CN106810604A - The method for extraction and purification of one boar relaxain - Google Patents
The method for extraction and purification of one boar relaxain Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/64—Relaxins
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Abstract
The invention provides the method for extraction and purification of a boar relaxain, comprise the following steps：(1) acetone gradient extracting prepares relaxain crude product；(2) cation-exchange chromatography；(3) hydrophobic chromatography；(4) freeze-drying；Wherein, the process of step (1) includes：Hydrochloric acid, acetone extraction, gained extract solution is used to add low-temperature treatment after acetone successively pig ovary, gained precipitation is dried to obtain relaxain crude product after being dehydrated through acetone after treatment.Not only high income, purity are high, yield is big for relaxain prepared by the method for the present invention, and the structure and bioactivity of gained relaxain preserve complete.
Description
Technical field
The invention belongs to biological pharmacy technical field, it is related to the method for extraction and purification of a boar relaxain, more specifically relates to
And one kind is extracted from pig ovary using acetone gradient, ion-exchange chromatography and hydrophobic chromatography technology isolate and purify pig relaxain
Method.
Background technology
Relaxain is the peptide hormone that a kind of preceding birth canal of being given a birth to dam has relexation, occurs mainly with mammal
Corpus luteum in gestation ovary, it is found initially as a kind of polypeptide hormone relevant with Mammalian Reproduction activity.With
Going deep into for research, it is found that relaxain has wider biological function：The formation for such as suppressing collagen is closed with clostridiopetidase A is promoted
Into, so as to promote the degraded of collagen, reproductive tract tissue is softened in childbirth, suppress uterine contractile, promote pelvis ligament to soften, thorn
Mammoplasia is swashed with differentiation；The blood vessel dilatation of various organs and tissue can be stimulated, suppress platelet aggregation；Stimulate islet cells
Excreting insulin, adjusts internal blood sugar；Promote chronic ulcer tissue healing；Influence pituitary hormone secretion.
As can be seen here, relaxain has complicated biological action and wide medical application prospect.It is external at present existing
Be used for relaxain in the middle of the treatment of general chorionitis by report, Connetics companies, and has completed I phase, II phase clinic
Experiment.And country's research in this regard is very few.
Relaxain is purified from many kind biologies, including pig, mouse, horse, shark, tiger, rat, spiny dogfish and the mankind.However,
The relaxain for how obtaining high-purity is still one is worth the problem of research.Content is low in animal body for relaxain, and stabilization
Property is poor, and extraction purification difficulty is high.High-purity relaxain need to extract the raw protein containing relaxain from ovary tissue, then go
Except almost all of foreign protein, there are many difficult points in its purifying process.As J.Doczi uses hydrochloric acid acetone extraction, chlorination zinc salt
The method of analysis, the relaxain purity for obtaining is not high, and the zinc chloride of high concentration also has an impact to relaxain activity
(O.D.Sherwood, Isolation and Characterization of Porcine and Rat Relaxin,
Advances in Experimental Medicine and Biology pp 115-138,1982, DOL 10.1007/
978-1-4613-3368-55).C.David Sherwood et al. are using hydrochloric acid acetone extraction, sieve chromatography and ion exchange
The method of chromatography, has obtained relaxain (C.D.SHERWOOD et al, the Purification and of higher degree
characterization ofporcine relaxin,Chemical Abstracts Band 80,Nr.13,1.April
1974,Columbus,Ohio,USA).But the method loses very big during sieve chromatography, and is unfavorable for extensive life
Produce；After ion exchange wash-out, there is the foreign protein of similar charge characteristic, influence the purity of final products.The sun for using later
Though the technology that ion-exchange chromatography and anion-exchange chromatography are combined can prepare the suitable sample of purity, because anion is handed over
Change the pH conditions high that chromatography is used in absorption and wash-out, the bioactivity of meeting heavy damage relaxain.
Other CN 102603888A disclose a kind of method that use Pichia yeast prepares RLN2, wherein
Need that filtering with microporous membrane for several times is repeated from broth extraction purifying human relaxain -2, and need 3 chromatogram post separations
Purification process, takes more long.CN 103788207A disclose a kind of side of the extraction purification RLN2 from zymotic fluid
Method, the method crosses the cellulose-acetafolic filtering of the molecular weight 3000 that dams after including being centrifuged zymotic fluid, go up successively
SephadexG-50 posts and SynchropakRP-PC18 reverse-phase HPLC chromatograms post carry out chromatographic purifying, are then rotated by low temperature
Concentration, freeze-drying obtains the RLN2 of purifying.The method expensive starting materials, are difficult to obtain, and are difficult to preserve relaxain knot
Structure and bioactivity it is complete.
