CN106518957A - Preparation method of spotted deer coronet small molecule protein polypeptide - Google Patents
Preparation method of spotted deer coronet small molecule protein polypeptide Download PDFInfo
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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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- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
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Abstract
The invention relates to a preparation method of a spotted deer coronet small molecule protein polypeptide. The preparation method comprises the following steps: extracting a coronet total peptide, desalting the total peptide crude extract, performing fractional separation on the total peptide, and freeze-drying. Thus, a small molecule polypeptide mixture of which the molecular weight is collectively distributed at 5kDa or below is obtained. By improving the traditional preparation method, the obtained small molecule protein polypeptide has a small molecular weight; and the small molecule polypeptide keeps a complete structure, has favorable activity and can be easily absorbed by a human body.
Description
Technical field
The present invention relates to bioprotein technical field, more particularly to a kind of system of CORNU CERVI disk small molecular protein polypeptide
Preparation Method.
Background technology
Antler refers to stag and remains in ossified thing on bone knob after saw young pilose antler, arrived Second Year it is new it is long and untidy go out before from
The dynamic plate-like material for coming off, is also named as " Cornu Cervi ", " Cornu Cervi cowl ", mainly contains various essential amino acids and several mineral materials
Element.Antler is in helmet shape or flat helmet shape, diameter 2-5cm, high 2-4.5cm, and surface is lark or taupe, in cellular,
Bottom surface is relatively put down and is rich in certain gloss, and top presents irregular hemispherical shape, and quality is more hard, and color is in greyish white
Color.In the last few years, for the composition of antler is mainly studied from following components, mainly amino acid, inorganic elements,
Protein and peptide material etc., while also have being reported in antler on a small quantity containing materials such as steroid, polysaccharides.
The albumen larger compared to molecular weight, small molecular protein and polypeptide are easier to be absorbed so as to have more by body
Obvious medical health effect, and the research of protein or polypeptide monomer and the acquisition of amino acid sequence can be protein engineering
Build bridge between research and genetic engineering research and the development for proteomics contributes, therefore, in recent years, increasingly
Many people are having concentrated on micromolecule polypeptide, polypeptide monomer to the sight that CORNU CERVI disk is studied.Jiang Wei is pre- with 5mM's
Cold SAS is stirred extraction to the soluble protein in CORNU CERVI disk powder, and by alcohol precipitation, revolving, freeze,
Sephadex desalination, ion-exchange chromatography and high-efficient liquid phase technique obtain the protein monomer for purifying, electrophoresis detection (SDS-PAGE)
Its relative molecular mass size is shown for 17.2kDa, Jing MALDI-TOF-MS identifications determine that it is peptidoglycan recognition protein 1
(PGRP 1), the sequence of matching is RLYEIIQ KWPHYRA, is named as cnPGRP1, and proves its tool by bacteriostatic test
There is obvious bacteriostatic activity (Shigella, staphylococcus aureus, saccharomycete, Escherichia coli, hay bacillus).Huang Fengjie et al.
Extraction, chromatographic column Resource S purifying, 75 gel filtration chromatographies of Superdex, anti-phase is homogenized by pH3.5 spirit of vinegars buffer solution
The methods such as efficient liquid phase obtain the CORNU CERVI disk polypeptide monomer that relative molecular mass size is 7.1276kDa, are delivered medicine to
KK-ay mouse observe its hypoglycemic activity, and set up insulin resistant model (inducing HepG2 cells using hyperinsulinism), research
Impact of the polypeptide to insulin resistance HepG2 cell sugar consumptions, as a result shows, the polypeptide can both reduce KK-ay mouse blood sugars
Level, can remarkably promote the sugar consumption of insulin resistant model again, illustrate that the protein monomer has certain hypoglycemic activity.Tang appoints
A relative molecular mass can be purified into by means such as ultrafiltration (hollow-fibre membrane), ion-exchange chromatography, gel permeation chromatographies
Small molecular protein monomer of the size for 17.0kDa, Jing MALDI-TOF-MS analyses find which is recognized with the peptide glycan of mammal
Albumen homology is higher, and finds that small molecular protein monomer propagation isocellular to L929 has a significant impact.Wang Zhibing et al.
