CN102372777B - Method for purifying natural serine protease inhibitors - Google Patents
Method for purifying natural serine protease inhibitors Download PDFInfo
- Publication number
- CN102372777B CN102372777B CN201110339033.5A CN201110339033A CN102372777B CN 102372777 B CN102372777 B CN 102372777B CN 201110339033 A CN201110339033 A CN 201110339033A CN 102372777 B CN102372777 B CN 102372777B
- Authority
- CN
- China
- Prior art keywords
- minutes
- supernatant liquor
- centrifugal
- per minute
- natural
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to a method for purifying natural serine protease inhibitors, which comprises the steps of silkworm pupa pretreatment, primary separation purification of the natural serine protease inhibitors, and the separation purification of the natural serine protease inhibitors. The method has the advantages that silkworm pupas are used as raw materials, the natural serine protease inhibitors with the medicinal value in the silkworm pupas are obtained through separation and purification, the method is simple and convenient to operate, the yield is high, the method is suitable for large-scale production, the purified natural serine protease inhibitors can be used as health-care medicine, and no toxic or side effect is caused.
Description
Technical field
The invention belongs to a kind of purification process of natural serpin.
Background technology
Silkworm chrysalis is the pupal cell stayed after the black silk of the silk cocoon of Bombycidae insect silkworm.Its wide material sources, be considered to the mankind's a kind of new nutrition source, is unique insects food in " as the new resource for food list of bread and cheese management " of Ministry of Health's approval.Silkworm chrysalis has high nutritive value, contains rich in protein, lipid acid, VITAMIN.Wherein contained serpin (serpin, abbreviation SP), can be used as proteinase inhibitor and participate in blood coagulation reaction, fibrolysis, vasculogenesis, complement activation, immunomodulatory and inflammatory reaction etc.Also have minority to carry out non-inhibit feature: to play a role as hormone transport molecule, molecular chaperones and tumor-inhibiting factor.They play critical regulating and controlling effect in multiple physiology system in human body, are the important factors that maintains homoiostasis.But at present, substantially also do not occur on market usining that in silkworm chrysalis, effective constituent, as the product of drug development, lacks the purification process of natural serpin.
Summary of the invention
The objective of the invention is to design a kind of purification process of natural serpin, utilize silkworm chrysalis as raw material, obtain the natural serpin with pharmaceutical use by separation and purification, the method is easy and simple to handle, and output is high, be applicable to scale operation, the natural serpin of purification can be used as health medicine, has no side effect.
For this reason, of the present inventionly comprise lower step:
(1) silkworm chrysalis pre-treatment, by the 50mM phosphoric acid buffer of fresh silkworm chrysalis and 4 ℃ of precoolings, pH7.4, by joining in juice extractor homogenate at 1: 1 30 seconds, ice bath on ice 5 minutes, continue homogenate, and triplicate, fully dissolve the albumen in silkworm chrysalis; Homogenate is transferred in Centrifuge Cup, and 4 ℃, per minute 15000 turns, and centrifugal 20 minutes, get supernatant liquor, supernatant liquor is crossed nine layers of filtered through gauze except degrease, repeated centrifugation and filtration step three times; Collect the supernatant liquor after filtering, 4 ℃, per minute 15000 turns, and centrifugal 20 minutes, 0.45 μ m filter membrane vacuum filtration for supernatant liquor, 4 ℃ of preservations;
(2) the initial gross separation purifying of natural serpin, be placed in 50 ℃ of waters bath with thermostatic control 30 minutes by supernatant liquor, then put whizzer per minute 15000 and turn, and centrifugal 20 minutes, gets supernatant;
(3) separation and purification of natural serpin, supernatant liquor is placed in to 100 ℃ of waters bath with thermostatic control 30 minutes, putting whizzer per minute 15000 turns again, centrifugal 20 minutes, get supernatant, 100 ℃ of waters bath with thermostatic control repeat this step for several times, until can obtain natural serpin without Precipitation again.
Several in described step (3) is 3 to 5 times.Aforesaid method has been realized purpose of the present invention.
Advantage of the present invention is to utilize silkworm chrysalis as raw material, obtain natural serpin in the silkworm chrysalis with pharmaceutical use by separation and purification, the method is easy and simple to handle, and output is high, be applicable to scale operation, the natural serpin of purifying can be used as health medicine, has no side effect.
