CN1284796C - Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment - Google Patents
Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment Download PDFInfo
- Publication number
- CN1284796C CN1284796C CN 200310121954 CN200310121954A CN1284796C CN 1284796 C CN1284796 C CN 1284796C CN 200310121954 CN200310121954 CN 200310121954 CN 200310121954 A CN200310121954 A CN 200310121954A CN 1284796 C CN1284796 C CN 1284796C
- Authority
- CN
- China
- Prior art keywords
- protein
- antifreeze protein
- alpine
- ice crystal
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a method for the tissue culture of an alpine periglacial plant and the production of cold induction secretory protein. Leaves of an alpine periglacial plant, which are refrigerated by liquid nitrogen, are ground, and the powder is mixed evenly with a phosphate buffer; after centrifuged with 12000g for a period of time, the powder becomes coarse extract of soluble protein; an ice crystal adsorber is utilized to enrich antifreeze protein twice; the enriched protein is desalted through a dialysis bag and is concentrated; an ice crystal inhibiting observing device is utilized to carry out recrystallization activity detection on the quantified protein. The present invention utilizes the characteristic that the alpine periglacial plant has the capability to resist repeated freeze thawing and adopts the ice crystal adsorber and a recrystallization inhibiting device to separate and detect the antifreeze protein of the alpine periglacial plant. The method which has quickness, effectiveness, simplicity, easiness and low cost is a short-cut for obtaining antifreeze protein from coarse extract of soluble protein of an alpine periglacial plant.
Description
Technical field
The invention belongs to protein engineering, relate to the method and the device thereof of initial gross separation of a kind of alpine periglacial plant antifreeze protein and detection.
Background technology
Antifreeze protein is found in the fish of the South Pole, be the special albumen that a class can suppress ice-crystal growth, can suppress recrystallization process and reduce freezing point of solution, from fish, insect, fungi and plant, find antifreeze protein so far, but homology is very low between these albumen, prove that this is the new albumen of evolving of a class, so the preferred plan of seeking is that the characteristic according to antifreeze protein is separated, detected from the species of low temperature environment.The freeze proof activity of plant antifreeze protein inhibition recrystallization is better than the antifreeze protein in fish or other source in rye, rye grass, the Radix Dauci Sativae; and the freeze proof main mechanism of more and more data proof plant is to suppress recrystallization; the membrane structure of protective plant; so existing technology concentrates on the cold hardy plant of inducing mostly, extract apoplast albumen and carry out cryoscopy and suppress recrystallization mensuration.But, be difficult to from specific several plant, find, and the difficult grasp of traditional apoplast extractive technique owing to the not ubiquity that antifreeze protein distributes.Cold cycle instrument temperature control and inversion phase contrast microscopy are had relatively high expectations to instrument, and common laboratory is difficult to accomplish.So set up simple high efficiency separation and detection system, in the low-temperature adaptation plant, screen on a large scale and be only preferred plan.
Summary of the invention
Purpose of the present invention is intended to can be adsorbed on according to antifreeze protein the characteristic of ice crystal surface, sets up initial gross separation of alpine periglacial plant antifreeze protein and detection method.
The ice crystal adsorption unit that the another object of the present invention utilization designs voluntarily adopts and freezes the concentration and separation that probe carries out antifreeze protein, and detects freeze proof activity with simple recrystallization observation device.
Purpose of the present invention can realize by following measure: 1, the alpine periglacial plant of liquid nitrogen cryopreservation is carried out utilizing ice crystal adsorption unit of the present invention to carry out the enrichment of twice antifreeze protein after total soluble protein extracts; 2, the albumen after the enrichment is passed through the dialysis tubing desalination, and utilize PEG8000 to concentrate, quantitative with Coomassie brilliant blue G250.Detailed content is as follows:
(1) the antifreeze protein ice crystal adsorption system of utilizing the present invention to set up is carried out the initial gross separation of alpine periglacial plant antifreeze protein.At first the alpine periglacial plant blade that liquid nitrogen is preserved grinds, and powder is with pH8.0 phosphoric acid buffer mixing, and centrifugal 10 minutes of 12000g, gets supernatant, and this is the soluble proteins crude extract, and it is stand-by that 45 milliliters of crude extracts are put into 4 ℃ of preservations of 50 milliliters of small beakers.Be connected with the outlet of elevated pressure nitrogen gas tank with 10 meters copper conduit one ends, the about 8 meters windings in conduit middle part are immersed in the liquid nitrogen container in the liquid nitrogen, and the conduit the other end is derived outside the liquid nitrogen container for about 1 meter, less than 50 centimetres of distant places conduit are turned back 180 ° going out liquid nitrogen container, and with curved 90 ° down of two-wires, as freezing probe.High pressure nitrogen carries out precooling by pressure loading valve along the conduit liquid nitrogen of flowing through, and the probe of flowing through is discharged in the air, and this probe immerses 50 milliliters of small beakers that contain protein crude extract and freezes absorption, in put rotor and carry out magnetic agitation and keep solution even.Freeze probe when liquid level in the beaker descends to taking out after 5 milliliters, the room temperature melted ice crystal is added phosphoric acid buffer to 45 milliliter, repeats to freeze absorption once with popping one's head in.