The content of the invention
In order to overcome shortcoming present in prior art, the extraction it is an object of the invention to provide a boar relaxain is pure
Change method.The method of the present invention prepare relaxain not only high income, purity are high, yield is big, and gained relaxain structure
Preserve complete with bioactivity.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
The method for extraction and purification of one boar relaxain, comprises the following steps：
(1) acetone gradient extracting prepares relaxain crude product；
(2) cation-exchange chromatography；
(3) hydrophobic chromatography；
(4) freeze-drying；
Wherein, the process of step (1) includes：Hydrochloric acid, acetone extraction, gained extract solution is used to add acetone successively pig ovary
Low-temperature treatment, to precipitate obtained by after treatment and be dried to obtain relaxain crude product after being dehydrated through acetone afterwards；Low temperature for example can be -10 DEG C --
40 DEG C, such as -15 DEG C, -19 DEG C, -21 DEG C, -26 DEG C, -30 DEG C, -35 DEG C, -38 DEG C etc.；
By pig ovary successively with hydrochloric acid, acetone extraction when the acetone and the ratio of amount of pig ovary be 1-5L/kg, for example,
1.3L/kg, 1.8L/kg, 2.5L/kg, 2.9L/kg, 3.3L/kg, 3.7L/kg, 4.1L/kg, 4.6L/kg, 4.9L/kg etc.；
The volume of the acetone is 1-10 times of the extract solution when extract solution adds low-temperature treatment after acetone, for example,
1.5 times, 2.3 times, 3.0 times, 3.5 times, 5.0 times, 7.0 times, 8.5 times, 9.0 times, 9.7 times etc..
Method for extraction and purification of the invention utilizes acidic organic solvent gradient, is dissolved in low-concentration organic solvent, high concentration
It is precipitated out in organic solvent, crude product is prepared to relaxain extracting, the acidic organic solvent of low ph value can not only keeps lax
Element is not destroyed by cathepsin, additionally it is possible to protect the distinctive disulfide bond pattern of relaxain；And by controlling organic solvent
Gradient, can more accurately remove the most of foreign protein and lipid material in ovary；In addition using cation-exchange chromatography and
Hydrophobic chromatography can to greatest extent remove the foreign protein beyond relaxain, and preserve the complete of relaxain structure and bioactivity.
Preferably, the process of step (1) includes：
A the pig ovary of frost is processed as fritter by (), add 0-10 DEG C, such as 1.5 DEG C, 2.6 DEG C, 4 DEG C, 6 DEG C, 7.5 DEG C,
8.7 DEG C, 9.5 DEG C etc. of hydrochloric acid, stir extracting 1-5 hours at 0-10 DEG C, add 0-10 DEG C, such as 1.5 DEG C, 2.6 DEG C, 4 DEG C,
6 DEG C, 7.5 DEG C, 8.7 DEG C, 9.5 DEG C etc. of acetone, stir extracting 2-10 hours at 0-10 DEG C, stand more than 5 hours, and preferably 10 is small
When；
B () will be centrifuged at step (a) 0-10 DEG C of extracted solution of gained；
(c) take step (b) gained supernatant add -10 DEG C -- 40 DEG C, such as -15 DEG C, -19 DEG C, -21 DEG C, -26 DEG C, -30
DEG C, -35 DEG C, -38 DEG C etc. of acetone, stir more than 10 hours, preferably 24 hours between rearmounted 0-10 DEG C of low-temperature operation；
D () will be centrifuged at step (c) 0-10 DEG C of bottom precipitation of gained, then precipitate described in being drying to obtain after being dehydrated through acetone
Relaxain crude product.
Preferably, pig ovary described in step (a) is processed through rubbing.
Preferably, the hydrochloric acid and the ratio of the amount of pig ovary are 0.1-1L/kg, preferably 0.3-0.5L/kg.
Preferably, the acetone and the ratio of the amount of pig ovary are 1-5L/kg, preferably 2-2.5L/kg.
Preferably, the concentration of the hydrochloric acid is 0.5-5mol/L, preferably 1.5mol/L.
Preferably, the temperature of the stirring extracting is 4-8 DEG C.
Preferably, the rotating speed being centrifuged described in step (b) is 3000rpm, the time of centrifugation is more than 5 minutes, preferably
It is 10 minutes.
Preferably, the volume of acetone described in step (c) is 1-10 times of the supernatant, preferably 4 times.