The inhibitor that to make by the inventive method is used gelatin-polyacrylamide gel active coloring method to carry out activity and detects.Run non-metagelatin-polyacrylamide gel after the inhibitor ultrafiltration and concentration, adhesive tape is cut off, respectively at 25 μ g/ml trypsinase, pH8.0, under the condition of 37 ℃ of water-baths, enzymolysis 30 minutes, 60 minutes, 2 hours, 3 hours, 12 hours, then, by the adhesive tape coomassie brilliant blue staining after enzymolysis, result proves that resulting inhibitor enzymolysis still keeps its biological activity after 3 hours.
Embodiment
A kind of purification process of natural serpin.Comprise lower step: (1) silkworm chrysalis pre-treatment, by the 50mM phosphoric acid buffer of fresh silkworm chrysalis and 4 ℃ of precoolings, pH7.4, by joining in juice extractor homogenate at 1: 1 30 seconds, ice bath on ice 5 minutes, continue homogenate, triplicate, fully dissolve the albumen in silkworm chrysalis; Homogenate is transferred in Centrifuge Cup, and 4 ℃, per minute 15000 turns, and centrifugal 20 minutes, get supernatant liquor, supernatant liquor is crossed nine layers of filtered through gauze except degrease, repeated centrifugation and filtration step three times; Collect the supernatant liquor after filtering, 4 ℃, per minute 15000 turns, and centrifugal 30 minutes, 0.45 μ m filter membrane vacuum filtration for supernatant liquor, 4 ℃ of preservations.(2) the initial gross separation purifying of natural serpin, be placed in 50 ℃ of waters bath with thermostatic control 30 minutes by supernatant liquor, then put whizzer per minute 15000 and turn, and centrifugal 20 minutes, gets supernatant.(3) separation and purification of natural serpin, supernatant liquor is placed in to 100 ℃ of waters bath with thermostatic control 30 minutes, putting whizzer per minute 15000 turns again, centrifugal 20 minutes, get supernatant, 100 ℃ of waters bath with thermostatic control repeat this step 3 to 5 times, until can obtain natural serpin without Precipitation again.The natural serpin obtained is aseptic subpackaged, and-50 ℃ of lyophilizes, make lyophilized powder.Detect with 12% polyacrylamide gel electrophoresis and detect sample, the band of target protein is arranged at the 42kDa place.
In a word, the present invention can utilize silkworm chrysalis as raw material, obtains the natural serpin with pharmaceutical use by separation and purification, the method is easy and simple to handle, and output is high, is applicable to scale operation, the natural serpin of purifying can be used as health medicine, has no side effect.
Claims (2)
1. the purification process of a natural serpin is characterized in that: comprise lower step,
(1) silkworm chrysalis pre-treatment, by the 50mM phosphoric acid buffer of fresh silkworm chrysalis and 4 ℃ of precoolings, pH7.4, by joining in juice extractor homogenate at 1: 1 30 seconds, ice bath on ice 5 minutes, continue homogenate, and triplicate, fully dissolve the albumen in silkworm chrysalis; Homogenate is transferred in Centrifuge Cup, and 4 ℃, per minute 15000 turns, and centrifugal 20 minutes, get supernatant liquor, supernatant liquor is crossed nine layers of filtered through gauze except degrease, repeated centrifugation and filtration step three times; Collect the supernatant liquor after filtering, 4 ℃, per minute 15000 turns, and centrifugal 30 minutes, 0.45 μ m filter membrane vacuum filtration for supernatant liquor, 4 ℃ of preservations;
(2) the initial gross separation purifying of natural serpin, be placed in 50 ℃ of waters bath with thermostatic control 30 minutes by supernatant liquor, then put whizzer per minute 15000 and turn, and centrifugal 20 minutes, gets supernatant;
(3) separation and purification of natural serpin, supernatant liquor is placed in to 100 ℃ of waters bath with thermostatic control 30 minutes, putting whizzer per minute 15000 turns again, centrifugal 20 minutes, get supernatant, 100 ℃ of waters bath with thermostatic control repeat this step for several times, until can obtain natural serpin without Precipitation again.