(2) with the ice crystal room temperature of freezing for the second time, utilize the desalination 12 hours in deionized water of the amount of damming 5000 daltonian dialysis tubings, changed water every 2 hours, dialysis tubing is imbedded in the powder of PEG8000 and is concentrated after the desalination.Protein solution after concentrating is done typical curve with Coomassie brilliant blue G250 colorimetric method for determining concentration with BSA.
Alpine periglacial plant antifreeze protein: 1, utilize inhibition recrystallization recording geometry of the present invention that albumen after quantitative is carried out the active detection of recrystallization.2, freeze proof active component vacuum-freeze-dry will be arranged.Detailed content is as follows:
(1) the inhibition recrystallization recording geometry of utilizing the present invention to set up is carried out the active detection of recrystallization to the albumen after quantitative.The temperature controllable refrigerator-freezer removes top cover, and interior placement Olympus BH/2 opticmicroscope removes microscope overhead camera, cover refrigerator-freezer with plastic foam plate, and borehole exposes the microphotograph eyepiece, utilizes photo eye-piece to mix up field luminance, focal length in advance, and the refrigerator-freezer temperature is made as centigradetemperature-5 ℃.Albumen quantitatively is diluted to 1mg/L and 5mg/L concentration with 20% sucrose solution, is added drop-wise to a slide glass, cover a cover glass and be pressed into even liquid film.After film-making being put into-70 ℃ of cryogenic refrigerators and freezing in 30 minutes, rapidly film-making is put into microscope stage by the plastic foam plate operation entry, by on cover glass, adding a melted paraxylene in the observation of operational observations mouth, 100 times of oily sem observations, adjust the visual field, recrystallization is put the photographic camera photographic recording after 15 minutes
(2) activated alpine periglacial plant antifreeze protein crude extract adopts the vacuum-freeze-dry machine to carry out putting into after the freeze-drying-70 ℃ of refrigerators preservations.
The present invention has following advantage compared to existing technology:
1. the existing technology of antifreeze protein concentrates on the cold hardy plant of inducing mostly, extracts apoplast albumen and carries out the antifreeze protein separation.Because the not ubiquity that antifreeze protein distributes is difficult to find target protein from specific several plant.Native system utilizes the many species of alpine periglacial plant to have the characteristics of the ability of low anti-multigelation, directly seeks from alpine periglacial plant soluble proteins crude extract, has improved to obtain the antifreeze protein possibility.
2. the isolating technology of traditional apoplast extractive technique and column chromatography system is difficult to be grasped, and the ice crystal adsorption system that the present invention sets up has been utilized the distinctive character of antifreeze protein, and common laboratory realizes easily, is a kind of separation means fast and effectively.
3. the method for traditional inhibition recrystallization adopts cold cycle instrument temperature control and is inverted phase contrast microscopy, and instrument is had relatively high expectations, and common laboratory is difficult to accomplish.The inhibition recrystallization observation device that the present invention sets up requires low to instrument, and more stable than cold cycle instrument temperature control, and object lens can be with 100 times, much larger than being inverted 40 times of micro-endoscope objective lenses.
