CN109942701A - A kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin - Google Patents
A kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin Download PDFInfo
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- CN109942701A CN109942701A CN201910311006.3A CN201910311006A CN109942701A CN 109942701 A CN109942701 A CN 109942701A CN 201910311006 A CN201910311006 A CN 201910311006A CN 109942701 A CN109942701 A CN 109942701A
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Abstract
The invention discloses a kind of isolation and purification methods of Chinese ring-necked pheasant egg white serpin, and Sephadex G-50 and Sephadex G-75 gel elution is respectively adopted after taking egg white liquid to be centrifugated freeze-drying twice and isolates and purifies to obtain Chinese ring-necked pheasant egg white serpin.The Chinese ring-necked pheasant egg white serpin that the present invention obtains, which uses, has stronger serpin activity.
Description
Technical field
The present invention relates to a kind of isolation and purification methods of protease inhibitors, more particularly to a kind of Chinese ring-necked pheasant egg white silk ammonia
The isolation and purification method of pepsin inhibitor.
Background technique
Protease inhibitors is a kind of polypeptide or protein, because it can inhibit enzyme in conjunction with proteinase activity position
Catalytic activity can be used as the modulator of protease.It is widely present in animals and plants and microbial body, with albumen corresponding in body
Hydrolase forms dynamic equilibrium, adjusts many important vital movements.The Chinese patent of Publication No. CN102372777A discloses
A kind of purification process of natural serine protease inhibitor, method include silkworm chrysalis pretreatment, natural serine protease suppression
The initial gross separation of preparation purifies, the step of isolating and purifying of natural serine protease inhibitor.Publication No.
The Chinese patent of CN103044542A discloses serpin and its gene and application in crucian ovum, from crucian ovum
It isolates and purifies to obtain serpin, molecular weight 42651.9Da, isoelectric point 5.61, crucian ovum serine protease
Inhibitor has the characteristics that the inhibitory activity strong to serine protease, and is not likely to produce drug resistance, can for preparation tumour,
The medicinal application of the diseases such as gastritis, pancreatitis, cancer.
Summary of the invention
The object of the present invention is to provide a kind of isolation and purification methods of Chinese ring-necked pheasant egg white serpin, to obtain
There must be the serpin of wide pH range stability.
Technical solution of the present invention is as follows: a kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin, packet
Include step: Chinese ring-necked pheasant egg white washes rear decladding and collects egg white liquid, and mixed phosphate hydrogen salt buffer solution is homogenized for the first time with mortar grinder
Centrifuge separation takes supernatant that freeze-dried powder is made, and freeze-dried powder is dissolved in hydrophosphate buffer solution second of centrifuge separation and is taken
Clear liquid；Sephadex G-50 solvent resistant column is balanced with hydrophosphate buffer solution, second of centrifuge separation is taken to take on supernatant
Sample carries out first time elution with hydrophosphate buffer solution, use is ultraviolet in treated Sephadex G-50 solvent resistant column
Spectrophotometer detects each collecting pipe protein content at wavelength 280mm and merges each eluting peak and be lyophilized；It is buffered with hydrophosphate
Previous step acquisition lyophilized products are dissolved in hydrophosphate buffer solution and carried out by solution equilibria Sephadex G-75 solvent resistant column
Third time centrifuging and taking supernatant is splined on that treated Sephadex G-75 solvent resistant column, with hydrophosphate buffer solution into
Second of elution of row, is detected each collecting pipe protein content at wavelength 280mm using ultraviolet specrophotometer and merges each eluting peak
And be lyophilized and obtain Chinese ring-necked pheasant egg white serpin, the hydrophosphate buffer concentration is 0.1M, and pH is
6.0。
Preferably, first time centrifugation and when being centrifuged for second, revolving speed 12000rpm, it is centrifuged 25 at 4 DEG C~
30min。
Preferably, when the first time is centrifuged, revolving speed 10000rpm is centrifuged 18~20min.
Preferably, when the first time elutes and elutes for the second time, elution flow rate 3mL/10min.
Technical solution of the present invention compared with prior art, has the advantages that
The serpin obtained from Chinese ring-necked pheasant egg white has stronger serpin activity,
When temperature is at 30~80 DEG C, Chinese ring-necked pheasant egg white serpin is maintained at 85% or more, in the model of pH 3~10
The activity for enclosing interior inhibitor remains at 90% or more, the apparent molecular weight point of Chinese ring-necked pheasant egg white serpin
It Yue Wei not 40kDa.
Detailed description of the invention
Fig. 1 is inhibitor activity curve of the Chinese ring-necked pheasant egg white serpin to trypsase.
Fig. 2 is time stability curve of the Chinese ring-necked pheasant egg white serpin to trypsase.
Fig. 3 is Chinese ring-necked pheasant egg white serpin activity and temperature relation schematic diagram.
Fig. 4 is Chinese ring-necked pheasant egg white serpin activity and pH relation schematic diagram.
Specific embodiment
Below with reference to embodiment, the invention will be further described, it should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention, after the present invention has been read, those skilled in the art are to various equal similar shapes of the invention
The modification of formula is fallen in the application range as defined in the appended claims.