Preferably, the time of the stirring is more than 2 minutes, preferably 5 minutes.
Preferably, the rotating speed being centrifuged described in step (d) is 4000rpm, the time of centrifugation is more than 5 minutes, preferably
It is 10 minutes.
Preferably, the process of step (2) includes：By step (1) gained relaxain dissolving crude product in level pad,
Supernatant liquid filtering is taken after centrifugation, regulation filtered fluid pH value and conductivity value are consistent with level pad, then by filtered fluid upper strata
Analysis post；After loading is finished, chromatographic column is rinsed with lavation buffer solution, then use elution destination protein, collect eluent.
Preferably, the level pad is NH4Ac, pH are 4-6.5, preferably 5.0-6.0.
Preferably, the level pad is the NH of 0.03-0.08mol/L4The NH of Ac, preferably 0.05mol/L4Ac。
Preferably, the ratio of the relaxain crude product and level pad is 1:10-20g/mL, for example, 1:12g/mL、
1:15g/mL、1:18g/mL etc..
Preferably, the lavation buffer solution is 0.03-0.08mol/L NH4Ac and 0.01-0.05mol/L NaCl's is mixed
Close liquid, preferably 0.05mol/LNH4The mixed liquor of Ac and 0.01-0.05mol/LNaCl.
Preferably, the eluent is 0.03-0.08mol/LNH4The mixed liquor of Ac and 0.1-0.3mol/LNaCl, preferably
It is 0.05mol/LNH4The mixed liquor of Ac and 0.1-0.3mol/LNaCl.
Preferably, the chromatography media of the chromatographic column is CM-Sepharose FF gels.
Preferably, the process of step (3) includes：Collect to add level pad in eluent to step (2), from
The heart, takes supernatant liquid filtering, then by chromatographic column on filtered fluid；After loading is finished, chromatographic column is rinsed with lavation buffer solution, then with washing
De- liquid wash-out destination protein, collects eluent.
Preferably, the level pad is NH4The mixed liquor of Ac and NaCl, pH is 4-6.5, preferably 5.0-6.0.
Preferably, the level pad is 0.02-0.08mol/LNH4The mixed liquor of Ac and 3mol/LNaCl, preferably
0.05mol/LNH4The mixed liquor of Ac and 3mol/LNaCl.
Preferably, the lavation buffer solution is 0.03-0.08mol/LNH4The mixed liquor of Ac and 0.5-3mol/LNaCl, it is excellent
Elect 0.05mol/LNH as4The mixed liquor of Ac and 1-1.5mol/LNaCl.
Preferably, the eluent is 0.03-0.08mol/LNH4The mixed liquor of Ac and 0.2-2mol/LNaCl, preferably
0.05mol/LNH4The mixed liquor of Ac and 0.5-0.8mol/LNaCl.
Preferably, the chromatography media of the chromatographic column is Phenyl-sepharose FF gels.
Preferably, the process of step (4) includes：The eluent that step (3) is collected is subsequently adding through milipore filter ultrafiltration
Acetum, the conductance to ultrafiltration filter liquor is not higher than the conductance of added acetum, then by filter liquor freeze-drying, also may be used
With freeze-drying after filter liquor is concentrated.
Preferably, the milipore filter is 2-10kd, preferably 5kd.
Preferably, the concentration of the acetum is 0.05-0.2mol/L, preferably 0.1mol/L.
