CN1076699A - The production method of calf skin factor and application - Google Patents
The production method of calf skin factor and application Download PDFInfo
- Publication number
- CN1076699A CN1076699A CN92107331.3A CN92107331A CN1076699A CN 1076699 A CN1076699 A CN 1076699A CN 92107331 A CN92107331 A CN 92107331A CN 1076699 A CN1076699 A CN 1076699A
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- egf
- calf
- production method
- filtration
- somatomedin
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- 244000309466 calf Species 0.000 title claims description 18
- 238000004519 manufacturing process Methods 0.000 title claims description 4
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims abstract description 5
- 102000013275 Somatomedins Human genes 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 239000000654 additive Substances 0.000 claims abstract description 3
- 230000000996 additive effect Effects 0.000 claims abstract description 3
- 239000003814 drug Substances 0.000 claims abstract description 3
- 238000001035 drying Methods 0.000 claims abstract description 3
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 238000002523 gelfiltration Methods 0.000 claims abstract description 3
- 239000000047 product Substances 0.000 claims description 4
- 210000001913 submandibular gland Anatomy 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims description 2
- 239000001913 cellulose Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 210000000214 mouth Anatomy 0.000 claims description 2
- 229920005654 Sephadex Polymers 0.000 claims 1
- 239000012507 Sephadex™ Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 230000002496 gastric effect Effects 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 4
- 241000283690 Bos taurus Species 0.000 abstract description 3
- 238000001962 electrophoresis Methods 0.000 abstract description 3
- 238000001502 gel electrophoresis Methods 0.000 abstract description 2
- 238000004255 ion exchange chromatography Methods 0.000 abstract 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000008351 acetate buffer Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 3
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 206010053615 Thermal burn Diseases 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 239000003699 antiulcer agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to preparation method's technical field of somatomedin.Its objective is exploitation EGF at home application and squeeze into the international market with cheap advantage.Technical scheme of the present invention is to get little bovine submaxillary to add the extract extracting, filtration, centrifugal, and operations such as ion exchange chromatography, gel-filtration, frost drying are extracted.Gained EGF finished product purity can reach electrophoresis one band, and sds gel electrophoresis is surveyed molecular weight about 6000, and iso-electric point is about 5.0.The EGF that the present invention produces can be used as the raw material and the additive of medicine, makeup, healthcare products etc.
Description
The invention belongs to preparation method's technical field of somatomedin.
Urogastron (Epidermal Growth Factor, be called for short EGF) is that nineteen sixty Cohen separates from mouse submandibular gland the earliest, has the newborn mice of stimulation and opens eyes in advance and go out the tooth reaction.Gregory in 1975 from people's urine, be separated to one identical with the EGF structure and have the bioactive peptide of gastric acid inhibitory secreting function, be named stomach chalone (Urogastrone).State such as Japan reports medically that EGF has antiulcer agent in recent years, short wound, burn-healing, cornea reparation and easing pain and diminishing inflammation effect.External EGF has been used for makeup according to business information, and external EGF price is about every 0.1mg100 dollar.
The objective of the invention is to develop EGF at home application and squeeze into the international market with cheap advantage.The raw material difficulty that consideration is urinated this material of preparation from mouse and people, with high costs, successfully from little bovine submaxillary, be separated to a kind of skins somatomedin through test of many times, the little CEGF of called after is called for short calf EGF.
Calf EGF has the character of EGF, can stimulate the culturing cell growth and breeding, and short DNA is synthetic.The whole animal test has healing of the burn surface of promotion and analgesic effect, and purity can reach electrophoresis one band, and sds gel electrophoresis is surveyed molecular weight about 6000, and iso-electric point is about 5.0.
Technical scheme of the present invention is to get little bovine submaxillary to add extract, homogenate extracting 6 hours, coarse filtration, 4000 rev/mins centrifugal 30 minutes, supernatant liquor is collected active ingredient through CM-Mierocrystalline cellulose and DEAE-cellulose chromatography, after concentrating, again after the SephadexG75 gel-filtration, collect active peak, frost drying is calf EGF finished product.
The determination of activity of the calf EGF of the present invention's preparation:
(1) short culturing cell DNA is synthetic: be mixed with 5 * 10 with the 3T3 cell of cultivating
4Cell/ml adds 24 well culture plates, every hole 1ml, add simultaneously different concns (5,10, calf EGF0.1ml 20.40ng), control group do not add EGF and add with volume physiological saline put 37 ℃ cultivate 18 hours after, add 3 hydrogen mark thymidines (
3H-TdR) continue to cultivate 6 hours in 2 μ Ci/20 μ l/ holes, abandons nutrient solution, washed 3 times with physiological saline after, fix 3 times with 5%TCA, add 0.1N NaOH200 μ l/ hole again.From the hole, quantitatively take out 100 μ l/ holes and put on the film of 4-9 type Lu, dry rearmounted liquid flashing determining bottle, add 5ml liquid sudden strain of a muscle liquid and on the liquid flashing determining instrument, survey count per minute (cpm) calculating
3H-TdR incorporation and control group relatively calculate calf EGF activity.Every sample is measured three parts simultaneously.