Each material source that the present embodiment is related to is Chinese ring-necked pheasant ovum, is bought in the local market of farm produce, quality 40g
~50g, for use.Trypsase (trypsin), chromophoric substrate N-Benzoyl-L-Arg-4-nitroanilide
Hydrochloride (B-3133) is purchased from Sigma company；Sephadex G-75 gel, it is public purchased from U.S. GE (Pharmacia)
Department.Other domestic reagents are to analyze pure, Pass Test requirement.Test key instrument and equipment: pH meter, high speed refrigerated centrifuge
Machine, Sephadex G-50, Sephadex G-75, chromatographic column (2.6 × 100cm), circulating water type vacuum pump, freeze drier,
Ultraviolet specrophotometer.
The isolation and purification method of Chinese ring-necked pheasant egg white serpin, comprising steps of Chinese ring-necked pheasant egg white is gone after washing
Shell collects egg white liquid, mixes suitable 0.1M hydrophosphate buffer solution (PBS, pH 6.0), is homogenized with mortar grinder, carries out the
Primary centrifugation, revolving speed 12000rpm are centrifuged 25~30min at 4 DEG C, collect supernatant, are made according to Conventional lyophilization processes
Freeze-dried powder.Freeze-dried powder is dissolved in suitable 0.1M hydrophosphate buffer solution (PBS, pH 6.0), second is carried out and is centrifuged,
Revolving speed is 12000rpm, and 25~30min is centrifuged at 4 DEG C, takes supernatant, is placed in EP pipe as Activity determination and Sephadex
G-50 sample is spare.
With 0.1M hydrophosphate buffer solution (PBS, pH6.0) balanced gel Filter column (2.6 × 100cm)；Take aforementioned sample
Product are splined on the Sephadex G-50 solvent resistant column handled well, are eluted for the first time with above-mentioned buffer solution, elution flow rate 3mL/
10min, fraction collector collect eluent (6 pipes/hr) automatically, are detected at wavelength 280mm using ultraviolet specrophotometer each
Collecting pipe protein content merges each eluting peak, is lyophilized spare.
With 0.1M hydrophosphate buffer solution (PBS, pH6.0) balanced gel Filter column (2.6 × 100cm), by back
Rapid lyophilized products are dissolved in 0.1M hydrophosphate buffer solution (PBS, pH6.0), progress third time centrifugation, revolving speed 10000rpm, from
18~20min of the heart takes supernatant to be splined on the Sephadex G-75 gel column balanced, is eluted with above-mentioned buffer solution, flow velocity
3mL/10min, fraction collector collect eluent (6 pipes/hr) automatically, are examined at wavelength 280mm using ultraviolet specrophotometer
Each collecting pipe protein content is surveyed, each eluting peak is merged, freeze-drying obtains Chinese ring-necked pheasant egg white serpin.In SDS-
Its apparent molecular weight of Chinese ring-necked pheasant egg white serpin respectively may be about 40kDa under PAGE reducing condition.
Protease inhibitory activity detection is as follows: in 50mM Tris-HCl buffer solution (containing 1mM CaCl2), pH
In 7.8 reaction system, by the different amounts of ovalbumin enzyme inhibitor sample isolated and purified and trypsase child care
Chromophoric substrate (B-3133) the starting reaction of final concentration of 20ul is added in 15min.Continuous 2min monitors light absorption at 410nm
Variation.The each step isolated and purified is all made of trypsase and B-3133 substrate detects inhibitor activity.The enzyme activity of 1 unit by
The amount of the paranitroanilinum discharged per minute indicates that the inhibiting effect of 1 unit refers to the p-nitrophenyl to release 1nmol per minute
The inhibition of amine tryptic activity.
The Chinese ring-necked pheasant ovum serpin isolated and purified is to the inhibition 4stability determination of protease
The ovum serpin that trypsase and reaction buffer, respective concentration purify is kept the temperature in room temperature condition, the time point
It Wei not 0.5h, 1h, 4h, 8h, 12h, 16h, 20h and for 24 hours.Enzyme activity when with no inhibitor is 100%, then calculates residual enzyme
It is living.
Chinese ring-necked pheasant ovum serpin according to the method measurement purifying of Dixon is normal to the inhibition of protease
Number: the Chinese ring-necked pheasant egg protein enzyme inhibitor that not same amount purifies is dissolved in 50mM Tris-HCl buffer solution, respectively with 20ul's
Trypsase (ultimate density 68nM) keeps the temperature 15min at room temperature, is eventually adding the starting reaction of 20ulB-3133 chromophoric substrate,
The variation of absorption value in 2min is monitored at 410nm, blank control adds the 50mMTris-HCl buffer solution of same inhibitor volume.
Inhibition constant KiBy formula Ki=[I]/(V0/V1+ 1) it calculates, wherein [I] is rubbing for Chinese ring-necked pheasant ovum serpin
That concentration, V0For the reaction speed of blank control (no inhibitor) reaction, V1For reaction speed when inhibitor is added.
Testing result surface, Chinese ring-necked pheasant ovum serpin inhibit the activity of trypsin hydrolysis substrate, should
It is 60.0nM that inhibitor, which inhibits the minimum concentration of trypsase, as Fig. 1 arrow is signified.When the inhibitor and trypsase are kept the temperature
When 15min, the activity of trypsase is totally constrained, even if holding time still can't detect the work of trypsase to for 24 hours
Property, as shown in Fig. 2, illustrating that it has comparable stability to the inhibitory effect of trypsase.The Chinese ring-necked pheasant ovum serine of purifying
The inhibition constant of protease inhibitors is 0.16nM.
Incorporated by reference to shown in Fig. 3 and Fig. 4, the thermal stability of Chinese ring-necked pheasant ovum serpin is in temperature 30~80
DEG C when, Chinese ring-necked pheasant egg white serpin is maintained at 85% or more, but when temperature is more than 80 DEG C, then loses to special
The hydrolysing activity of property substrate.Chinese ring-necked pheasant egg white serpin has extensive ph stability, in pH 3~10
In the range of the activity of inhibitor remain at 90% or more, show that the protease inhibitors has preferable pH stability.
Claims (4)
1. a kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin, which is characterized in that comprising steps of ring neck
Pheasant egg white washes rear decladding and collects egg white liquid, and mixed phosphate hydrogen salt buffer solution is homogenized centrifuge separation for the first time with mortar grinder and takes
Freeze-dried powder is made in supernatant, and freeze-dried powder is dissolved in hydrophosphate buffer solution second of centrifuge separation and takes supernatant；Use phosphorus
Sour hydrogen salt buffer solution balances Sephadex G-50 solvent resistant column, takes second of centrifuge separation that supernatant is taken to be splined on processing
Sephadex G-50 solvent resistant column afterwards, carries out first time elution with hydrophosphate buffer solution, uses uv-spectrophotometric
It counts and detects each eluting peak of each collecting pipe protein content merging at wavelength 280mm and be lyophilized；It is balanced with hydrophosphate buffer solution
Sephadex G-75 solvent resistant column, by previous step acquisition lyophilized products be dissolved in hydrophosphate buffer solution carry out third time from
The heart takes the supernatant Sephadex G-75 solvent resistant column that is splined on that treated, is carried out second with hydrophosphate buffer solution
Elution is detected each collecting pipe protein content at wavelength 280mm using ultraviolet specrophotometer and merges each eluting peak and be lyophilized
To Chinese ring-necked pheasant egg white serpin, the hydrophosphate buffer concentration is 0.1M, pH 6.0.
2. the isolation and purification method of Chinese ring-necked pheasant egg white serpin according to claim 1, feature exist
In when the first time is centrifuged and is centrifuged for the second time, revolving speed 12000rpm is centrifuged 25~30min at 4 DEG C.
3. the isolation and purification method of Chinese ring-necked pheasant egg white serpin according to claim 1, feature exist
In when the first time is centrifuged, revolving speed 10000rpm is centrifuged 18~20min.
4. the isolation and purification method of Chinese ring-necked pheasant egg white serpin according to claim 1, feature exist
In, when the first time elutes and elutes for the second time, elution flow rate 3mL/10min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910311006.3A CN109942701A (en) | 2019-04-18 | 2019-04-18 | A kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin |
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| CN201910311006.3A CN109942701A (en) | 2019-04-18 | 2019-04-18 | A kind of isolation and purification method of Chinese ring-necked pheasant egg white serpin |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4902509A (en) * | 1985-01-16 | 1990-02-20 | Krka, Tovarna Zdravil, N.Sol.O. | Process for the isolation of chicken egg cystatin, antiviral agents containing it and its use as viral protease inhibitor |
| CN103044542A (en) * | 2012-08-17 | 2013-04-17 | 常熟理工学院 | Serine protease inhibitor in crucian egg as well as gene and application thereof |
| CN104086639A (en) * | 2014-07-21 | 2014-10-08 | 天津商业大学 | Purification process of chicken ovomucoid |
-
2019
- 2019-04-18 CN CN201910311006.3A patent/CN109942701A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4902509A (en) * | 1985-01-16 | 1990-02-20 | Krka, Tovarna Zdravil, N.Sol.O. | Process for the isolation of chicken egg cystatin, antiviral agents containing it and its use as viral protease inhibitor |
| CN103044542A (en) * | 2012-08-17 | 2013-04-17 | 常熟理工学院 | Serine protease inhibitor in crucian egg as well as gene and application thereof |
| CN104086639A (en) * | 2014-07-21 | 2014-10-08 | 天津商业大学 | Purification process of chicken ovomucoid |
Non-Patent Citations (3)
| Title |
|---|
| I. SAXENAA等: "Protein proteinase inhibitors from avian egg whites", 《CELLULAR AND MOLECULAR LIFE SCIENCES》 * |
| 欧雪等: "乌鳢鱼卵中胰蛋白酶抑制剂的分离纯化及性质研究", 《常熟理工学院学报（自然科学）》 * |
| 陈春良等: "《新编特种经济动物饲养手册》", 30 April 2000 * |
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Application publication date: 20190628 |