CN109897094A - A kind of preparation method and application of epithelical cell growth factor - Google Patents
A kind of preparation method and application of epithelical cell growth factor Download PDFInfo
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- CN109897094A CN109897094A CN201711285671.7A CN201711285671A CN109897094A CN 109897094 A CN109897094 A CN 109897094A CN 201711285671 A CN201711285671 A CN 201711285671A CN 109897094 A CN109897094 A CN 109897094A
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- epidermal growth
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Abstract
The present invention relates to a kind of preparation method and applications of epithelical cell growth factor, after 4 DEG C of milk sample of freezing are thawed, it is centrifuged, dialysis, it extracts, purification step, obtain purifying epidermal growth factor factor solutions, with operating method simplicity, the advantages that biocompatibility of the EGF of extraction is high, and the epidermal growth factor solution of this method extraction purification can determine the dosage form of its preparation according to form of medication, excipient in acceptable pharmacopeia, diluent, carrier etc., the effect of mode being administered orally enables EGF acts on whole body, change the traditional administration mode of EGF, preferably play anti-aging effects.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to it is a kind of improve human body cell growth speed, have anti-aging
The preparation method and application of the epithelical cell growth factor (EGF) of effect.
Background technique
Epithelical cell growth factor (EGF) is strong mitogenic factor, and short time effect can enhance cell pair
The transfer of inorganic ions and amino acid, long duration of action can promote cell nucleic acid and protein, promote cell division.Research
Show denier EGF can intense stimulus cell growth, inhibit aging gene expression, prevent skin aging, make skin each group
Divide and keeps best physiological status.The discovery of EGF discloses the secret of human skin aging, is that the mankind recognize skin for the first time
Aging can reverse, skin can juvenescence again.
EGF is one of the necessary material for maintaining organism normal physiological condition, and pilose antler is as the most active growing part of deer body
Position is stored with a large amount of EGF, has vast potential for future development in terms of clinical treatment, however extracts in pilose antler, will cause pair
The injury of deer.In addition, being mostly currently on the market to be mainly used for postoperative wound by percutaneous drug delivery form about the product of EGF
Healing and scar reparation, effect limitation are too big.
Summary of the invention
The present invention is in view of the deficienciess of the prior art, providing a kind of preparation method of epithelical cell growth factor and answering
With, the epidermal growth factor easy to operate for obtaining nontoxic residue-free, processing step is as follows:
(1) material selection
The lotion of healthy milch goat, hand milking's mode sample, and when sampling discards preceding 5 milk, cold at -40 DEG C immediately after sampling
Jelly saves backup；
(2) centrifugal treating
After 4 DEG C of milk sample of freezing are thawed, milk sample is drawn with liquid-transfering gun and is placed in centrifuge tube, it is then low in 5000r/min
Warm (4 DEG C) are centrifuged 10min in high speed freezing centrifuge, remove upper layer butterfat, take supernatant；
(3) it dialyses
The supernatant is packed into the bag filter of molecular cut off 3500Da, 4 DEG C to 0.02mol/L Tris-HCL (8.0)
Then dialysis 30~50 minutes replaces buffer and repeats to dialyse, obtains dialyzate；
(4) it extracts
The dialyzate is crossed into Q Sepharose Fast Flow column (5.5cm*20cm), is eluted, flow velocity 2~
4ml/ minutes flow velocitys first elute foreign protein peak, 450 with 300~500ml 0.002mol/LTris-HCL (pH8.0) buffer
The buffer of the 0.002mol/LTris-HCL (pH8.0) of~650ml 0.5mol/L NaCl is eluted, and target protein is collected
Peak, and detected at 280nm wavelength with Ultraviolet Detector.
Preferably, in the dialysis step, dialysis number is 4-8 times.
Preferably, in the dialysis step, dialysis number is 6 times.
It preferably, further include purification step, the target protein peak i.e. epidermal growth factor that will be collected into is packed into retention molecule
The bag filter of 3500Da is measured, 4 DEG C are dialysed to 0.02mol/LTris-HCL (8.0) 30~50 minutes, are then replaced buffer and are repeated
Dialysis 4~8 times obtains purifying epidermal growth factor factor solutions.
It is new by obtaining epidermal growth factor with freeze drying technology and liposome embedded technology to epithelical cell growth factor
Dosage form determines that the dosage form of its preparation: excipient, diluent, carrier etc., the mode being administered orally make according to form of medication
The effect of EGF, can act on whole body.
The beneficial effects of the present invention are: 1, epidermal growth factor is extracted in goat milk, avoid the injury to deer, while sheep
Milk acquisition modes are easier.2, change the traditional administration mode of EGF, EGF is prepared by freeze drying technology and liposome embedded technology
Novel form, it is intended to which the effect of mode being administered orally enables EGF acts on whole body, preferably plays anti-aging effects.