2. by the purification process of natural serpin claimed in claim 1, it is characterized in that: the several in described step (3) is 3 to 5 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110339033.5A CN102372777B (en) | 2011-11-01 | 2011-11-01 | Method for purifying natural serine protease inhibitors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110339033.5A CN102372777B (en) | 2011-11-01 | 2011-11-01 | Method for purifying natural serine protease inhibitors |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102372777A CN102372777A (en) | 2012-03-14 |
| CN102372777B true CN102372777B (en) | 2013-12-25 |
Family
ID=45792046
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110339033.5A Expired - Fee Related CN102372777B (en) | 2011-11-01 | 2011-11-01 | Method for purifying natural serine protease inhibitors |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102372777B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103044542B (en) * | 2012-08-17 | 2015-08-19 | 常熟理工学院 | Serpin and gene thereof and application in crucian ovum |
| CN104382098B (en) * | 2014-11-17 | 2017-05-03 | 江南大学 | Method for improving gel strength of minced fish product by adding silkworm chrysalis and silkworm chrysalis containing minced fish product processed through method |
| CN106478812B (en) * | 2016-10-21 | 2020-11-20 | 沈阳药科大学 | Serine protease inhibitor-3, and function, preparation method and application thereof |
-
2011
- 2011-11-01 CN CN201110339033.5A patent/CN102372777B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102372777A (en) | 2012-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10414795B2 (en) | Techniques of preparing collagen active peptides | |
| CN104745663B (en) | A kind of method of PINPROL comprehensive utilization | |
| CN103052717B (en) | Industrial production method for producing antihypertensive bioactive peptide | |
| US10005823B2 (en) | Peptides having effects on promoting wound healing, collagen production, angiogenesis, activation of immunocytes and applications thereof | |
| CN111892644B (en) | Memory improving active peptide for protecting oxidative stress of nerve cells and preparation method thereof | |
| CN101974589B (en) | Method for preparing immunocompetent soybean peptide by enzymolysis and membrane separation | |
| CN104313099B (en) | A kind of preparation method of the active egg white powder peptide of high anti-oxidation | |
| CN102372777B (en) | Method for purifying natural serine protease inhibitors | |
| CN104513843A (en) | Joint preparation method of polysaccharide and protein peptide | |
| RU2019122797A (en) | REMOVAL OF NUCLEIC ACIDS AND THEIR FRAGMENTS FROM BIOMASS MATERIAL | |
| CN109468357A (en) | A kind of preparation method of spleen aminopeptide | |
| CN104610430A (en) | Antioxidant peptide prepared by utilizing lucid ganoderma albumen and preparation method thereof | |
| CN109355337A (en) | Method for preparing active collagen polypeptide from fresh pigskin | |
| CN106146644A (en) | Antithrombotic peptide and directional enzymatic preparation method thereof | |
| CN101353687A (en) | Melissa powder peptides having angiotensin transferase inhibitory activity and preparation thereof | |
| CN106589068B (en) | Sea bream antioxidant polypeptide and preparation method thereof | |
| CN103740797B (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| CN103695513A (en) | Method for improving yield of soybean peptide with low molecular weight | |
| CN103320488B (en) | Method for preparing ACE (Angiotensin Converting Enzyme) inhibitory peptides through proteolysis of eucommia seed cakes | |
| CN110042139A (en) | A kind of preparation method and applications of animal blood activity complex peptides | |
| CN106434816B (en) | A method of reducing blood lipid peptide is produced with leech | |
| CN108191991A (en) | A kind of method of purification of Polysaccharide in Pleurotus eryngii | |
| CN103184261A (en) | Co-production processing process of pharmaceutical gelatin and high-grade collagen | |
| CN112625120B (en) | Process for continuous large-scale production of recombinant collagen | |
| CN106146608A (en) | A kind of extraction preparation method of apidaecin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: TIANJIN?INTERNATIONAL?JOINT?ACADEMY?OF?BIOTECHNOLO Effective date: 20130531 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130531 Address after: 300457 No. 220 Dongting Road, Tianjin economic and Technological Development Zone, Binhai New Area, Tianjin Applicant after: TEFEI (TIANJIN) BIOMEDICINE TECHNOLOGY CO.,LTD. Applicant after: Tianjin International Joint Academy of Biotechnology & Medicine Address before: 300457 No. 220 Dongting Road, Tianjin economic and Technological Development Zone, Binhai New Area, Tianjin Applicant before: TEFEI (TIANJIN) BIOMEDICINE TECHNOLOGY CO.,LTD. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131225 Termination date: 20211101 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |