CN110818773B - Method for purifying active substance in amniotic fluid of non-human animal - Google Patents
Method for purifying active substance in amniotic fluid of non-human animal Download PDFInfo
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- CN110818773B CN110818773B CN201810909193.0A CN201810909193A CN110818773B CN 110818773 B CN110818773 B CN 110818773B CN 201810909193 A CN201810909193 A CN 201810909193A CN 110818773 B CN110818773 B CN 110818773B
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- C07K1/16—Extraction; Separation; Purification by chromatography
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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
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Abstract
The invention relates to a method for purifying an active substance in amniotic fluid of a non-human animal, comprising the step of isolating a neutral fraction having a molecular weight of 500-1200 daltons from said amniotic fluid. The invention also provides an extract which is the neutral fraction with the molecular weight of 500-1200 daltons. The extract of the present invention can promote cell proliferation.
Description
Technical Field
The invention relates to a method for purifying active ingredients in amniotic fluid of non-human animals.
Background
At present, the purification method of amniotic fluid is various, and the purification method can be classified into precipitation technology, chromatography technology, double liquid phase extraction technology and the like according to the major categories. The principle of separation and purification is to utilize the differences of physical and chemical properties, wherein the physical properties include the size, shape and solubility of molecules, and the chemical properties include isoelectric point, hydrophobicity and affinity with other molecules. Different separation and purification methods can be adopted according to different physical or chemical properties of the target protein.
However, in the case of amniotic fluid with complex components, the current protein purification technology is not mature enough, the cost is high, the steps are complex, and the content of active substances is lost or even inactivated in the recovery process.
Thus, there remains a need in the art for a method of purifying amniotic fluid.
Disclosure of Invention
The invention aims to provide a method for purifying active substances in non-human animal amniotic fluid, aiming at the defects of complex steps, long time consumption and the like in the existing amniotic fluid purification and separation process, and the method is feasible and simple.
The method of the invention comprises the steps of separating a neutral fraction having a molecular weight of 500-1200 daltons from amniotic fluid; preferably, the method comprises a step of separating the fraction having a molecular weight of 500 to 1200 daltons using a gel chromatography method and a step of separating the neutral fraction using an ion exchange method.
In one or more embodiments, the method comprises the steps of:
(1) Separating neutral fraction with molecular weight of 500-2000 daltons from amniotic fluid; and
(2) Separating the neutral fraction with molecular weight of 500-1200 Dalton from the neutral fraction with molecular weight of 500-2000 Dalton.
In one or more embodiments, the amniotic fluid is from an avian egg.
In one or more embodiments, the avian is poultry, such as chickens, ducks and geese.
In one or more embodiments, the avian eggs are eggs having an age of 5-20 days old, preferably 6-15 days old.
In one or more embodiments, the avian egg is an egg with an age of 5 to 12 days old, preferably an egg with an age of 6 to 11 days old, more preferably an egg with an age of 7 to 9 days old, more preferably an egg with an age of 7 to 8 days old, or an egg of another avian species other than a chicken at a development stage corresponding to the development stage at which the egg with an age of 5 to 12 days old is present.
In one or more embodiments, the amniotic fluid is from a non-human mammal.
In one or more embodiments, the amniotic fluid is from a rodent.
In one or more embodiments, the amniotic fluid is from an embryo from a rodent with an embryo age of 8-14 days, or from a non-human mammal with a developmental stage corresponding to that of a rodent with an embryo age of 8-14 days.
In one or more embodiments, step (1) uses a gel chromatography column and an ion exchange column for the separation.
In one or more embodiments, the gel chromatography packing has a separation range of 500 to 10000
And D, dalton.
In one or more embodiments, the ion exchange column is an anion exchange column, and when anion exchange is performed, the anion column is equilibrated with a buffer solution having a pH of 7.5 to 8.5, and then loaded, and the unbound fraction is collected.
In one or more embodiments, the ion exchange column is a cation exchange column, and is performed by equilibrating the cation column with a buffer having a pH of 5.8 to 7.0, loading the column, and collecting the unbound fraction
In one or more embodiments, step (2) uses a gel chromatography column to perform the separation, wherein the separation range of the gel chromatography packing is 500 to 10000 daltons.
The invention also provides an extract isolated from amniotic fluid of a non-human animal, said extract not being bound to an ion exchange column at a ph between 5.8 and 8.0 and having a molecular weight in the range of 500 to 1200 daltons.
