protein and nucleic acid by using the device. The device for extracting the protein and the nucleic acid by breaking the walls of the fungi adopts the driving device and the grinding head, so that the walls of the fungi are broken more thoroughly, the method is economical and practical, the operation is simple and convenient, the instrument is portable, the grinding head can be repeatedly sterilized under high pressure and repeatedly used, the energy is saved, the environment is protected, the cell walls of the filamentous fungi can be conveniently and quickly broken, the protein and the nucleic acid can be obtained, the purity and the yield of the protein and the nucleic acid extracted by the method are higher, and the device can be used for subsequent mass spectrum identification and nucleic acid extraction and can be popularized and used.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a device for extracting protein and nucleic acid by breaking walls of fungi and a method for extracting protein and nucleic acid by applying the device.
Background
Fungal cells are typically eukaryotic cells having a cell wall, a cell membrane, a nucleus, organelles, a cytoplasm. The cell wall is the outermost structure of the fungal cell and has the functions of maintaining the cell shape and resisting osmotic pressure, the wall thickness of the fungal cell is about 100-250nm and accounts for 30 percent of the dry weight of the cell, and the main chemical components of the fungal cell are as follows: chitin, cellulose, dextran, mannan, protein, lipid, inorganic salt, etc. The fungal cell wall breaking technology is a high-tech means, is widely applied and can be divided into a mechanical method and a non-mechanical method according to the existence of external acting force. The currently common mechanical wall breaking methods include liquid nitrogen grinding with a grinder, direct grinding with a quartz glass grinder, a magnetic bead wall breaking oscillation method, an ultrasonic crushing method, shearing force wall breaking and the like; the non-mechanical method comprises a chemical reagent method, repeated freezing and thawing by liquid nitrogen-hot water, a biological enzymolysis method and the like.
The traditional fungus wall breaking method is long in time consumption, liquid nitrogen or other chemical reagents, magnetic beads, special grinders and the like are required to be prepared in the grinding process, the limitation of laboratory conditions is large, the method is not environment-friendly, and an enzymatic method or a large-scale grinder and the like are expensive, so that the method is not suitable for clinical popularization.
Disclosure of Invention
Therefore, the invention provides a device for extracting protein and nucleic acid by breaking the wall of fungi and a method for extracting protein and nucleic acid by using the device.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention also provides a device for extracting protein and nucleic acid by breaking the wall of fungi, which comprises a grinding head and a driving device for driving the grinding head, wherein the grinding head is provided with a conical end part and a rotating shaft connected with the bottom end of the conical end part, and a plurality of convex parts are arranged on the outer side surface of the conical end part.
In one embodiment of the invention, the rotating shaft is provided with a cover plate with a circular cross section.
In one embodiment of the present invention, the driving device includes a driving motor, a power supply connected to the driving motor, and a switch controlling the power supply.
In one embodiment of the present invention, the rotation shaft of the drive motor is connected to the rotation shaft of the grinding bit through a sleeve.
In one embodiment of the present invention, the sleeve is a rubber sleeve.
In one embodiment of the invention, the tapered end is sized to fit in a 1.5ml EP tube.
The embodiment of the invention also provides a method for extracting protein and nucleic acid by using the device for extracting protein and nucleic acid by breaking the wall of fungi.
In one embodiment of the invention, the fungus is placed in an EP tube and the conical end of the abrading head is inserted into the EP tube and abraded to obtain an abraded bacterial suspension.
In one embodiment of the present invention, the rotation speed of the polishing head is 2000-3000rpm, and the polishing time is 2-3 min.
The invention has the following advantages:
the device for extracting the protein and the nucleic acid by breaking the walls of the fungi adopts the driving device and the grinding head, so that the walls of the fungi are broken more thoroughly, the method is economical and practical, the operation is simple and convenient, the instrument is portable, the grinding head can be repeatedly sterilized under high pressure and repeatedly used, the energy is saved, the environment is protected, the cell walls of the filamentous fungi can be conveniently and quickly broken, the protein and the nucleic acid can be obtained, the purity and the yield of the protein and the nucleic acid extracted by the method are higher, and the device can be used for subsequent mass spectrum identification and nucleic acid extraction and can be popularized and used.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a schematic structural diagram of an apparatus for extracting proteins and nucleic acids by breaking walls of fungi, provided by an embodiment of the invention;
fig. 2 is a schematic structural diagram of a driving device according to an embodiment of the present invention;
FIG. 3 is a schematic view of a polishing head according to an embodiment of the present invention;
fig. 4 is a comparison chart of before and after extracting Cladosporium carisonii from the apparatus for extracting proteins and nucleic acids by breaking the walls of fungi according to the embodiment of the present invention, wherein A is the effect before grinding, the fungus thallus is complete, the thallus and solute are clearly defined, and the thallus and solute cannot be dissolved by themselves; b is the effect after grinding, the liquid which is visible to naked eyes is turbid, and the liquid has no large block or granular shape;
FIG. 5 is a comparison chart of the extraction of Cladosporium carisonii from the apparatus for extracting proteins and nucleic acids by breaking the wall of fungi provided by the embodiment of the invention, wherein A is a protein peak diagram of mass spectrum before grinding, and has disordered baseline, no characteristic peak and no result; and B is a protein peak image of the ground mass spectrum, the baseline is stable, the characteristic peak is obvious, the result can indicate the seed level, and the requirement of strain identification can be met.