Preferably, method for extraction and purification of the invention comprises the following steps：
(1) acetone gradient extracting prepares relaxain crude product
The pig ovary that will be freezed is rubbed, and adds 4 DEG C of 1.5mol/L hydrochloric acid, and extracting 3 hours is stirred at 4-8 DEG C, adds 4
DEG C acetone, at 4-8 DEG C stir extracting 5 hours, stand 10 hours；4 DEG C of centrifugations of extracted solution, centrifugal rotational speed is 3000rpm, from
10 minutes heart time；Take centrifuged supernatant and add 4 times -20 DEG C of volume of acetone, and be stirred continuously 5 minutes, put 4-8 DEG C of low temperature
Operation room 24 hours, siphon supernatant takes 4 DEG C of centrifugations of bottom precipitation, and centrifugal rotational speed is 4000rpm, and centrifugation time 10 minutes sinks
Shallow lake prepares relaxain crude product through acetone dewatering and vacuum drying；
(2) cation-exchange chromatography
By step (1) gained relaxain dissolving crude product in level pad, centrifugation takes supernatant liquid filtering, regulation filtering
Liquid pH value and conductivity value are consistent with level pad, upper chromatographic column；After loading is finished, chromatographic column is rinsed with lavation buffer solution,
Elution destination protein is used again, collects eluent；
The level pad is the NH of 0.05mol/L4Ac, pH are 5.0-6.0；
The lavation buffer solution is 0.05mol/LNH4The mixed liquor of Ac and 0.01-0.05mol/LNaCl；
The eluent is 0.05mol/LNH4The mixed liquor of Ac and 0.1-0.3mol/LNaCl；
The chromatography media of the chromatographic column is CM-Sepharose FF gels；
(3) hydrophobic chromatography
To level pad is added in the eluent that step (2) is collected, it is centrifuged, supernatant liquid filtering is taken, by filtered fluid upper strata
Analysis post；After loading is finished, chromatographic column is rinsed with lavation buffer solution, then use elution destination protein, collect eluent；
The level pad is 0.05mol/LNH4The mixed liquor of Ac and 3mol/LNaCl, pH is 5.0-6.0；
The lavation buffer solution is 0.05mol/LNH4The mixed liquor of Ac and 1-1.5mol/LNaCl；
The eluent is 0.05mol/LNH4The mixed liquor of Ac and 0.5-0.8mol/LNaCl；
The chromatography media of the chromatographic column is Phenyl-sepharose FF gels；
(4) freeze-drying
The eluent that step (3) is collected constantly adds 0.1mol/L acetums through 5kd milipore filter ultrafiltration, extremely super
Filter filter liquor conductance is equal to 0.1mol/L acetums, takes the concentrate freeze-drying after ultrafiltration.
Extracting and chromatography process are carried out preferably between 4-8 DEG C of low-temperature operation in the inventive method, purified water used and preparation
Solution is preferably cooled to 4-8 DEG C in advance in advance, and ammonium acetate used, sodium chloride, glacial acetic acid, hydrochloric acid is pure for analysis, and acetone can be industry
Level.
The method of the present invention can not only keep relaxain not destroyed by cathepsin, additionally it is possible to protect relaxain special
Some disulfide bond patterns；Relatively foreign protein and lipid material beyond relaxain can be removed precisely and to greatest extent, and preserved
Relaxain structure and bioactivity it is complete.While prepared by the method for the present invention, product yield is high, purity is high, yield is big, preferably
Solve shortcoming present in prior art.
Brief description of the drawings
Fig. 1 is the molecular sieve HPLC collection of illustrative plates of the pig relaxain standard items of the amino acid of B chains 29；
Fig. 2 is the molecular sieve HPLC collection of illustrative plates of the products obtained therefrom of embodiment 1；
Fig. 3 is Coomassie brilliant blue electrophoresis and Westernblot electrophoresis patterns；
Fig. 4 is the molecular sieve HPLC collection of illustrative plates of the products obtained therefrom of embodiment 2.
Specific embodiment
For ease of understanding the present invention, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is used only for help and understands the present invention, is not construed as to concrete restriction of the invention.
Embodiment 1
The method for extraction and purification of one boar relaxain, comprises the following steps：
(1) pig ovary for freezing 10.2kg is rubbed, the 1.5mol/L hydrochloric acid 5L for adding prior precooling to be 4 DEG C, is stirred at 4 DEG C
Extracting 3 hours is mixed, 4 DEG C of acetone 25L is added, extracting 5 hours is stirred at 4 DEG C, stand 10 hours；4 DEG C of centrifugations of extracted solution,
Centrifugal rotational speed is 3000rpm, centrifugation time 10 minutes；The acetone that centrifuged supernatant adds 4 times -20 DEG C of volume is taken, and constantly
Stirring 5 minutes, puts 24 hours between 8 DEG C of low-temperature operations, siphon supernatant, takes 4 DEG C of centrifugations of bottom precipitation, and centrifugal rotational speed is
4000rpm, centrifugation time 10 minutes, precipitation prepares relaxain crude product 102g through acetone dewatering and vacuum drying；
(2) step (1) gained relaxain crude product 102g is dissolved in 2.0L level pads (0.05mol/L NH4Ac, pH
In 5.0-6.0), supernatant liquid filtering is taken after centrifugation, regulation filtered fluid pH value and conductivity value are consistent with level pad, then will
CM-Sepharose FF gel chromatography columns on filtered fluid；After loading is finished, with lavation buffer solution (0.05mol/L NH4Ac,
0.01mol/L NaCl) chromatographic column is rinsed, then with eluent (0.05mol/L NH4Ac, 0.3mol/L NaCl) wash-out purpose egg
In vain, eluent is collected；
(3) collect to add level pad (0.05mol/L NH in eluent to step (2)4Ac, 3mol/L NaCl,
PH 5.0-6.0), centrifugation takes supernatant liquid filtering, then by Phenyl-sepharose FF gel chromatography columns on filtered fluid；On
After sample is finished, with lavation buffer solution (0.05mol/L NH4Ac, 1mol/L NaCl) chromatographic column is rinsed, then use eluent
(0.05mol/L NH4Ac, 0.8mol/L NaCl) wash-out destination protein, collect eluent；
(4) eluent for collecting step (3) is through 5kd milipore filter ultrafiltration, and constantly adds 0.1mol/L acetums, extremely
Ultrafiltration filter liquor conductance is equal to 0.1mol/L acetums, takes the concentrate freeze-drying after ultrafiltration and prepares relaxain sterling
2.05g, lot number 090502.