In one or more embodiments, the extract is from poultry egg amniotic fluid.
In one or more embodiments, the avian is poultry, such as chickens, ducks and geese.
In one or more embodiments, the avian eggs are avian eggs having an embryo age of 5-20 days, preferably 6-15 days.
In one or more embodiments, the avian egg is an egg with an age of 5 to 12 days old, preferably an egg with an age of 6 to 11 days old, more preferably an egg with an age of 7 to 9 days old, more preferably an egg with an age of 7 to 8 days old, or an egg of another avian species other than a chicken at a development stage corresponding to the development stage at which the egg with an age of 5 to 12 days old is present.
In one or more embodiments, the amniotic fluid is from a non-human mammal.
In one or more embodiments, the amniotic fluid is from a rodent.
In one or more embodiments, the amniotic fluid is from an embryo from a rodent with an embryo age of 8-14 days, or from a non-human mammal with a developmental stage corresponding to that of a rodent with an embryo age of 8-14 days.
In one or more embodiments, the extract is extracted using a method as described in any of the embodiments herein.
In certain embodiments, the present invention also provides a pharmaceutical composition comprising an extract or active ingredient as described in any of the embodiments herein, and optionally a pharmaceutically acceptable carrier.
In certain embodiments, the present invention also provides a kit comprising:
(1) Gel chromatography column; and
(3) An ion exchange column.
In one or more embodiments, the kit further comprises a buffer and optionally degassed double distilled water.
In one or more embodiments, the buffer is a phosphate buffer.
In one or more embodiments, the pH of the buffer is in the range of 5.8 to 8.5.
In one or more embodiments, the separation range of the packing in the gel chromatography column is 500 to 10000 daltons.
In one or more embodiments, the ion exchange column is an anion exchange column or a cation exchange column.
In one or more embodiments, the gel chromatography column comprises a first gel chromatography column and a second gel chromatography column, wherein the resolution of the second gel chromatography column is higher than the resolution of the first gel chromatography column.
Drawings
FIG. 1: gel column GE Sephacryl S-200 chromatogram.
FIG. 2: cell viability assay GE Sephacryl S-200 fractions were separated. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; H20-A represents the fraction of the A peak; H20-B represents the fraction of the B peak; H20-C represents the fraction of the C peak.
FIG. 3: anion exchange column GE HiPrep Q separation chromatogram.
FIG. 4: desalting column HiPrep 26/10 desaling Desalting anion unbound fraction chromatogram.
FIG. 5: desalting column HiPrep 26/10 desaling Desalting anion binding partial chromatogram.
FIG. 6: cell viability assay Desalting column HiPrep 26/10 desaling separation fraction. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; hiprep Q-Bind represents the anion column bound fraction; hiprep A-U1 represents the unbound fraction of the anion column.
FIG. 7: gel column GE HiLoad 16/600 Superdex75pg separation chromatogram.
FIG. 8: cell viability assay gel column GE HiLoad 16/600 Superdex75pg fractions were separated. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; S-200B represents the fraction of the B peak; q UNBOUND denotes the UNBOUND fraction of the anion column; 3-1 to 3-6 represent the equal volumes of fractions 1 to 6, respectively, in the third purification step.
FIG. 9: and (4) separating a chromatogram map of the cation exchange column GE HiPrep SP. Wherein UV represents an ultraviolet absorption value; cond represents the electrical conductivity; conc B represents the elution concentration.
FIG. 10: and (4) separating chromatogram map of HiPrep Q of anion exchange column. Wherein UV represents an ultraviolet absorption value; cond represents the electrical conductivity; conc B represents the elution concentration.
FIG. 11: cell viability assay unbound fractions from cation exchange column GE HiPrep SP and anion exchange column HiPrep Q were separated. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; hiprep SP-UN represents fractions not bound to Hiprep SP column; hiprep Q-UN represents fractions not bound to a Hiprep Q column; hiprep Q-Bound represents fractions Bound to the Hiprep Q column.
Detailed Description
It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the examples) may be combined with each other, and the combination order between the respective techniques may be adjusted to each other, thereby constituting a preferred embodiment.
The invention provides a purification and extraction method for active ingredients in amniotic fluid of non-human animal embryos, and a growth factor group with the function of promoting cell proliferation is obtained. The process herein comprises the step of separating a neutral fraction having a molecular weight of 500-1200 daltons from the amniotic fluid. Gel columns and ion exchange columns known in the art may be used to carry out the methods herein. For example, a fraction having a molecular weight of 500 to 1200 daltons can be separated from amniotic fluid using a well-known gel chromatography column (e.g., various gel chromatography columns described below), and then a neutral fraction can be separated from the fraction using an ion exchange method (e.g., using an ion exchange column described below). Alternatively, the neutral fraction may be separated from the amniotic fluid by ion exchange means (e.g. using an ion exchange column as described below) and then the neutral fraction may be separated using a gel chromatography column (e.g. various gel chromatography columns as described below) to obtain a fraction having a molecular weight in the range of 500 to 1200 daltons.
In certain embodiments, the invention provides for the separation of a neutral fraction having a molecular weight in the range of 500 to 2000 daltons from amniotic fluid and then a fraction having a molecular weight in the range of 500 to 1200 daltons from the neutral fraction. In particular, the method may comprise the steps of:
(1) Separating neutral fraction with molecular weight of 500-2000 daltons from amniotic fluid; and
(2) Separating the neutral fraction with molecular weight of 500-1200 Dalton from the neutral fraction with molecular weight of 500-2000 Dalton.
Step (1) can be achieved by using gel chromatography and ion exchange methods. The fractions with molecular weight of 500-2000 daltons are separated from the amniotic fluid by means of a gel chromatography column, whereas the fraction without electric charge (neutral) is obtained by means of ion exchange.
<xnotran> , , GE SephacrylS-100, sephacrylS-200, sephacrylS-300, sephacryl S-400, superose 12, superose 6, </xnotran>Superdex 12 and Superdex 6, and the like. It will be appreciated that any other gel chromatography packing with a separation range of 500-10000 daltons may be used. In general, when a gel column is used, ddH may be used first 2 The flow rate of the O-balanced gel chromatographic column can be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1ml/min. Typically, the UV absorption is between 200-300nm, such as 280nm. And after the ultraviolet absorption curve is stable and the base line is returned, ending the balance. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation conditions. After the sample loading is finished, degassing ddH can be used 2 And O, eluting the crude product, and collecting fractions with the molecular weight of 500-2000 daltons. If desired, the separation by gel chromatography can be repeated several times, and fractions with the same peak time for each separation are mixed.
Herein, charged components can be separated from uncharged components using methods well known in the art. This can be achieved, for example, using ion exchange methods. Both anion exchange and cation exchange can be used in the process of the present invention. In certain embodiments, an anion exchange process is employed herein. Commercially available anion exchange columns can be used, including but not limited to DEAE Sepharose, ANX Sepharose, Q Sepharose, capto DEAE, capto Q, mono Q, and Mini Q from GE corporation. It should be understood that other brands of anion exchange packing may also be used. Alternatively, commercially available cation exchange columns may be used, including but not limited to CM Sepharose, SP Sepharose, capto S, mono S, mini S, and the like.
Typically, when ion exchange is performed, the ion exchange column is first equilibrated with a buffer. The buffer may be a buffer conventional in the art, for example, a phosphate buffer, especially a sodium phosphate buffer, may be used. The pH of the buffer can be determined according to the ion exchange column used. For example, when an anion exchange column is used, the anion exchange column may be equilibrated with a buffer solution having a pH of 7.5 to 8.5, preferably 7.5 to 8.0; when a cation exchange column is used, the cation exchange column can be equilibrated with a buffer solution having a pH of 5.8 to 7.0, preferably 5.8 to 6.5. In certain embodiments, the sodium phosphate buffer contains Na 2 HPO 4 And NaH 2 PO 4 The pH was about 5.8 or 8.0. Book (I)The invention preferably uses an anion exchange column for the separation. The flow rate may be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1ml/min. Generally, after the 280nm UV absorption curve has stabilized and the baseline has returned, the equilibrium is terminated. After equilibration is complete, the sample can be loaded and the effluent fraction (i.e., the fraction not bound to the column) collected. The sample flow rate is determined according to the actual preparation conditions.
In the step (1), gel chromatography can be carried out firstly to separate out a fraction with the molecular weight of 500-2000 daltons, and then ion exchange is carried out to separate out a neutral fraction; alternatively, ion exchange can be carried out to separate neutral fraction from amniotic fluid, and then gel chromatography is carried out to separate active ingredients with molecular weight of 500-2000 Dalton from the neutral fraction to obtain neutral fraction with molecular weight of 500-2000 Dalton.
The main purpose in step (2) is to further separate the neutral fraction obtained in step (1) to obtain active ingredients with molecular weight in the range of 500-1200 daltons. Herein, commercially available gel chromatography columns may be used to separate fractions having molecular weights in the range of 500-1200 daltons. Suitable gel chromatography columns include, but are not limited to, hiLoad Superdex 16/600 Superdex75pg, superdex Peptide, superdex 200, superdex 30, and the like from GE. It will be appreciated that other brands of gel chromatography packing with separation ranges between 500 and 10000 daltons may also be used.
In general, ddH can be used first 2 O balance gel column, the flow rate can be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1ml/min. Generally, after the 280nm UV absorption curve is stabilized and the baseline is returned, the equilibrium is ended. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation conditions. After the sample loading is finished, degassing ddH can be used 2 And O, eluting the crude product, and collecting fractions to obtain fractions containing components with molecular weights in the range of 500-1200 daltons, namely the extract obtained by the method.
Herein, amniotic fluid may be derived from avian eggs and non-human mammals. Fowl eggs are referred to as poultry eggs. Preferred birds are poultry, such as chickens, ducks and geese. Preferably, the present invention uses eggs having an age of 5-20 days, preferably 6-15 days old. It will be appreciated that the appropriate age of the embryo need not be the same for different eggs. For example, when eggs are used, eggs having an age of 5 to 12 days are preferably used, eggs having an age of 6 to 11 days are more preferably used, eggs having an age of 7 to 9 days are more preferably used, and eggs having an age of 7 to 8 days are more preferably used. When eggs of other birds are used, eggs whose development period corresponds to the development period of the above-described embryonated egg may be used. For example, when using duck eggs, duck eggs having an embryo age of 8-10 days, especially 8-9 days, may be the best.
The poultry egg amniotic fluid can be obtained by adopting a conventional method. For example, the blunt end of an egg of the corresponding embryo age may be knocked to break the shell and peel it open to form an opening of approximately 2 cm in diameter. The shell membrane and yolk membrane were then carefully torn apart with forceps, taking care not to disrupt the amniotic membrane. The amniotic membrane and the associated tissue, which are wrapped with the embryo, are poured from the shell into a culture dish, and the amniotic membrane is punctured with an injector to extract amniotic fluid until the amniotic membrane is tightly attached to the embryo, thereby obtaining the amniotic fluid used in the present invention.
Herein, amniotic fluid may also be derived from a non-human mammal, particularly a rodent, such as from a mouse. In certain embodiments, the amniotic fluid is from an embryo of a rodent with a gestational age of 8-14 days, or from a non-human mammal whose developmental stage corresponds to the developmental stage at which a rodent with a gestational age of 8-14 days is located. The amniotic fluid can be obtained by conventional methods. For example, the amniotic fluid used in the present invention can be obtained by cutting the abdominal cavity of a mouse pregnant for 8-14 days with surgical scissors, carefully removing and cutting the uterus, and puncturing the amniotic membrane with a syringe to extract the amniotic fluid until the amniotic membrane is attached to the embryo.
It will be appreciated that, if necessary, the amniotic fluid may be centrifuged to separate impurities that may be contained, such as egg yolk and the like, to obtain as pure an amniotic fluid as possible.
Typically, the elution system used in the methods described herein comprises ddH 2 O and a buffer. The buffer is preferably phosphate buffer, more preferably sodium phosphate buffer, and the pH of the buffer may be in the range of 5.8 to 8.5 depending on the ion exchange column. For example, in certain embodimentsThe pH of the buffer is about 5.8 or 8.0. In the separation and purification process, the elution system of the gel chromatography is degassed ddH 2 O, other salt components are not introduced, so that on one hand, the operation can be reduced, namely, the process of buffer solution replacement is saved, the consumed time of the sample preparation can be shortened in the process of sample preparation, the costs of materials, instruments, consumables and the like are saved, meanwhile, the biological activity of the sample can be better protected, and the risk brought by the addition of a purification step is reduced.
It was found herein that the extract obtained by the above method, which was formulated into a solution having a pH of 5.8 to 8.0, was passed through various ion exchange columns (DEAE Sepharose, Q Sepharose, mono Q, CM Sepharose, SP Sepharose, and Mono S) which contained no active ingredient bound to these ion exchange columns.
Also provided herein is an extract of amniotic fluid of a non-human animal, which extract does not bind to an ion exchange column at a ph of 5.8 to 8.0, and which extract contains components having a molecular weight in the range of 500 to 1200 daltons.