In the figure: 100-grinding head; 110-a projection; 120-a cover plate; 130-a rotating shaft; 200-a drive device; 210-a rotation axis; 220-a cannula; 230-a power supply; 240-switch; 250-driving the motor.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in FIGS. 1 to 3, the present invention provides a device for extracting protein and nucleic acid by breaking the wall of fungus, the device comprises a grinding head 100 and a driving device 200 for driving the grinding head 100, wherein the grinding head 100 has a conical end and a rotating shaft 130 connected with the bottom end of the conical end, a plurality of protrusions 110 are arranged on the outer side surface of the conical end, the protrusions 110 are uniformly arranged on the outer surface of the conical end, and when rotating, the protrusions 110 can generate friction action with the inner side wall of an EP tube, so that the cell walls of the fungus can be better broken.
The driving device 200 includes a driving motor 250, a power supply 230 connected to the driving motor 250, and a switch 240 for controlling the power supply 230, wherein the power supply 230 adopts a storage battery or an external power supply 230 with a USB interface, and when in use, the switch 240 is directly turned on or the external power supply 230 is turned on. The rotating shaft 210 of the driving motor 250 is connected with the rotating shaft 130 of the grinding bit 100 through a sleeve 220, the sleeve 220 can be a rubber sleeve 220, the rotating shaft 210 and the rotating shaft 130 are in interference fit, and when the device is not used, the sleeve 220, the rotating shaft 130 and the rotating shaft 210 can be detached.
The size of the cone-shaped end part is matched with that of a 1.5ml EP tube, when in use, the cone-shaped end part can be directly inserted into the EP tube, and due to the small gap between the outer surface of the cone-shaped end part and the inner side surface of the EP tube, after the thalli are added, the walls of the thalli are broken under the action of friction force between the outer surface of the cone-shaped end part and the inner side surface of the EP tube. The rotating shaft 130 can generate larger shearing force to the conical end during the high-speed rotation, and the crushing effect on the fungal cell wall is better.
Preferably, the rotating shaft 130 is provided with a cover plate 120 having a circular cross section, and the diameter of the cover plate 120 is equal to the inner diameter of the EP tube, so that the cover plate can prevent fungal liquid from splashing in the EP tube to a certain extent when in use.
Example 2
The present invention also provides a method for extracting proteins and nucleic acids using the apparatus for fungal wall-broken extraction of proteins and nucleic acids of example 1, which comprises: putting the fungus into an EP tube added with sterile water in advance, adding an absolute ethyl alcohol solution after the fungus is added into the EP tube, centrifuging to remove supernatant, adding 40 mu l of 70% methanol solution, inserting the conical end part of a grinding head 100 into the EP tube for grinding, wherein the rotating speed of the grinding head 100 is 2000rpm-3000rmp, and the grinding time is 2-3 min. Obtaining the grinding bacterial suspension. And standing the ground bacterial suspension for 5min, adding 40 mu l of acetonitrile, uniformly mixing to obtain bacterial suspension for identification, and adding 1 mu l of bacterial suspension into a target plate for subsequent identification of a time-of-flight mass spectrometer.
Example 3
The invention also provides a method for extracting protein and nucleic acid by using the device for extracting protein and nucleic acid by breaking the wall of fungus in example 1, which comprises the following steps: the fungus is placed in an EP tube, 400 mu l of sterile water is added after the fungus is added into the EP tube, then the conical end part of the grinding head 100 is inserted into the EP tube for grinding, the rotating speed of the grinding head 100 is 2000-3000rpm, and the grinding time is 2-3 min. Obtaining the grinding bacterial suspension. And standing the ground bacterial suspension for 5min, adding 40 mu l of acetonitrile, uniformly mixing to obtain bacterial suspension for identification, and adding 1 mu l of bacterial suspension into a target plate for subsequent identification of a time-of-flight mass spectrometer.
Test examples
By using the device for extracting proteins and nucleic acids by breaking the walls of fungi in example 1 and according to the extraction method in example 2, the Cladosporium carbolonii (Cladosporium carrionii) is extracted, as shown in FIGS. 4 and 5, and FIG. 4 is a front and back comparison graph of the Cladosporium carbolonii (Cladosporium carrionii) extracted by the device for extracting proteins and nucleic acids by breaking the walls of fungi in the example of the invention, wherein A is the effect before grinding, the fungi thallus is complete, the thallus and solute are clearly defined and cannot be dissolved by themselves; b is the effect after grinding, the liquid is turbid and has no large block or granular shape.