(2) scalding healing test: small white mouse 20～40g20 male and female half and half, be divided into two groups, cut off the hair 2 * 2cm at back, put 80 ℃ of water-bath preheatings with the boiling hot tool of 15mm diameter, every mouse scalded for 20 seconds, cause the II degree to scald, scald began in back second day with calf EGF different concns (2,5,10ng/ml) exterior coating treatment, control group is coated with physiological saline and controls, calf EGF treatment group forms crust after 10 days, and contrast then can not form calf EGF treatment group decrustation wound healing after 14 days, incrustation just appears in control group after 17 days, the activity of contrast statistical confirmation calf EGF treatment group.
The EGF cost that the present invention produces is lower; price is 1mg/400 yuan at present; can be used for the raw material of burn curative and some skin and mucous membrane (oral cavity, digestive tube etc.) protection medicine, also can be used for the additive of makeup and healthcare products and the biochemical reagents of cell and tissue culture.
Be embodiments of the invention below:
Get calf submaxillary gland 500g, add 1500mlpH3.9,0.05M acetate buffer, homogenizer homogenate, put and stir extracting 6 hours in the ice bath, standing over night, after 4 layers of filtered through gauze, centrifugal 30 minutes of 4000rpm, collect supernatant, last CM52 post (having used pH3.9 acetate buffer balance) column volume 5 * 15cm collects the protein peak that passes, and uses NH
4OH transfers pH to 7.0, then goes up the DE52 post and (has used pH7.0,0.05M the column volume 3 * 10cm Ammoniom-Acetate cushioning balance) uses 100mlpH7.0 earlier, after the 0.05M Ammoniom-Acetate is washed post, with pH7.0,0.05M acetate buffer → pH3.9,0.05M acetate buffer, each 200ml straight line gradient elution of 0.15MNacl, fraction collection and detection vigor, merging vigor peak, freeze-drying concentrate the back and go up SephadexG75 fraction collection detection vigor, collect the freeze-drying again of vigor peak.Get about calf EGF20mg at last.
SDS electrophoretic examinations purity and supposition molecular weight.Get calf EGF product 2mg and carry out SDS-PAGE by the Laemmli method, gum concentration 12%.Did standard protein (the biochemical standard molecular weight that provides in Shanghai is 94000,64000,34000 and 12500) electrophoresis simultaneously 6 hours, and calculated according to standard molecular weight, molecular weight is about 6000.
Claims (3)
1, a kind of skins somatomedin calf EGF production method is characterized in that with calf submaxillary gland separation and Extraction.
2, production method according to claim 1, it is characterized in that getting calf submaxillary gland oiling extract homogenate extracting, coarse filtration, 4000 rev/mins are centrifugal 30 minutes, supernatant liquor is through CM-Mierocrystalline cellulose and DEAE cellulose chromatography, collect active ingredient concentrate after again through Sephadex G75, gel-filtration is collected active peak, frost drying.
3, a kind of skins somatomedin calf EGF is characterized in that, can be used for the raw material of burn curative, the additive of producing medicine, makeup and the healthcare products of protecting skin and oral cavity and gastrointestinal mucosal and the biochemical reagents of cell and tissue culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92107331.3A CN1076699A (en) | 1992-03-21 | 1992-03-21 | The production method of calf skin factor and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92107331.3A CN1076699A (en) | 1992-03-21 | 1992-03-21 | The production method of calf skin factor and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1076699A true CN1076699A (en) | 1993-09-29 |
Family
ID=4942823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92107331.3A Pending CN1076699A (en) | 1992-03-21 | 1992-03-21 | The production method of calf skin factor and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1076699A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1113896C (en) * | 1998-01-04 | 2003-07-09 | 刘荣秀 | Preparation of epidermal growth factor with marine products and relevant discard |
| CN108014036A (en) * | 2016-11-04 | 2018-05-11 | 无锡法瑞雅生物细胞科学有限公司 | A kind of peel off pack and preparation method thereof |
-
1992
- 1992-03-21 CN CN92107331.3A patent/CN1076699A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1113896C (en) * | 1998-01-04 | 2003-07-09 | 刘荣秀 | Preparation of epidermal growth factor with marine products and relevant discard |
| CN108014036A (en) * | 2016-11-04 | 2018-05-11 | 无锡法瑞雅生物细胞科学有限公司 | A kind of peel off pack and preparation method thereof |
| CN108014036B (en) * | 2016-11-04 | 2020-10-09 | 无锡法瑞雅生物细胞科学有限公司 | Peel-off facial mask and preparation method thereof |
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