Herein, non-human animals include avian animals and non-human mammals. Birds are, in particular, poultry, such as chickens, ducks and geese. Preferably, embryos aged between 5 and 20 days, preferably between 6 and 15 days, are used in the present invention. It will be appreciated that the appropriate age of the embryos need not be the same from egg to egg. For example, when eggs are used, eggs having an age of 5 to 12 days are preferably used, eggs having an age of 6 to 11 days are more preferably used, eggs having an age of 7 to 9 days are more preferably used, and eggs having an age of 7 to 8 days are more preferably used. When eggs of other birds are used, eggs whose development period corresponds to the development period of the above-mentioned embryonated egg may be used. For example, when using duck eggs, duck eggs having an embryo age of 8-10 days, especially 8-9 days, may be the best. Herein, the non-human mammal includes various mammals other than human, and particularly refers to rodents such as mice. In certain embodiments, the amniotic fluid is from an embryo from a rodent with a gestational age of 8-14 days, or from a non-human mammal with a developmental stage corresponding to the developmental stage in which a rodent with a gestational age of 8-14 days is located.
In certain embodiments, the extract of the invention is an amniotic fluid extract of chicken eggs having an embryo age of 7-9 days, which extract is not bound to an ion exchange column at a pH of 5.8-8.0, and which extract contains components having a molecular weight in the range of 500-1200 daltons.
In certain embodiments, the extract of the present invention is an extract obtained by extraction using a method as described in any of the embodiments herein.
The extract can effectively enhance the speed of cell division and improve the regeneration function of aged cells; helps to reduce wrinkles, makes skin ruddy, tender and glossy, and simultaneously promotes wound healing and repairs organ injuries. Thus, in certain embodiments, also provided herein is a composition for promoting cell proliferation. In certain embodiments, the composition is a pharmaceutical composition comprising an extract or active ingredient as described in any of the embodiments herein, and optionally a pharmaceutically acceptable carrier. Suitable carriers may be those known in the art, for example, carriers suitable for use in formulating the active ingredient into tablets, injections, lyophilizates, pastes or the like.
In certain embodiments, the present invention also provides a kit comprising:
(1) Gel chromatography column; and
(2) An ion exchange column.
The gel column in the kit may be a gel column known in the art, the separation range of the packing of which is typically 500-10000 dalton. The ion exchange column may be an anion exchange column or a cation exchange column. Both the gel chromatography column and the ion exchange column described in any of the embodiments herein may be comprised in a kit as described herein. Preferably, the gel columns in the kit comprise two gel columns, one for separating components having a molecular weight of 500-2000 daltons from amniotic fluid, such columns including, but not limited to, sephacryl S-100, sephacryl S-200, sephacryl S-300, sephacryl S-400, superose 12, superose 6, superdex 12 and Superdex 6, etc., and columns having the same function as the same. The other has higher resolution compared with the former one, and is used for separating the fraction with the molecular weight of 500-1200 daltons from the fraction with the molecular weight of 500-2000 daltons, and the chromatographic columns comprise, but are not limited to, hiLoadSuperdex 16/600 Superdex75pg, superdex Peptide, superdex 200, superdex 30 and the like, and the chromatographic columns with the same functions as the chromatographic columns. Thus, in certain embodiments, the kit contains an ion exchange column, a first gel chromatography column and a second gel chromatography column having a higher resolution than the first gel chromatography column; wherein the first gel chromatography column is operable to separate components having a molecular weight in the range of 500 to 2000 daltons and the second gel chromatography column is operable to separate components having a molecular weight in the range of 500 to 1200 daltons.
The kit further comprises a buffer (preferably a phosphate buffer) and optionally degassed double distilled water. The phosphate buffer may be a sodium phosphate buffer. In certain embodiments, the pH of the buffer is in the range of 5.8 to 8.5. Buffers in different pH ranges may be provided depending on the different ion exchange columns provided in the kit.
In certain embodiments, the kit may further comprise reagents necessary to carry out the cell viability assay CCK-8, such as reagents comprising WST-8 [ 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt ].
Herein, purification is used interchangeably with extraction and isolation, and means the separation or extraction of an active ingredient from amniotic fluid.
The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.