By enzymolysis obtain CORNU CERVI disk PROTEIN C PP, electrophoresis detection (SDS-PAGE) its relative molecular mass between 8-15kDa,
Activity test proves that CPP can resist rat mammary gland hyperplasia, and the phagocytic function of mouse is made a significant impact.Qiu Fangping et al. will
Into after the micro mist of granularity 0.01mm, 45 DEG C are distilled floodings suction filtration leaching liquor to CORNU CERVI disk low-temperature grinding, and supernatant is carried out
Saltout and obtain albumen precipitation and renaturation is carried out to albumen precipitation, then obtained by dialysing and chromatographing (Sephadex G-100) means
Antler albumin A PPB, electrophoresis detection (SDS-PAGE) its relative molecular mass is between 20.1-31.0kDa, and which is carried out
Acetic acid causes mouse writhing experiment, as a result shows which has analgesic activity.
The content of the invention
In order to obtain small molecular protein polypeptide and keep the structural integrity of micromolecule polypeptide so as to which activity is good, it is easier to quilt
Absorption of human body, the present invention provide a kind of preparation method of CORNU CERVI disk small molecular protein polypeptide.
A kind of preparation method of CORNU CERVI disk small molecular protein polypeptide, is carried out as steps described below：
Step one：The extraction of the total peptide of antler；
Step 2：The desalination of total peptide crude extract；
Step 3：The classification of total peptide is separated；
Step 4：Freeze-drying.
Preferably, the extraction of the total peptide of antler, comprises the following steps：
Step one：Weigh 450-550g antler powder, add 3000mL to be 3.2- in the pH of the abundant precooling of 4 DEG C of refrigerators
3.8 Acetic acid-sodium acetate buffer solution, after magnetic stirring apparatus is sufficiently stirred for, is centrifuged to homogenate, takes supernatant；Delay thereto
Slowly, after 95% ethanol of the abundant precooling of 4 DEG C of refrigerators of addition reaches 55% up to supernatant final concentration, 4 DEG C of refrigerators are placed and every 1h
Stirring once, is centrifuged after 3.5-5h, takes supernatant；Operation above is carried out under the conditions of 4 DEG C in addition to rotary evaporation；
Step 2：Supernatant vacuum rotary evaporator rotary evaporation under the conditions of 40 DEG C -45 DEG C to the volume of concentrate is
400mL or so；
Step 3：Concentrate is placed in big culture dish after filtering, and vacuum freeze drier is freezed, and obtains the total peptide of CORNU CERVI disk
Crude extract, it is stand-by after -20 DEG C of Refrigerator stores loaded on valve bag；
The speed being centrifuged twice in described step one is 5000-6500rpm, and the time is 25min.
Preferably, the desalination of total peptide crude extract, comprises the following steps：
Step one：Take the total peptide crude extract 1.2-1.8g of CORNU CERVI disk, with 10mL deionized water dissolvings it is abundant after, cross 0.45
μm filter, is sample solution；
Step 2：Sample solution is slowly instilled in chromatographic column along column wall with glue head dropper and is careful not to impact glue surface,
Deionized water is eluted, and stable in 0.8-1.2mL/min, nucleic acid-protein detector by adjusting wriggling revolution speed coutroi velocity
Light absorption value is detected at 280nm, is collected with clean beaker after appearance, stop collecting when light absorption value returns baseline, and continue to use
Three times column volume deionized water is eluted, and delivery port is closed after wash-out (chromatographic column after wash-out can be reused)；
Step 3：Collection liquid is freezed, stand-by after -20 DEG C of Refrigerator stores loaded on valve bag.