It is 95.2%, appearance time and the ammonia of B chains 29 that the present embodiment is obtained into product through molecular sieve HPLC collection of illustrative plates display content
Base acid standard items collection of illustrative plates is consistent, and standard items collection of illustrative plates is shown in Fig. 1, and the collection of illustrative plates of the present embodiment product is shown in Fig. 2.
The yield of the present embodiment product is 0.02%, far above the 0.01% of C.David Sherwood
(C.D.SHERWOOD et al,Purification and characterization ofporcine relaxin,
Chemical Abstracts Band 80, Nr.13,1.April 1974, Columbus, Ohio, USA), reach international neck
First level.
Coomassie brilliant blue electrophoresis non-reducing sample is single band, and sample is presented two band, western- after reduction
The specific binding anti-with relaxain of blot electrophoresis is obvious, shows that relaxain structure and bioactivity keep complete, sees Fig. 3.
From Sigma companies, content is 99.5% for the wherein amino acid standard items of B chains 29 purchase；Western-blot rabbit-anti pigs
How anti-relaxain is buys from Sigma companies.
Embodiment 2
The method for extraction and purification of one boar relaxain, comprises the following steps：
(1) pig ovary for freezing 10kg is rubbed, and adds the 1.5mol/L hydrochloric acid 3L that prior precooling is 4 DEG C, is stirred at 8 DEG C
Extracting 3 hours, adds 4 DEG C of acetone 20L, and extracting 5 hours is stirred at 8 DEG C, stands 10 hours；4 DEG C of centrifugations of extracted solution, from
Heart rotating speed is 3000rpm, centrifugation time 10 minutes；Take centrifuged supernatant and add 4 times -20 DEG C of volume of acetone, and constantly stir
Mix 5 minutes, put 24 hours between 4 DEG C of low-temperature operations, siphon supernatant, take 4 DEG C of centrifugations of bottom precipitation, centrifugal rotational speed is 4000rpm,
Centrifugation time 10 minutes, precipitation prepares relaxain crude product 95g through acetone dewatering and vacuum drying；
(2) step (1) gained relaxain crude product 95g is dissolved in 1.0L level pads (0.05mol/L NH4Ac, pH
In 5.0-6.0), supernatant liquid filtering is taken after centrifugation, regulation filtered fluid pH value and conductivity value are consistent with level pad, then will
CM-Sepharose FF gel chromatography columns on filtered fluid；After loading is finished, with lavation buffer solution (0.05mol/L NH4Ac,
0.05mol/L NaCl) chromatographic column is rinsed, then with eluent (0.05mol/L NH4Ac, 0.1mol/L NaCl) wash-out purpose egg
In vain, eluent is collected；
(3) collect to add level pad (0.05mol/L NH in eluent to step (2)4Ac, 3mol/L NaCl,
PH 5.0-6.0), centrifugation takes supernatant liquid filtering, then by Phenyl-sepharose FF gel chromatography columns on filtered fluid；On
After sample is finished, with lavation buffer solution (0.05mol/L NH4Ac, 1.1mol/L NaCl) chromatographic column is rinsed, then use eluent
(0.05mol/L NH4Ac, 0.5mol/L NaCl) wash-out destination protein, collect eluent；
(4) eluent for collecting step (3) is through 5kd milipore filter ultrafiltration, and constantly adds 0.1mol/L acetums, extremely
Ultrafiltration filter liquor conductance is equal to 0.1mol/L acetums, takes the concentrate freeze-drying after ultrafiltration and prepares relaxain sterling
2.03g, lot number 090801.