Description of drawings
Fig. 1 is an antifreeze protein ice crystal adsorption unit diagrammatic cross-section of the present invention
Fig. 2 suppresses recrystallization observation device diagrammatic cross-section for the present invention
Fig. 3 is a plastic foam plate lid synoptic diagram of the present invention
Fig. 4 observes the recrystallization process synoptic diagram of sucrose solution for the present invention
Fig. 5 be high mountain ion mustard in the right side for be the high mountain annual bluegrass in the left side of the present invention, and the lower-left is a serpentgrass, the bottom right be Radix Rhodiolae and upper left be the sucrose blank, the upper right 1mg/LBSA of being contrasts synoptic diagram
Embodiment
The present invention also will be described in further detail in conjunction with the accompanying drawings: at first alpine periglacial plant blade Radix Rhodiolae, high mountain annual bluegrass, high mountain ion mustard, the strain bud knotweed that liquid nitrogen is preserved utilizes liquid nitrogen to grind, the powder that grinding obtains pH8.0 phosphoric acid buffer mixing, and centrifugal 10 minutes of 12000g, get supernatant, this is the soluble proteins crude extract, and it is stand-by 4 ℃ of preservations that 45 milliliters of crude extracts are put into 50 milliliters of small beakers 5.With high pressure nitrogen by pressure loading valve 2 with 0.1 MPa pressure along conduit 3 liquid nitrogen 7 precoolings of popping one's head in of flowing through, probe 7 immerses 50 milliliters of small beakers 5 that contain protein crude extract and freezes absorption, in put rotor and carry out magnetic agitation and keep solution evenly (referring to Fig. 1).Freeze probe 7 when liquid level in the beaker descends to taking out after 5 milliliters, the room temperature melted ice crystal is added phosphoric acid buffer to 45 milliliter, repeats to freeze absorption once with popping one's head in.The ice crystal room temperature of freezing is for the second time melted, and utilizes the desalination 12 hours in deionized water of the amount of damming 5000 daltonian dialysis tubings, changes water every 2 hours, and dialysis tubing is imbedded in the powder of PEG8000 and concentrated after the desalination.Protein solution after concentrating is done typical curve with Coomassie brilliant blue G250 colorimetric method for determining concentration with BSA.Albumen quantitatively is diluted to 1mg/L and 5mg/L concentration with 20% sucrose solution, is added drop-wise to a slide glass, cover a cover glass and be pressed into even liquid film.After film-making being put into-70 ℃ of cryogenic refrigerators and freezing in 8 30 minutes, rapidly film-making 11 is put into microscope 9 Stage microscopes by plastic foam plate 13 operation entries, by on cover glass, adding a melted paraxylene (referring to Fig. 2,4) in the observation of photo eye-piece 10 operational observations mouths, 100 times of oily sem observations, adjust the visual field, recrystallization is put the photographic camera photographic recording after 15 minutes, find that high mountain ion mustard and strain bud knotweed have the activity (referring to Fig. 5) that suppresses recrystallization.
Observation finishes, and lays down overhead camera 12, covers handle hole lid 17 and vision slit lid 18.
Claims (5)
1, a kind of alpine periglacial plant antifreeze protein separates and the method that detects, and comprises the steps:
(1) the alpine periglacial plant blade with liquid nitrogen cryopreservation grinds, and powder phosphoric acid buffer mixing, and the centrifugal 10-15 of 12000g minute is the crude extract of soluble proteins;
(2) 45 milliliters of crude extracts are put into 50 milliliters small beaker (5) 4 ℃ of preservations, utilized the ice crystal adsorption unit to carry out the enrichment of twice antifreeze protein;
(3) with the ice crystal room temperature of freezing for the second time, the utilization amount of damming 5000 daltonian dialysis tubings were deionized water desalination 12 hours, changed water every two hours, dialysis tubing is imbedded in the powder of PEG8000 and is concentrated after the desalination, and the protein solution after concentrating is with Coomassie brilliant blue G250 colorimetric method for determining concentration;
(4) utilize inhibition recrystallization recording geometry that the albumen after quantitative is carried out the active detection of recrystallization; Albumen quantitatively is diluted to 1mg/L and 5mg/L concentration with 20% sucrose solution, be added drop-wise to a slide glass, cover a cover glass and be pressed into even liquid film, with film-making (11) put into subzero 70 ℃ of low temperature refrigerators (8) freezing after, film-making (11) is put on microscope (9) Stage microscope rapidly, add a melted paraxylene on the cover glass, the activity of carrying out antifreeze protein detects.
2, alpine periglacial plant antifreeze protein according to claim 1 separates and the method that detects, it is characterized in that described antifreeze protein ice crystal adsorption unit carries out the enrichment of twice antifreeze protein, be probe (7) to be immersed the 50 milliliters of small beakers (5) that contain protein crude extract adsorb, the interior liquid level of beaker (5) descended take out freezing probe (7) after 5 milliliters, the room temperature melted ice crystal, add phosphoric acid buffer to 45 milliliter, repeat once with probe (7) freezing absorption.
3, alpine periglacial plant antifreeze protein according to claim 1 separates and the method that detects, and it is characterized in that the alpine periglacial plant blade is Radix Rhodiolae, high mountain annual bluegrass, high mountain ion mustard, strain bud knotweed.
4, a kind of ice crystal adsorption unit of the method that is used for the described alpine periglacial plant antifreeze protein separation of claim 1 and detects, it is characterized in that copper conduit (3) one ends are connected with the outlet of elevated pressure nitrogen gas tank, conduit (3) is by pressure loading valve (2), its middle part is twined and is immersed in the interior liquid nitrogen (4) of liquid nitrogen container, and conduit (3) the other end is derived outside the liquid nitrogen container (4); Less than 50 centimetres of distant places conduit (3) is turned back 180 ° going out liquid nitrogen container (4), and with turn back curved 90 ° down of the two-wires that form of conduit,, and immerse and contain crude extract small beaker (5) and carry out freezing absorption as freezing probe (7); Put rotor (6) in the beaker (5) and carry out the moving stirring of magnetic.