Example 1
This example is aimed at stepwise purification and follow-up of biologically active compounds in the extraction of fresh crude embryos by analytical column gel column Sephacryl S-200, anion exchange column HiPrep Q, desalting column HiPrep 26/10 desaling (Desalting was aimed at testing the activity of the fractions), hiLoad 16/600 Superdex75 pg. Make full use of
The system combines the characteristics and advantages of each analytical column according to the chemical and physical properties and biological activity of the embryo extract (amniotic fluid) to finally obtain the growth factor group with the function of promoting cell proliferation.
1. Material
1.1 purification of samples: fresh eggs aged 7 days were amniotic fluid, 50ml.
1.2 Main Experimental Equipment and consumables
1)GE
Pure；
2) Gel column GE Sephacryl S-200;
3) Anion exchange column GEHiPrep Q;
4) Desalting column GEHiPrep 26/10 desaling;
5) Gel column GEHiLoad 16/600 Superdex75pg;
6)Superloop 10ml。
2. method of producing a composite material
2.1 preparation of the solution
(1) Sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 8.0):
46.6ml 1mol/l Na 2 HPO 4 with 3.4ml 1mol/l NaH 2 PO 4 Mixing, adding ddH 2 And O is metered to 1L.
(2) Sodium phosphate buffer B (50 mM Na) 2 HPO 4 +NaH 2 PO 4 And 500mmnacl, ph 8.0):
46.6ml 1mol/l Na 2 HPO 4 with 3.4ml 1mol/l NaH 2 PO 4 Mixing with 250ml of 2mol/l NaCl, adding ddH 2 And O is metered to 1L.
2.2 Experimental methods
Sample treatment: 50ml fresh amniotic fluid was filtered through a 0.22 μm filter. Or adding appropriate amount of hexane into fresh amniotic fluid, centrifuging at 2500rpm and 4 deg.C for 20min to obtain water phase, and filtering with 0.22 μm filter membrane.
Sample purification
The first step is as follows: gel column GE Sephacryl S-200
ddH 2 O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: the flow rate is 1ml/min, and the sample loading amount is 10ml;
and (3) elution: by degassing ddH 2 The crude product was eluted with a flow rate of 2ml/min and the fractions were collected in equal volumes, 3 ml/tube. 2 column volumes (240 ml) elute;
repeating the separation and purification for 5 times, and fully mixing the parts with the same peak-off time in each time;
freeze drying each portion of the sample;
and (3) measuring the cell activity: better growing AC16 was digested and plated in 96-well plates at 8000 wells, with five replicates per group. In 5% of CO 2 The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. DMEM, DMEM and medium containing 20% of the different fractions were replaced with 10% FBS after 24 hours of starvation culture with DMEM. After 24 hours of incubation, 10. Mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader.
The second step is that: anion exchange column GEHiPrep Q
Sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 8.0) equilibrium anion exchange column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: taking the part with biological activity after the first step of purification, using a pump to load the sample with the flow rate of 1.5ml/min and the sample amount of 250ml, and simultaneously collecting the non-binding part of the anion column with the same volume, 2 ml/tube;
and (3) column washing: sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 8.0) to elute the anion column at a flow rate of 2ml/min until the ultraviolet absorption curve at 280nm is stable and returns to the baseline;
gradient elution: 0-1.5 column volume (30 ml), sodium phosphate buffer B (50 mM Na) 2 HPO 4 +NaH 2 PO 4 500mM NaCl, pH 8.0) percentage from 0 to 100%, sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 8.0) percent from 100% to 0. Elution was continued for 3 column volumes (60 ml) with sodium phosphate buffer B. The flow rate is 2ml/min, and the fraction is collected in equal volume, 2 ml/tube;
and (3) column washing: sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 8.0) eluting the anion column at a flow rate of 2ml/min until the ultraviolet absorption curve at 280nm is stable and returns to the baseline;
desalting: the bound and unbound fractions from the ion column were separately replaced with GEHiPrep 26/10 desaling to degassed ddH 2 O, collecting the desalted fraction;
freeze drying;
and (3) measuring the cell activity: better growing AC16 was digested and plated in 96-well plates at 8000 wells, with five replicates per group. In 5% of CO 2 The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. DMEM, DMEM and medium containing 20% of the different fractions were replaced with 10% FBS after 24 hours of starvation culture with DMEM. After 24 hours of incubation, 10. Mu.l of CCK-8 reagent was added per well. After incubation for 2 hours, the absorbance was measured at 450nm in a microplate reader.