It is 93.0%, appearance time and the ammonia of B chains 29 that the present embodiment is obtained into product through molecular sieve HPLC collection of illustrative plates display content
Base acid standard diagram is unanimously shown in Fig. 4, and yield is 0.0203%, and Coomassie brilliant blue electrophoresis and western-blot electrophoresis are presented allusion quotation
Type relaxain feature, is shown in Fig. 3.
Embodiment 3
The method for extraction and purification of one boar relaxain, comprises the following steps：
(1) pig ovary for freezing 10.4kg is rubbed, and the 4.5mol/L hydrochloric acid 1.2L that prior precooling is 2 DEG C is added, at 2 DEG C
Stirring extracting 5 hours, adds 2 DEG C of acetone 17L, and extracting 10 hours is stirred at 2 DEG C, stands 5 hours；2 DEG C of extracted solution from
The heart, centrifugal rotational speed is 3000rpm, centrifugation time 5 minutes；The acetone that centrifuged supernatant adds 8 times -10 DEG C of volume is taken, not
Disconnected stirring 3 minutes, puts 10 hours between 2 DEG C of low-temperature operations, siphon supernatant, takes 8 DEG C of centrifugations of bottom precipitation, and centrifugal rotational speed is
4000rpm, centrifugation time 5 minutes, precipitation prepares relaxain crude product 108g through acetone dewatering and vacuum drying；
(2) step (1) gained relaxain crude product 108g is dissolved in 1.5L level pads (0.08mol/L NH4Ac, pH
In 5.0-6.0), supernatant liquid filtering is taken after centrifugation, regulation filtered fluid pH value and conductivity value are consistent with level pad, then will
CM-Sepharose FF gel chromatography columns on filtered fluid；After loading is finished, with lavation buffer solution (0.03mol/L NH4Ac,
0.01mol/L NaCl) chromatographic column is rinsed, then with eluent (0.03mol/L NH4Ac, 0.3mol/L NaCl) wash-out purpose egg
In vain, eluent is collected；
(3) collect to add level pad (0.02mol/L NH in eluent to step (2)4Ac, 3mol/L NaCl,
PH 5.0-6.0), centrifugation takes supernatant liquid filtering, then by Phenyl-sepharose FF gel chromatography columns on filtered fluid；On
After sample is finished, with lavation buffer solution (0.08mol/L NH4Ac, 0.9mol/L NaCl) chromatographic column is rinsed, then use eluent
(0.03mol/L NH4Ac, 0.4mol/L NaCl) wash-out destination protein, collect eluent；
(4) eluent for collecting step (3) is through 3kd milipore filter ultrafiltration, and constantly adds 0.2mol/L acetums, extremely
Ultrafiltration filter liquor conductance is equal to 0.2mol/L acetums, takes the concentrate freeze-drying after ultrafiltration and prepares relaxain sterling
2.17g。
It is 94.3% that the present embodiment is obtained into product through molecular sieve HPLC collection of illustrative plates display content, and yield is 0.0217%, is examined
Mas bright blue electrophoresis and western-blot electrophoresis are presented typical relaxain feature.
Embodiment 4
The method for extraction and purification of one boar relaxain, comprises the following steps：
(1) pig ovary for freezing 9.9kg is rubbed, the 0.5mol/L hydrochloric acid 9L for adding prior precooling to be 8 DEG C, is stirred at 8 DEG C
Extracting 1 hour is mixed, 8 DEG C of acetone 45L is added, extracting 2 hours is stirred at 8 DEG C, stand 12 hours；8 DEG C of centrifugations of extracted solution,
Centrifugal rotational speed is 3000rpm, centrifugation time 15 minutes；The acetone that centrifuged supernatant adds 2 times -10 DEG C of volume is taken, and constantly
Stirring 8 minutes, puts 32 hours between 8 DEG C of low-temperature operations, siphon supernatant, takes 2 DEG C of centrifugations of bottom precipitation, and centrifugal rotational speed is
4000rpm, centrifugation time 15 minutes, precipitation prepares relaxain crude product 93g through acetone dewatering and vacuum drying；
(2) step (1) gained relaxain crude product 93g is dissolved in 1.8L level pads (0.03mol/L NH4Ac, pH
In 5.0-6.0), supernatant liquid filtering is taken after centrifugation, regulation filtered fluid pH value and conductivity value are consistent with level pad, then will
CM-Sepharose FF gel chromatography columns on filtered fluid；After loading is finished, with lavation buffer solution (0.08mol/L NH4Ac,
0.01mol/L NaCl) chromatographic column is rinsed, then with eluent (0.08mol/L NH4Ac, 0.2mol/L NaCl) wash-out purpose egg
In vain, eluent is collected；
(3) collect to add level pad (0.08mol/L NH in eluent to step (2)4Ac, 3mol/L NaCl,
PH 5.0-6.0), centrifugation takes supernatant liquid filtering, then by Phenyl-sepharose FF gel chromatography columns on filtered fluid；On
After sample is finished, with lavation buffer solution (0.03mol/L NH4Ac, 1.2mol/L NaCl) chromatographic column is rinsed, then use eluent
(0.08mol/L NH4Ac, 0.7mol/L NaCl) wash-out destination protein, collect eluent；
(4) eluent for collecting step (3) is through 3kd milipore filter ultrafiltration, and constantly adds 0.05mol/L acetums,
0.05mol/L acetums are equal to ultrafiltration filter liquor conductance, the concentrate freeze-drying after ultrafiltration are taken and is prepared relaxain sterling
2.00g。
It is 95.3% that the present embodiment is obtained into product through molecular sieve HPLC collection of illustrative plates display content, and yield is 0.0200%, is examined
Mas bright blue electrophoresis and western-blot electrophoresis are presented typical relaxain feature.
Obviously, above-described embodiment is only intended to clearly illustrate example, and not to the restriction of implementation method.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need and unable to be exhaustive to all of implementation method.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. the method for extraction and purification of a boar relaxain, comprises the following steps：
(1) acetone gradient extracting prepares relaxain crude product；
(2) cation-exchange chromatography；
(3) hydrophobic chromatography；
(4) freeze-drying；
Wherein, the process of step (1) includes：Pig ovary is used into hydrochloric acid, acetone extraction successively, it is low after gained extract solution addition acetone
Temperature treatment, gained precipitation is dried to obtain relaxain crude product after being dehydrated through acetone after treatment；
By pig ovary successively with hydrochloric acid, acetone extraction when the acetone and the ratio of amount of pig ovary be 1-5L/kg；
The volume of the acetone is 1-10 times of the extract solution when extract solution adds low-temperature treatment after acetone.
2. method according to claim 1, it is characterised in that the process of step (1) includes：
A the pig ovary of frost is processed as fritter by (), add 0-10 DEG C of hydrochloric acid, extracting 1-5 hours is stirred at 0-10 DEG C, then add
Stirring is extracted 2-10 hours at entering 0-10 DEG C of acetone, 0-10 DEG C, stands more than 5 hours, preferably 10 hours；
B () will be centrifuged at step (a) 0-10 DEG C of extracted solution of gained；
C () takes step (b) gained supernatant and adds -10 DEG C -- and 10 is small between 40 DEG C of acetone, the rearmounted 0-10 DEG C of low-temperature operation of stirring
When more than, preferably 24 hours；
D () will be centrifuged at step (c) 0-10 DEG C of bottom precipitation of gained, then precipitate and described relaxing is drying to obtain after being dehydrated through acetone
Plain crude product.
3. method according to claim 2, it is characterised in that pig ovary described in step (a) is processed through rubbing；
Preferably, the hydrochloric acid and the ratio of the amount of pig ovary are 0.1-1L/kg, preferably 0.3-0.5L/kg；
Preferably, the acetone and the ratio of the amount of pig ovary are 2-2.5L/kg；
Preferably, the concentration of the hydrochloric acid is 0.5-5mol/L, preferably 1.5mol/L；
Preferably, the temperature of the stirring extracting is 4-8 DEG C.
4. according to the method in claim 2 or 3, it is characterised in that the rotating speed being centrifuged described in step (b) is 3000rpm,
The time of centrifugation is more than 5 minutes, preferably 10 minutes；
Preferably, the volume of acetone described in step (c) is 4 times of the supernatant；
Preferably, the time of the stirring is more than 2 minutes, preferably 5 minutes；
Preferably, the rotating speed being centrifuged described in step (d) is 4000rpm, and the time of centrifugation is more than 5 minutes, preferably 10 points
Clock.
5. the method according to claim any one of 2-4, it is characterised in that the process of step (2) includes：By step (1)
Gained relaxain dissolving crude product takes supernatant liquid filtering in level pad after centrifugation, adjust filtered fluid pH value and conductivity value
It is consistent with level pad, then by chromatographic column on filtered fluid；After loading is finished, chromatographic column is rinsed with lavation buffer solution, then use
Elution destination protein, collects eluent.