5, a kind of inhibition recrystallization observation device of the method that is used for the described alpine periglacial plant antifreeze protein separation of claim 1 and detects, it is characterized in that placing in the temperature controllable refrigerator-freezer (8) Olympus BH/2 opticmicroscope (9), film-making (11) is placed into microscope (9) Stage microscope, photo eye-piece (10) stretches out outside the plastic covering cystose lid (13), cover and be provided with photo eye-piece hole (14), handle hole (15), operational observations hole (16), add overhead camera (12) when taking pictures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310121954 CN1284796C (en) | 2003-12-08 | 2003-12-08 | Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310121954 CN1284796C (en) | 2003-12-08 | 2003-12-08 | Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1627055A CN1627055A (en) | 2005-06-15 |
| CN1284796C true CN1284796C (en) | 2006-11-15 |
Family
ID=34761612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310121954 Expired - Fee Related CN1284796C (en) | 2003-12-08 | 2003-12-08 | Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1284796C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106883293A (en) * | 2017-04-26 | 2017-06-23 | 昆明学院 | A kind of method of quick separating antifreeze protein |
| CN111795909B (en) * | 2019-04-09 | 2022-04-08 | 中国科学院化学研究所 | Method for screening ice control material |
| CN114689874B (en) * | 2022-06-01 | 2022-11-08 | 深圳市帝迈生物技术有限公司 | D-dimer reagents for liquid stabilization and freeze-thaw resistance |
-
2003
- 2003-12-08 CN CN 200310121954 patent/CN1284796C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1627055A (en) | 2005-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Stangoulis et al. | Kinetic analysis of boron transport in Chara | |
| Pokrovsky et al. | Fate of colloids during estuarine mixing in the Arctic | |
| CN102645680B (en) | Cloud chamber for atmospheric ice nucleus activation counting and cloud chamber system | |
| Larsson et al. | Continuous flow hollow fiber liquid-phase microextraction and monitoring of NSAID pharmaceuticals in a sewage treatment plant effluent | |
| CN1284796C (en) | Method for separating and detecting antifreeze protein of alpine periglacial plant, and equipment | |
| CN104764827B (en) | A kind of assay method of tomato plant terminal bud Endogenous Hormone Contents in Vitro | |
| CN101255103A (en) | Marine penicillium antitumor reactive compound as well as preparation method and utilization thereof | |
| AU2020103868A4 (en) | Preparation method of apoplast antifreeze protein standard sample of winter rapeseed leaves and protein lysis buffer | |
| CN102311435A (en) | Preparation method for high purity rhynchophylline | |
| CN107727776B (en) | Extraction and high performance liquid detection method of beta-ecdysone of euphausia superba | |
| Sun et al. | Differentiation analysis of boron isotopic fractionation in different forms within plant organ samples | |
| McAndrews | Paleobotany of a wild rice lake in Minnesota | |
| CN205665208U (en) | Multichannel map shows freezing non -methane total hydrocarbon on -line monitoring of collection appearance of second grade | |
| CN102353567B (en) | Method for abstracting and enriching organic phosphorus in substrate sludge | |
| CN102830183A (en) | Sample pre-treatment method for determining endogenesis hormone content of towel gourd seeds by adopting HPLC (High Performance Liquid Chromatography) | |
| CN111220736B (en) | Method for determining benzalkonium chloride in plants | |
| CN207649987U (en) | The piece-rate system of suspended particulate substance and planktonic organism suitable for water body | |
| Ishii | Extraction and ion chromatographic determination of free and combined oxalic acids in vegetables | |
| CN102747034A (en) | Kit for in-vitro separation purification of karyocytes and use method thereof | |
| AU2020101821A4 (en) | Method for extracting apoplastic antifreeze proteins from winter rape roots | |
| CN111239298B (en) | Method for measuring decamethylammonium chloride in plant | |
| Bai et al. | Na+ and water uptake in relation to the radial reflection coefficient of root in arrowleaf saltbush under salt stress | |
| CN106518957A (en) | Preparation method of spotted deer coronet small molecule protein polypeptide | |
| Marcin et al. | A simple technique for cryopreservation of the rumen protozoon Entodinium caudatum | |
| Pokrovsky et al. | Transformation of organic carbon, trace element, and organo-mineral colloids in the mixing zone of the largest European Arctic river. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: LANZHOU UNIV.; AN LIZHE Free format text: FORMER OWNER: AN LIZHE Effective date: 20080307 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20080307 Address after: 222, Tianshui South Road, Lanzhou, Gansu, zip code: 730000 Co-patentee after: An Lizhe Patentee after: Lanzhou University Address before: Lanzhou University, Lanzhou, Gansu, China: 730000 Patentee before: An Lizhe |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20061115 Termination date: 20100108 |