Specific embodiment
Most preferred embodiment:
(1) material selection
The lotion of healthy milch goat, hand milking's mode sample, and when sampling discards preceding 5 milk, cold at -40 DEG C immediately after sampling
Jelly saves backup；
(2) centrifugal treating
After 4 DEG C of milk sample of freezing are thawed, milk sample is drawn with liquid-transfering gun and is placed in centrifuge tube, it is then low in 5000r/min
Warm (4 DEG C) are centrifuged 10min in high speed freezing centrifuge, remove upper layer butterfat, take supernatant；
(3) it dialyses
It is packed into the bag filter of molecular cut off 3500Da, 4 DEG C are dialysed to 0.02mol/L Tris-HCL (8.0) 40 minutes,
Then replacement buffer repeats to dialyse, and carries out 6 times altogether, obtains pretreated lotion；
(4) it extracts
The dialyzate is crossed into QSepharose Fast Flow column (5.5cm*20cm), is eluted, 2~4ml/ of flow velocity
Minute flow velocity, first with 300~500ml 0.002mol/LTris-HCL (pH8.0) buffer elute foreign protein peak, 450~
The buffer of the 0.002mol/LTris-HCL (pH8.0) of 650ml 0.5mol/L NaCl is eluted, and target protein is collected
Peak, and detected at 280nm wavelength with Ultraviolet Detector；
(5) it purifies
Target protein peak, that is, epidermal growth factor is collected, is packed into the bag filter of molecular cut off 3500Da, 4 DEG C right
0.02mol/L Tris-HCL (8.0) dialyses 30~50 minutes, then replaces buffer and repeats to dialyse, carries out 6 times, obtain pure altogether
Change epidermal growth factor factor solutions.
Best mode for carrying out the invention above described embodiment only expresses, the description thereof is more specific and detailed, but not
It can therefore be construed as limiting the scope of the patent.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of epithelical cell growth factor, it is characterised in that comprise the steps of:
(1) material selection
The lotion for choosing healthy milch goat is sampled, spare in -40 DEG C of freezen protectives immediately after sampling；
(2) centrifugal treating
After 4 DEG C of milk sample of freezing are thawed, milk sample is drawn with liquid-transfering gun and is placed in centrifuge tube, then in 5000r/min low temperature (4
DEG C) centrifugation 10min, upper layer butterfat is removed, supernatant is taken；
(3) it dialyses
The supernatant is packed into the bag filter of molecular cut off 3500Da, 4 DEG C are dialysed to 0.02mol/LTris-HCL (8.0)
It 30~50 minutes, then replaces buffer and repeats to dialyse, obtain dialyzate；
(4) it extracts
The dialyzate is crossed into Q Sepharose Fast Flow column (5.5cm*20cm), is eluted, 2~4ml/ of flow velocity points
The flow velocity of clock, first with 300~500ml 0.002mol/LTris-HCL (pH8.0) buffer elution foreign protein peak, 450~
The buffer of the 0.002mol/LTris-HCL (pH8.0) of 650ml 0.5mol/L NaCl is eluted, and target protein is collected
Peak, and detected at 280nm wavelength with Ultraviolet Detector.
2. preparation method as described in claim 1, it is characterised in that: it further include purification step, the target protein that will be collected into
Peak, that is, epidermal growth factor is packed into the bag filter of molecular cut off 3500Da, and 4 DEG C saturating to 0.02mol/L Tris-HCL (8.0)
Then analysis 30~50 minutes replaces buffer and repeats to dialyse, obtains purifying epidermal growth factor factor solutions.
3. preparation method as claimed in claim 1 or 2, which is characterized in that the material selection step, it is preferred to use artificial to squeeze
Milk mode is sampled, and when sampling discards preceding 5 milk.
4. preparation method as claimed in claim 1 or 2, which is characterized in that select high speed to freeze in the centrifugal treating step
Centrifuge is centrifuged.
5. preparation method as claimed in claim 1 or 2, which is characterized in that in the dialysis step, dialysis number is 4-8 times.
6. preparation method as claimed in claim 5, which is characterized in that in the dialysis step, dialysis number is 6 times.
7. preparation method as claimed in claim 2, which is characterized in that in the purification step, dialysis number is 4-8 times.
8. preparation method as claimed in claim 2, which is characterized in that in the purification step, dialysis number is 6 times.
9. using freeze-drying skill by the epithelical cell growth factor obtained to any one of claim 1,2,7,8 preparation method
The epidermal growth factor novel form that art and liposome embedded technology obtain, which is characterized in that the mode being administered orally makes EGF
The effect of can act on whole body.
10. epidermal growth factor novel form as claimed in claim 9, which is characterized in that determine its system according to form of medication
The dosage form of agent, excipient, diluent, carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711285671.7A CN109897094A (en) | 2017-12-07 | 2017-12-07 | A kind of preparation method and application of epithelical cell growth factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711285671.7A CN109897094A (en) | 2017-12-07 | 2017-12-07 | A kind of preparation method and application of epithelical cell growth factor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109897094A true CN109897094A (en) | 2019-06-18 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201711285671.7A Pending CN109897094A (en) | 2017-12-07 | 2017-12-07 | A kind of preparation method and application of epithelical cell growth factor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358538A (en) * | 2020-11-09 | 2021-02-12 | 甘肃黑驴王子生物科技有限公司 | Enrichment method of epidermal growth factor in fresh donkey milk |
-
2017
- 2017-12-07 CN CN201711285671.7A patent/CN109897094A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358538A (en) * | 2020-11-09 | 2021-02-12 | 甘肃黑驴王子生物科技有限公司 | Enrichment method of epidermal growth factor in fresh donkey milk |
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Application publication date: 20190618 |