Preferably, the classification of total peptide is separated, and is comprised the following steps：
Step one：Take the total peptide desalination product 80-125mg of CORNU CERVI disk, with 2mL deionized water dissolvings it is abundant after, cross 0.45 μ
M millipore filters, are sample solution；
Step 2：Sample solution is slowly instilled in chromatographic column along column wall with glue head dropper and is careful not to impact glue surface,
Deionized water is eluted, and stable in 0.3-0.6mL/min, nucleic acid-protein detector by adjusting wriggling revolution speed coutroi velocity
Light absorption value is detected at 215nm, automatic collector collects the test tube at the simultaneously each peak of label collection, stops when light absorption value returns baseline
Collect, and continue to be eluted with three times column volume deionized water, gel column delivery port is closed after wash-out, and (gel column after wash-out can be with
Secondary use).
Preferably, freeze-drying, comprises the following steps：Collection liquid is placed in big culture dish, vacuum freeze drier freezes
It is dry, obtain CORNU CERVI disk small molecular protein polypeptide.
The preparation method of CORNU CERVI disk small molecular protein polypeptide provided by the present invention, including the total peptide of antler carries
Take；The desalination of total peptide crude extract；Four steps of classification separation and freeze-drying of total peptide, concentrate on 5kDa so as to obtain molecular weight
Following micromolecule polypeptide mixture, by the improvement to traditional preparation method, the small molecular protein polypeptide for obtaining, molecular weight
Little, it is possible to keep the structural integrity of micromolecule polypeptide, activity is good, it is easy to be absorbed by the body.
Description of the drawings
Fig. 1 is the SDS-PAGE of component 3 in embodiment 1.
Specific embodiment
Embodiment
A kind of preparation method of CORNU CERVI disk small molecular protein polypeptide, is carried out as steps described below：
Step one：The extraction of the total peptide of antler：500g antler powder is weighed, adds 3000mL fully pre- in 4 DEG C of refrigerators
The Acetic acid-sodium acetate buffer solution of cold pH 3.6, after magnetic stirring apparatus is sufficiently stirred for, homogenate is centrifuged (4 DEG C,
6000rpm, 25min), supernatant is taken, and 95% ethanol of the abundant precooling of 4 DEG C of refrigerators is slowly added to thereto until supernatant final concentration
After reaching 55%, 4 DEG C of refrigerators are placed and are stirred once every 1h, (4 DEG C, 6000rpm, 25min) are centrifuged, take supernatant after 4h；On
Clear liquid vacuum rotary evaporator rotary evaporation to volume of concentrate under the conditions of 45 DEG C is 400mL or so；After concentrate is filtered
It is placed in big culture dish, vacuum freeze drier is freezed, and obtains the total peptide crude extract of CORNU CERVI disk, loaded on valve bag after -20 DEG C
Refrigerator store is stand-by；More than operation carry out under the conditions of 4 DEG C in addition to rotary evaporation, the precipitation that obtains is centrifuged twice respectively at 37
Valve bag, Cord blood are loaded on after drying in DEG C baking oven；
Step 2：The desalination of total peptide crude extract：The total peptide crude extract 1.5g of CORNU CERVI disk is taken, 10mL deionized water dissolvings are used
After fully, 0.45 μm of filter is crossed, is sample solution；Sample solution is slowly instilled in chromatographic column and noted along column wall with glue head dropper
Glue surface, deionized water wash-out should not be impacted, and is stablized in 1mL/min, nucleic acid egg by adjusting wriggling revolution speed coutroi velocity
White detector detects light absorption value at 280nm, is collected with clean beaker after appearance, stops collecting when light absorption value returns baseline,
And continue to be eluted with three times column volume deionized water, delivery port is closed after wash-out (chromatographic column after wash-out can be reused)；
Collection liquid is freezed, stand-by after -20 DEG C of Refrigerator stores loaded on valve bag；
The chromatographic column of described step two need to be balanced using before, comprised the following steps：Weigh 30g Sephadex
G-25 gel dry powder is placed in 1000mL beakers, is added thereto to 500mL deionized waters, the swelling 2h in boiling water bath, and period is used
Glass bar is gently mixed and promotes gel swelling fully, and has seen whether inhomogenous fine particle or impurity, if any need to topple over