6. method according to claim 5, it is characterised in that the level pad is NH4Ac, pH are 4-6.5, preferably
It is 5.0-6.0；
Preferably, the level pad is the NH of 0.03-0.08mol/L4The NH of Ac, preferably 0.05mol/L4Ac；
Preferably, the ratio of the relaxain crude product and level pad is 1:10-20g/mL；
Preferably, the lavation buffer solution is 0.03-0.08mol/L NH4The mixed liquor of Ac and 0.01-0.05mol/LNaCl, it is excellent
Elect 0.05mol/L NH as4The mixed liquor of Ac and 0.01-0.05mol/L NaCl；
Preferably, the eluent is 0.03-0.08mol/L NH4The mixed liquor of Ac and 0.1-0.3mol/L NaCl, preferably
0.05mol/L NH4The mixed liquor of Ac and 0.1-0.3mol/L NaCl；
Preferably, the chromatography media of the chromatographic column is CM-Sepharose FF gels.
7. the method according to claim any one of 2-6, it is characterised in that the process of step (3) includes：To step (2)
Collect to add level pad in eluent, be centrifuged, supernatant liquid filtering is taken, then by chromatographic column on filtered fluid；Loading is finished
Afterwards, chromatographic column is rinsed with lavation buffer solution, then uses elution destination protein, collect eluent.
8. method according to claim 7, it is characterised in that the level pad is NH4The mixed liquor of Ac and NaCl,
PH is 4-6.5, preferably 5.0-6.0；
Preferably, the level pad is 0.02-0.08mol/L NH4The mixed liquor of Ac and 3mol/L NaCl, preferably
0.05mol/L NH4The mixed liquor of Ac and 3mol/L NaCl；
Preferably, the lavation buffer solution is 0.03-0.08mol/L NH4The mixed liquor of Ac and 0.5-3mol/L NaCl, preferably
It is 0.05mol/L NH4The mixed liquor of Ac and 1-1.5mol/L NaCl；
Preferably, the eluent is 0.03-0.08mol/L NH4The mixed liquor of Ac and 0.2-2mol/L NaCl, preferably
0.05mol/L NH4The mixed liquor of Ac and 0.5-0.8mol/L NaCl；
Preferably, the chromatography media of the chromatographic column is Phenyl-sepharose FF gels.
9. the method according to claim any one of 2-8, it is characterised in that the process of step (4) includes：By step (3)
The eluent of collection is subsequently adding acetum through milipore filter ultrafiltration, and to the conductance of ultrafiltration filter liquor, to be not higher than added acetic acid molten
The conductance of liquid, then by filter liquor freeze-drying, it is also possible to freeze-drying after filter liquor is concentrated.
10. method according to claim 9, it is characterised in that the milipore filter is 2-10kd, preferably 5kd；
Preferably, the concentration of the acetum is 0.05-0.2mol/L, preferably 0.1mol/L.
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| CN201710126424.6A CN106810604A (en) | 2017-03-06 | 2017-03-06 | The method for extraction and purification of one boar relaxain |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551704A (en) * | 2019-08-30 | 2019-12-10 | 集美大学 | Method for separating and purifying angiotensin converting enzyme from pig lungs |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3096246A (en) * | 1960-05-24 | 1963-07-02 | Warner Lambert Pharmaceutical | Process for the extraction and purification of relaxin |
| CN103788207A (en) * | 2014-02-10 | 2014-05-14 | 济南环肽医药科技有限公司 | Method for extracting and purifying human relaxin-2 from fermentation liquor |
-
2017
- 2017-03-06 CN CN201710126424.6A patent/CN106810604A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3096246A (en) * | 1960-05-24 | 1963-07-02 | Warner Lambert Pharmaceutical | Process for the extraction and purification of relaxin |
| CN103788207A (en) * | 2014-02-10 | 2014-05-14 | 济南环肽医药科技有限公司 | Method for extracting and purifying human relaxin-2 from fermentation liquor |
Non-Patent Citations (3)
| Title |
|---|
| C. DAVID SHERWOOD等: "Purification and Characterization of Porcine Relaxin", 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 * |
| R.H. RENEGAR等: "Purification and Partial Characterization of Relaxin and Relaxin Precursors from the Hamster Placenta", 《BIOL REPROD.》 * |
| SELENA S. LAYDEN 等: "Purification and characterization of porcine prorelaxin", 《J. B&HEM. BIOPHYS. METHODS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551704A (en) * | 2019-08-30 | 2019-12-10 | 集美大学 | Method for separating and purifying angiotensin converting enzyme from pig lungs |
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