The third step: gel column GEHiLoad 16/600 Superdex75pg
ddH 2 O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
sampling: the flow rate is 1ml/min, and the sample loading amount is 10ml;
and (3) elution: by degassing ddH 2 The sample was eluted with a flow rate of 1ml/min and fractions were collected in equal volumes, 2 ml/tube. Elute 1.5 column volumes (240 ml);
freeze drying;
and (3) measuring the cell activity: better growing AC16 was digested and plated in 96-well plates at 8000 wells, with five replicates per group. In 5% of CO 2 The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. DMEM, DMEM and medium containing 20% of the different fractions were replaced by 10% FBS after starvation for 24 hours with the medium DMEM. After 24 hours of incubation, 10. Mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader.
3. Results of the experiment
1) The chromatogram of fresh amniotic fluid separated by gel column GE Sephacryl S-200 is shown in FIG. 1. Cell viability assay found that the B peak in figure 1 had major activity, and the results are shown in figure 2.
2) The B peak was further separated by anion exchange column GE HiPrep Q, and the results are shown in FIG. 3. The bound and unbound fractions were desalted using Desalting column HiPrep 26/10 desaling, and the chromatograms of desalted unbound fraction and bound fraction are shown in FIGS. 4 and 5, respectively. The cell viability assay found that the unbound fraction was biologically active, and the results are shown in FIG. 6.
3) The unbound fraction was further separated by gel column GE HiLoad 16/600 Superdex75pg and the chromatogram is shown in FIG. 7. Cell viability assays followed groups of biologically active growth factors, the results are shown in figure 8.
Example 2
Using the same gel column, elution system and amniotic fluid as in example 1, the following separation and purification were performed:
1. separating and purifying active ingredients
The first step is as follows: gel column GE Sephacryl S-200
ddH 2 O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: the flow rate is 1ml/min, and the sample loading amount is 10ml;
and (3) elution: by degassing ddH 2 Eluting the crude product with 2ml/min of flow rate, and collecting fractions with molecular weight of 500-2000 daltons;
repeating the separation and purification for 5 times, and fully mixing the parts with the same peak-off time in each time;
the second step is that: cation exchange column GE HiPrep SP
Sodium phosphate buffer A (50 mM Na) 2 HPO 4 +NaH 2 PO 4 pH 5.8) equilibrium cation exchange column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: taking the fraction with molecular weight of 500-2000 daltons obtained in the first step, loading with pump at flow rate of 1.5ml/min and loading amount of 250ml, and collecting the unbound part of cation exchange column; the chromatogram is shown in FIG. 9;
the third step: gel column GE HiLoad 16/600 Superdex75pg
ddH 2 O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: sampling the non-combined part obtained in the second step, wherein the flow rate is 1ml/min, and the sampling amount is 10ml;
and (3) elution: by degassing ddH 2 O eluting the sample at a flow rate of 1ml/min, and collecting fractions with molecular weights in the range of 500-1200 daltons.
2. Active ingredient detection
Better growing AC16 was digested and plated in 96-well plates at 8000 wells, with five replicates per group. In 5% of CO 2 The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. DMEM, DMEM and medium containing 20% of the fraction were replaced with 10% FBS after 24 hours of starvation culture with DMEM. After 24 hours of incubation, 10. Mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader. Cell viability of the unbound regions after cation exchange column GE HiPrep SP treatment is shown in figure 11.
Example 3
Using the same gel column, elution system and amniotic fluid as in example 2, the following separation and purification were performed:
1. separating and purifying active ingredients
The first step is as follows: an ion exchange column, namely an anion exchange column HiPrep Q can be used, the pH of each solution is respectively 5.8 and 8.0, then the solutions are respectively loaded on the ion exchange column at the flow rate of 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the base line is returned;
loading: sampling amniotic fluid with a pump at a flow rate of 1.5ml/min and a sample volume of 50ml, and collecting unbound fraction from the ion column; the chromatogram obtained using HiPrep Q is shown in fig. 10;
the second step is that: gel column GE Sephacryl S-200
ddH 2 O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: the sample is the unbound fraction of the first step, the flow rate is 1ml/min, and the sample loading amount is 10ml;
and (3) elution: by degassing ddH 2 Eluting the crude product with 2ml/min of flow rate, and collecting fractions with molecular weight of 500-2000 daltons;
the third step: gel column GEHiLoad 16/600 Superdex75pg
ddH 2 O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;
loading: sampling the fraction of 500-2000 daltons obtained in the second step at a flow rate of 1ml/min and a sample loading amount of 10ml;
and (3) elution: by degassing ddH 2 O eluting the sample at a flow rate of 1ml/min, and collecting fractions having a molecular weight in the range of 500-1200 daltons.
2. Active ingredient detection
Better growing AC16 was digested and plated in 96-well plates at 8000 wells, with five replicates per group. In 5% of CO 2 The cells are adhered to the wall after being cultured for 2 hours in an incubator with the saturated humidity of 37 ℃. DMEM, DMEM and medium containing 20% of the fraction were replaced by 10% FBS after starvation culture for 24 hours with the medium DMEM. After 24 hours of incubation, 10. Mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader. Cell viability of the unbound regions after anion exchange column GE HiPrep Q treatment is shown in fig. 11.
Claims (8)
1. A method for purifying the active principle of amniotic fluid of a non-human animal, comprising:
(1) Separating neutral fraction with molecular weight of 500-2000 daltons from amniotic fluid, wherein the separation is carried out by using gel chromatography column and ion exchange column; and
(2) Separating neutral fraction with molecular weight of 500-1200 Dalton from the neutral fraction with molecular weight of 500-2000 Dalton;
wherein the separation range of the filler of the gel chromatography column is 500-12000 daltons;
wherein the ion exchange column is an anion exchange column or a cation exchange column; wherein, when anion exchange is carried out, the anion column is balanced by buffer solution with pH of 7.5-8.5, then sample is loaded, and the fraction which is not combined is collected; when cation exchange is carried out, firstly, a buffer solution with the pH value of 5.8-7.0 is used for balancing a cation column, then, sample loading is carried out, and unbound fraction is collected;
wherein the amniotic fluid is amniotic fluid from eggs with embryo age of 5-12 days, or the amniotic fluid is amniotic fluid from duck eggs or goose eggs with embryo age of 6-15 days, or the amniotic fluid is from embryos of rodents with embryo age of 8-14 days.
2. The method of claim 1, wherein the eggs are eggs having a 6-11 day old embryo.
3. The method of claim 1, wherein said egg is a 7-9 day old egg.
4. The method of claim 1, wherein the egg is an egg having an age of 7-8 days old.
5. The method of claim 1, wherein the ion exchange column is an anion exchange column.
6. The method according to any one of claims 1 to 5, wherein the step (2) is performed using a gel chromatography column; wherein the separation range of the gel chromatography filler is 500-12000 daltons.
7. An active ingredient from amniotic fluid of a non-human animal purified by the method of any one of claims 1 to 6.
8. A pharmaceutical composition comprising the active ingredient of claim 7, and optionally a pharmaceutically acceptable carrier.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250216A (en) * | 2008-04-10 | 2008-08-27 | 童兴龙 | Method for extracting tumour-inhibiting protein from amniotic fluid of pregnant woman |
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN102850450A (en) * | 2011-07-01 | 2013-01-02 | 齐鲁制药有限公司 | Purification method of pegylated recombinant human granulocyte colony stimulating factor |
| CN104231037A (en) * | 2014-09-17 | 2014-12-24 | 哈德逊（天津）生物技术有限责任公司 | Rapid protein purification combination chromatographic column, kit and preparation method |
| CN105769926A (en) * | 2016-02-28 | 2016-07-20 | 汉中领维生物科技发展有限公司 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
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| US8551538B2 (en) * | 2011-08-18 | 2013-10-08 | Golden Pearl Investment LLC | Skin formulation, preparation and uses thereof |
| US8895081B2 (en) * | 2011-08-18 | 2014-11-25 | Golden Pearl Investment LLC | Formulation for treatment of dry mouth and mouth sores |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250216A (en) * | 2008-04-10 | 2008-08-27 | 童兴龙 | Method for extracting tumour-inhibiting protein from amniotic fluid of pregnant woman |
| CN102850450A (en) * | 2011-07-01 | 2013-01-02 | 齐鲁制药有限公司 | Purification method of pegylated recombinant human granulocyte colony stimulating factor |
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN104231037A (en) * | 2014-09-17 | 2014-12-24 | 哈德逊（天津）生物技术有限责任公司 | Rapid protein purification combination chromatographic column, kit and preparation method |
| CN105769926A (en) * | 2016-02-28 | 2016-07-20 | 汉中领维生物科技发展有限公司 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| The effect of chick embryo amniotic fluid on sciatic nerve regeneration of rats;Farjah G H等;《 Iranian Journal of Veterinary Research》;20150101;第16卷(第02期);167-171 * |
| 周国庆.生长因子.《生物化学》.2004,152-153. * |
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