Remove, make supernatant no longer muddy；After gel fully swelling and supernatant no longer muddiness, vacuum decompression bubble removing fills post；Chromatographic column
(Φ 16mm × 40cm) before dress post needs wash with 0.2M NaOH one time, then is rinsed well with water, finally with distilled water with
Deionized water respectively cleaning three times；Pillar is perpendicularly fixed in chromatography freezer, peristaltic pump, nucleic acid-protein inspection is sequentially connected in order
Survey instrument；One is done at the 5cm of distance post top to be labeled as weighing the foundation of filling gel bed；Chromatographic column delivery port is sealed,
The deionized water of 1/4 column volume is poured into post bottom, unnecessary deionized water in the beaker of splendid attire gel is slowly toppled over away directly
Ratio to remaining deionized water and gel volume is 1: 1, and glass bar is gently mixed after making gel even suspension in deionized water,
Gel homogenate is slowly poured in post, after the gel sedimentation that 1～2cm or so occurs in column bottom, chromatographic column delivery port is opened, it is compacted
Dynamic pump coutroi velocity is 1mL/min；Constantly gel homogenate is slowly poured in post to supplement the deionized water for flowing out until gel
Stop when sedimentation is higher than mark 2cm or so, deionized water is mobile phase, makes chromatography column equilibration 12h；
Step 3：The classification of total peptide is separated：The total peptide desalination product 100mg of CORNU CERVI disk is taken, is filled with 2mL deionized water dissolvings
After point, 0.45 μm of millipore filter is crossed, is sample solution；Sample solution is slowly instilled in chromatographic column and noted along column wall with glue head dropper
Meaning should not impact glue surface, deionized water wash-out, and be stablized in 0.5mL/min, core by adjusting wriggling revolution speed coutroi velocity
Acid albumin detector detects light absorption value at 215nm, and automatic collector collects the test tube at the simultaneously each peak of label collection, treats that light absorption value is returned
Stop collecting when returning baseline, and continue to be eluted with three times column volume deionized water, closing gel column delivery port is (after wash-out after wash-out
Gel column can be with secondary use)；
Described step three chromatographic column need to be balanced using before, comprised the following steps：Chromatographic column specification is Φ 20mm
×100cm.Weigh 20g Sephadex G-50 gel dry powder to be placed in 1000mL beakers, be added thereto to 1000mL go from
Sub- water, the swelling 2h in boiling water bath, period are gently mixed with glass bar and promote gel swelling fully, and have seen whether heterogeneity
Fine particle or impurity, if any removing need to be toppled over, make supernatant no longer muddy.Treat that gel is fully swelling and supernatant is no longer muddy
Afterwards, vacuum decompression bubble removing, fills post, and chromatographic column before dress post needs to be washed with 0.2M NaOH one time, then is rinsed well with water,
Respectively cleaning three times of distilled water and deionized water are used finally；Pillar is perpendicularly fixed in chromatography freezer, is sequentially connected in order compacted
Dynamic pump, nucleic acid-protein detector；One is done at the 5cm of distance post top to be labeled as weighing the foundation of filling gel bed；Envelope
Firmly chromatographic column delivery port, pours into the deionized water of 1/4 column volume to post bottom, by deionized water unnecessary in the beaker of splendid attire gel
Slowly topple over away until the ratio of remaining deionized water and gel volume is 1: 1, glass bar is gently mixed makes gel even suspension
After in deionized water, gel homogenate is slowly poured in post, after the gel sedimentation that 1-2cm or so occurs in column bottom, opened
Chromatographic column delivery port, peristaltic pump coutroi velocity are 1mL/min；Constantly gel homogenate is slowly poured in post to supplement going for outflow
Ionized water is until stop when gel sedimentation is higher than mark 2cm or so；With deionized water as mobile phase, chromatographic column 24h is balanced；
Step 4：The freeze-drying of antler micromolecule polypeptide：Collection liquid containing purpose thing is placed in big culture dish, vacuum
Freeze drier is freezed, and obtains CORNU CERVI disk micromolecule polypeptide.
The electrophoresis detection of separation product：Using tri- layers of gel electrophoresis of Tricine-SDS-PAGE, electrophoresis purpose is to find plum blossom
Antler micromolecule polypeptide mixture place peak.Specific method is as follows：
(1) fixation of vertical gel mould：Glass plate interlayer width used by electrophoresis is 1.0mm, uses clean water glass plate
And with distilled water and deionized water rinse, glass plate is fixed in electrophoresis tank, it is seated on glue folder.
(2) preparation of gel：The preparation (5.0mL) of separation gel：Sequentially add in clean small beaker 1.620mL go from
Sub- water, 1.675mL gel buffer liquid, 1.675mL glue stock solution II, 25 μ L, 10% ammonium persulfates and 2.5 μ L TEMED, liquid relief
Encapsulating after rifle piping and druming is uniform, encapsulating stop encapsulating when being highly 4cm or so, pour into 1mL isopropanol to glue surface upper strata with liquid-transfering gun,
1h is solidified under room temperature.The preparation (2.5mL) of interbed glue：Filter paper removes isopropanol.Sequentially add in clean small beaker
1.149mL deionized waters, 0.838mL gel buffer liquid, 0.500mL glue stock solution I, 12.5 μ L, 10% ammonium persulfates and 1.25 μ
L TEMED, encapsulating after liquid-transfering gun piping and druming is uniform, encapsulating stop encapsulating when being highly 1.5cm or so, with liquid-transfering gun to glue surface upper strata
Pour into 1h is solidified under 1mL isopropanols, room temperature.The preparation (2.5mL) of concentration glue：Filter paper removes isopropanol.To clean small beaker
In sequentially add 1.677mL deionized waters, 0.620mL gel buffer liquid, 0.200mL glue stock solution I, 30 μ L, 10% persulfuric acid
Ammonium and 1.20 μ L TEMED, encapsulating after liquid-transfering gun piping and druming is uniform, insert glue comb solidify 1h under room temperature.
(3) sample treatment and electrophoresis：Take classification and separate and collect 50 μ L of peak liquid, add equal-volume 2 × electrophoresis loading buffer
Liquid, liquid-transfering gun piping and druming mix 10s.Sample is processed after 5min with the common boiling water baths of electrophoresis Marker, loading (10 μ of albumen Marker
L, 25 μ L of sample).Electrophoresis starting voltage is 60V, when bromophenol blue instruction line is migrated to concentration glue and interbed glue line of demarcation by electricity
Pressure is adjusted to 100V, when bromophenol blue instruction line is migrated to distance separation glue bottom 0.2cm, closes electrophoresis apparatus, stops electrophoresis.Electrophoresis
During buffer solution keep ice bath.
(4) fixed, dyeing and decolouring：Film is transferred in clean big culture dish, appropriate fixer is added, on shaking table
Fixed 1h.After fixation is finished, outwell fixer and film is cleaned with distilled water, add dyeing liquor (coomassie brilliant blue R_250) dye
Color 5h.After dyeing is finished, remove dyeing liquor and film is cleaned with distilled water, destainer decolourizes to clean background.Film in
Take pictures in ultraviolet imager and preserve.
These three components are further analyzed by isolated 3 components of Jing Sephadex G-50 gel chromatographies.Electricity
Swimming testing result finds that the protein major part in component 1 and component 2 is relative molecular mass size more than 20.0kDa's
High molecular weight protein material, is not inconsistent with the target of the present invention；And material contained in component 3 for molecular weight concentrate on 5kDa with
Under micromolecule polypeptide mixture, meet the target of the present invention, that is, (Fig. 1 is to obtain CORNU CERVI disk micromolecule polypeptide
Tri- layers of gel electrophoresis figures of Tricine-SDS-PAGE).
Below with reference to the embodiment 6 in Chinese invention patent CN102408477A disclosed in Jilin Agriculture University, deer is detected
Impact of the angle disk polypeptide to the mouse mastopathy cell cycle (detection method is entirely by reference to the comparative example).
By above detection data it is recognised that the CORNU CERVI disk small molecular protein polypeptide in the present invention is to mouse breast cancer
Cell has the effect of significant Inhibit proliferaton, and this inhibition to be significantly better than the data disclosed in comparative example.
It is presumed that, improvement of the present invention to traditional preparation method is exactly based on, the small molecular protein polypeptide for obtaining divides
Son amount is little, it is possible to keep the structural integrity of micromolecule polypeptide, and activity is good, higher to the inhibitory action of mouse mastopathy cell.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (7)
1. a kind of preparation method of CORNU CERVI disk small molecular protein polypeptide, it is characterised in that carry out as steps described below：
Step one：The extraction of the total peptide of antler；
Step 2：The desalination of total peptide crude extract；
Step 3：The classification of total peptide is separated；
Step 4：Freeze-drying.
2. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 1, it is characterised in that antler is total
The extraction of peptide, comprises the following steps：
Step one：Weigh 450-550g antler powder, add 3000mL in the pH of the abundant precooling of 4 DEG C of refrigerators for 3.2-3.8's
Acetic acid-sodium acetate buffer solution, after magnetic stirring apparatus is sufficiently stirred for, is centrifuged to homogenate, takes supernatant；It is slowly added to thereto
After 95% ethanol of the abundant precooling of 4 DEG C of refrigerators is until supernatant final concentration reaches 55%, 4 DEG C of refrigerators are placed and simultaneously stir one every 1h
It is secondary, it is centrifuged after 3.5-5h, takes supernatant；Operation above is carried out under the conditions of 4 DEG C in addition to rotary evaporation；
Step 2：Supernatant vacuum rotary evaporator rotary evaporation to volume of concentrate under the conditions of 40 DEG C -45 DEG C is 400mL
Left and right；
Step 3：Concentrate is placed in big culture dish after filtering, and vacuum freeze drier is freezed, and is obtained the total peptide of CORNU CERVI disk and is slightly carried
Thing, it is stand-by after -20 DEG C of Refrigerator stores loaded on valve bag；
The speed being centrifuged twice in described step one is 5000-6500rpm, and the time is 25min.
3. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 1, it is characterised in that described is total
The desalination of peptide crude extract, comprises the following steps：
Step one：Take the total peptide crude extract 1.2-1.8g of CORNU CERVI disk, with 10mL deionized water dissolvings it is abundant after, cross 0.45 μm filter
Device, is sample solution；
Step 2：Sample solution is slowly instilled in chromatographic column along column wall with glue head dropper and be careful not to impact glue surface, spent
Ion water elution, and it is stable in 0.8-1.2mL/min by adjusting wriggling revolution speed coutroi velocity, and nucleic acid-protein detector exists
Light absorption value is detected at 280nm, is collected with clean beaker after appearance, stopped collecting when light absorption value returns baseline, and continue to use three
Times column volume deionized water wash-out, closes delivery port after wash-out；
Step 3：Collection liquid is freezed, stand-by after -20 DEG C of Refrigerator stores loaded on valve bag.
4. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 1, it is characterised in that described is total
The classification of peptide is separated, and is comprised the following steps：
Step one：Take the total peptide desalination product 80-125mg of CORNU CERVI disk, with 2mL deionized water dissolvings it is abundant after, cross 0.45 μm it is micro-
Hole filter, is sample solution；
Step 2：Sample solution is slowly instilled in chromatographic column along column wall with glue head dropper and be careful not to impact glue surface, spent
Ion water elution, and it is stable in 0.3-0.6mL/min by adjusting wriggling revolution speed coutroi velocity, and nucleic acid-protein detector exists
Light absorption value is detected at 215nm, automatic collector collects the test tube at the simultaneously each peak of label collection, stop receiving when light absorption value returns baseline
Collection, and continue to be eluted with three times column volume deionized water, gel column delivery port is closed after wash-out.
5. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 1, it is characterised in that described is cold
It is lyophilized dry, comprise the following steps：Collection liquid is placed in big culture dish, vacuum freeze drier is freezed, and obtains CORNU CERVI disk little
Molecule protein polypeptide.
6. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 3, it is characterised in that described layer
Analysis post need to be balanced using before, comprised the following steps：Weigh 30g Sephadex G-25 gel dry powder and be placed in 1000mL burnings
In cup, 500mL deionized waters are added thereto to, the swelling 2h in boiling water bath, period are gently mixed with glass bar and promote gel molten
It is swollen abundant, and inhomogenous fine particle or impurity have been seen whether, if any removing need to be toppled over, make supernatant no longer muddy；Wait to coagulate
After glue fully swelling and supernatant no longer muddiness, vacuum decompression bubble removing fills post；Chromatographic column (Φ 16mm × 40cm) is needed before dress post
Washed with 0.2M NaOH one time, then rinsed well with water, finally with distilled water and deionized water respectively cleaning three times；By pillar
It is perpendicularly fixed in chromatography freezer, is sequentially connected peristaltic pump, nucleic acid-protein detector in order；Do at the 5cm of distance post top
One is labeled as weighing the foundation of filling gel bed；Seal chromatographic column delivery port, to post bottom pour into 1/4 column volume go from
Sub- water, unnecessary deionized water in the beaker of splendid attire gel is slowly toppled over away until remaining deionized water and gel volume
Than for 1: 1, glass bar is gently mixed after making gel even suspension in deionized water, gel homogenate is slowly poured in post, is treated
After the gel sedimentation of 1～2cm or so occurs in column bottom, chromatographic column delivery port is opened, peristaltic pump coutroi velocity is 1mL/min；No
It is disconnected gel homogenate slowly to be poured in post to supplement the deionized water for flowing out until stopping when gel sedimentation is higher than mark 2cm or so
Only, deionized water is mobile phase, makes chromatography column equilibration 12h.
7. the preparation method of CORNU CERVI disk small molecular protein polypeptide as claimed in claim 4, it is characterised in that described layer
Analysis post need to be balanced using before, comprised the following steps：Chromatographic column specification is Φ 20mm × 100cm；Weigh 20g
Sephadex G-50 gel dry powder is placed in 1000mL beakers, is added thereto to 1000mL deionized waters, molten in boiling water bath
Swollen 2h, period are gently mixed with glass bar and promote gel swelling fully, and have seen whether inhomogenous fine particle or impurity,
If any removing need to be toppled over, make supernatant no longer muddy；After gel fully swelling and supernatant no longer muddiness, vacuum decompression bubble removing,
Dress post, chromatographic column before dress post need wash with 0.2M NaOH one time, then are rinsed well with water, finally with distilled water with go from
Respectively cleaning three times of sub- water；Pillar is perpendicularly fixed in chromatography freezer, peristaltic pump, nucleic acid-protein detection is sequentially connected in order
Instrument；One is done at the 5cm of distance post top to be labeled as weighing the foundation of filling gel bed；Chromatographic column delivery port is sealed, to
Post bottom pours into the deionized water of 1/4 column volume, by contain gel beaker in unnecessary deionized water slowly topple over away until
The ratio of remaining deionized water and gel volume is 1: 1, and glass bar is gently mixed after making gel even suspension in deionized water, will
Gel homogenate is slowly poured in post, after the gel sedimentation that 1～2cm or so occurs in column bottom, is opened chromatographic column delivery port, is wriggled
Pump coutroi velocity is 1mL/min；Constantly gel homogenate is slowly poured in post to supplement the deionized water for flowing out until gel sinks
Stop when drop is higher than mark 2cm or so；With deionized water as mobile phase, chromatographic column 24h is balanced.
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| CN201611069257.8A CN106518957A (en) | 2016-11-17 | 2016-11-17 | Preparation method of spotted deer coronet small molecule protein polypeptide |
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| CN201611069257.8A CN106518957A (en) | 2016-11-17 | 2016-11-17 | Preparation method of spotted deer coronet small molecule protein polypeptide |
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