FIG. 5 is a comparison chart of the extraction of Cladosporium carisonii from the apparatus for extracting proteins and nucleic acids by breaking the wall of fungi provided by the embodiment of the invention, wherein A is a protein peak diagram of mass spectrum before grinding, and has disordered baseline, no characteristic peak and no result; and B is a protein peak image of the ground mass spectrum, the baseline is stable, the characteristic peak is obvious, the result can indicate the seed level, and the requirement of strain identification can be met.
The method for extracting the protein and the nucleic acid by using the device for extracting the protein and the nucleic acid by breaking the walls of the fungi has the advantages that the driving device drives the grinding head to mechanically grind and break the walls, 1.5EP tubes commonly used in clinical laboratories are used in a matching manner, filamentous fungi are cultured, thalli are collected, mechanical grinding is carried out, the cell walls of the fungi can be conveniently and quickly broken, and the obtained protein and nucleic acid are used for mass spectrum identification or nucleic acid extraction and are suitable for identification of clinical conventional flight mass spectrometers and molecular biology research.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. The device for extracting protein and nucleic acid by breaking the wall of fungi comprises a grinding head and a driving device for driving the grinding head, wherein the grinding head is provided with a conical end and a rotating shaft connected with the bottom end of the conical end, and a plurality of bulges are arranged on the outer side surface of the conical end.
2. The device for extracting protein and nucleic acid by fungus wall breaking as claimed in claim 1,
and a cover plate with a circular cross section is arranged on the rotating shaft.
3. The device for extracting protein and nucleic acid by fungus wall breaking as claimed in claim 1,
the driving device comprises a driving motor, a power supply connected with the driving motor and a switch for controlling the power supply.
4. The device for extracting protein and nucleic acid by fungus wall breaking as claimed in claim 3,
the rotating shaft of the driving motor is connected with the rotating shaft of the grinding head through a sleeve.
5. The device for extracting protein and nucleic acid by fungus wall breaking as claimed in claim 4,
the sleeve is a rubber sleeve.
6. The device for extracting protein and nucleic acid by fungus wall breaking as claimed in claim 1,
the size of the tapered end was compatible with a 1.5ml EP tube.
7. A method for extracting protein and nucleic acid by using the device for extracting protein and nucleic acid by fungus wall breaking as claimed in any one of claims 1 to 6.
8. The method of claim 7,
and (3) placing the fungus into an EP tube, and inserting the conical end part of the grinding head into the EP tube for grinding to obtain ground fungus suspension.
9. The method of claim 8,
the rotation speed of the grinding head is 2000rpm, and the grinding time is 2-3 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110500265.8A CN113122439A (en) | 2021-05-08 | 2021-05-08 | Device for extracting protein and nucleic acid by breaking wall of fungus and method for extracting protein and nucleic acid by applying device |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110500265.8A CN113122439A (en) | 2021-05-08 | 2021-05-08 | Device for extracting protein and nucleic acid by breaking wall of fungus and method for extracting protein and nucleic acid by applying device |
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| Publication Number | Publication Date |
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| CN113122439A true CN113122439A (en) | 2021-07-16 |
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| CN202110500265.8A Pending CN113122439A (en) | 2021-05-08 | 2021-05-08 | Device for extracting protein and nucleic acid by breaking wall of fungus and method for extracting protein and nucleic acid by applying device |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988062A (en) * | 2017-12-28 | 2018-05-04 | 广州聚能纳米生物科技股份有限公司 | A kind of cell breaking plant explosion-protection equipment |
| CN108102882A (en) * | 2018-02-06 | 2018-06-01 | 江苏阿拉丁环保科技有限公司 | Microbial cell wall-breaking machine |
| CN109182105A (en) * | 2018-09-27 | 2019-01-11 | 瑞智康生物技术（昆山）有限公司 | A kind of rotation detent hand held tissue dismembyator |
| CN111366434A (en) * | 2013-03-15 | 2020-07-03 | 赛拉诺斯知识产权有限责任公司 | Automated assay device for disrupting cells in a sample |
-
2021
- 2021-05-08 CN CN202110500265.8A patent/CN113122439A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111366434A (en) * | 2013-03-15 | 2020-07-03 | 赛拉诺斯知识产权有限责任公司 | Automated assay device for disrupting cells in a sample |
| CN107988062A (en) * | 2017-12-28 | 2018-05-04 | 广州聚能纳米生物科技股份有限公司 | A kind of cell breaking plant explosion-protection equipment |
| CN108102882A (en) * | 2018-02-06 | 2018-06-01 | 江苏阿拉丁环保科技有限公司 | Microbial cell wall-breaking machine |
| CN109182105A (en) * | 2018-09-27 | 2019-01-11 | 瑞智康生物技术（昆山）有限公司 | A kind of rotation detent hand held tissue dismembyator |
Non-Patent Citations (1)
| Title |
|---|
| ""电动组织匀浆器"", Retrieved from the Internet <URL:https://mp.weixin.qq.com/s/5oSdy_Kn_XUCIJ2e3ybs0g> * |
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