CN106834395A - A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified - Google Patents
A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified Download PDFInfo
- Publication number
- CN106834395A CN106834395A CN201710244487.1A CN201710244487A CN106834395A CN 106834395 A CN106834395 A CN 106834395A CN 201710244487 A CN201710244487 A CN 201710244487A CN 106834395 A CN106834395 A CN 106834395A
- Authority
- CN
- China
- Prior art keywords
- zymotic fluid
- ludon
- lepirudin
- purified
- isolated
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
Abstract
The invention discloses a kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified, belong to biotechnological pharmaceuticses field.Take HV3Glycerine strain cultivation and fermentation obtains zymotic fluid, zymotic fluid is centrifuged, obtain fermented liquid supernatant liquid, fermented liquid supernatant liquid 12M salt acid for adjusting pH, the processing mode heated in fermentation tank is put into afterwards, foreigh protein removing is effectively removed, the difficulty of subsequent purification technique is substantially reduced, the operability for producing technique in enormous quantities is improved.Gained zymotic fluid takes out the method using filter filtering after being kept for 28 DEG C stand from tank, removal foreign protein and lipid material, improve the operability for producing technique in enormous quantities, the present invention is effective according to the separation purifying technique that hirudin design feature is designed, pigment content is substantially reduced while improving purity of protein, technological operation is easy, cost is relatively low.
Description
Technical field
The invention belongs to biological pharmacy technical field, and in particular to the lepirudin 023 ludon for being adapted to industrialized production is isolated and purified
Method.
Background technology
At present, hirudin is a kind of Acid polypeptide of leeches animal saliva glandular secretion, containing 65~66 amino acid residues,
Mainly there is HV1、HV2、HV3This 3 kinds of isomers, have structural homology very high between them.Hirudin is the work for finding so far
With most strong thrombin inhibitor, hirudin (HV3) protein expression engineering bacteria bacterial activity depend on anticoagulation enzyme activity
Property intensity, it is with fibrin ferment with equimolar than forming the stable compound that non-covalent bond is combined closely.Pharmacology and clinical research
Show, hirudin can be used for the related thrombopenia and disseminated intravascular coagulation of DVT, II type heparin, and
Antithrombotic etc. when haemodialysis and extracorporal circulatory system.Compared with traditional anticoagulant such as heparin, aspirin, hirudin
Consumption is small, curative effect is high, adverse reaction is few and it is light, will not cause allergic reaction.Lepirudin 023 ludon turned into it is a kind of it is efficient, single-minded,
New anti-freezing, the anti-bolt medicine of safety, with wide potential applicability in clinical practice.
The processing mode in fermentation tank is put into after being heated using large quqntitics of fermentation liquid when being isolated and purified due to current hirudin,
It is unsuitable for mass industrialized production, and when foreign protein and lipid material is removed, the method for taking low-temperature centrifugation, high-volume
The poor operability of production, and purity of protein is low, pigment content is higher, high cost.Albumen need to be improved when isolating and purifying hirudin
Pigment content is substantially reduced while purity, makes technological operation simplicity, cost relatively low, and suitable industrial amplification production is depended on
New technology and method.
The content of the invention
It is an object of the invention to provide a kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified, using hair
The processing mode that zymotic fluid supernatant is adjusted by pH and upper tank is heated, pigment content is substantially reduced while improving purity of protein,
Make technological operation easy；Foreign protein and lipid material, reduces cost are removed using the method filtered after stand at low temperature, and improves work
The operability of skill, is adapted to industrialized production.
To achieve these goals, technical solution of the present invention is as follows：
The present invention provides a kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified, and step is as follows：
(1) hirudin (HV is taken3) protein expression engineering bacteria Spawn incubation fermentation obtain zymotic fluid, zymotic fluid is centrifuged, obtain
Fermented liquid supernatant liquid, it is standby；
(2) pH is adjusted to 3.8-4.2 by above-mentioned fermented liquid supernatant liquid hydrochloric acid, is put into afterwards in fermentation tank and is heated to 78-
It is 82 DEG C, standby；
(3) gained zymotic fluid in step (2) is taken out into 2-8 DEG C of standing of holding from tank, fermented liquid supernatant is taken after being layered
Liquid, it is standby；
(4) fermented liquid supernatant liquid in step (3) is filtered with 0.45 μm of filter；Zymotic fluid after filtering is carried out
Isolate and purify and obtain destination protein active part.
Preferably, it is adapted to the method that the lepirudin 023 ludon of industrialized production is isolated and purified, Spawn incubation fermentation in step (1)
The step of it is as follows：
A, the hirudin (HV that will be preserved3) protein expression engineering bacteria strain keeps constant temperature static on LBA solid mediums
Culture 11-13 hours, obtains single bacterium colony, standby；
B, above-mentioned single bacterium colony is chosen, be inoculated in LBA fluid nutrient mediums and keep constant-temperature table culture 14-16 hours, obtain one
Level seed liquor, it is standby；
C, the above-mentioned primary seed solution of absorption, continuation is cultivated in being inoculated in LBA fluid nutrient mediums, keeps constant-temperature table culture
14-16 hours, secondary seed solution is obtained, it is standby；
D, the OD600nm values for monitoring above-mentioned secondary seed solution, when OD600nm values reach 4.0-4.4, start stream plus mend carbon
Culture medium, by dissolved oxygen control in 20%-40% during feed supplement, when continuous 4.5-5.5 hour of OD600 no longer increases, stopping
Carbon is mended, is started slow stream plus is mended nitrogen culture medium, treat hirudin (HV3) protein expression engineering bacteria bacterial activity when no longer increasing, stops
Nitrogen is only mended, fermentation to thalline terminates fermentation, zymotic fluid is obtained when entering self-dissolving stage, the decline of Product formation ability, standby；
Preferably, it is adapted to the method that isolates and purifies of lepirudin 023 ludon of industrialized production, A-C the step of Spawn incubation ferments
In, thermostatic control is at 36.5-37.5 DEG C.
Preferably, it is adapted to the method that isolates and purifies of lepirudin 023 ludon of industrialized production, A-C the step of Spawn incubation ferments
In, thermostatic control is at 37 DEG C.
Preferably, it is adapted to the method that the lepirudin 023 ludon of industrialized production is isolated and purified, in step (2), fermented supernatant fluid
PH is adjusted to 4.0 with 12M hydrochloric acid, zymotic fluid is put into 80 DEG C is heated in tank.
It is highly preferred that the method that the lepirudin 023 ludon for being adapted to industrialized production is isolated and purified, zymotic fluid is put into tank and is added
Hot to 80 DEG C are kept for 50-70 minutes, effectively except foreigh protein removing.
Compared with prior art, the invention has the advantages that：
Key to the invention is that being adapted to the design of the lepirudin 023 ludon separation purifying technique of industrialized production.It is experimentally confirmed that
Using designed separation purifying technique, the processing mode that fermented liquid supernatant liquid is adjusted by pH and upper tank is heated improves albumen
Pigment content is substantially reduced while purity, makes technological operation easy；Foreign protein is removed using the method filtered after stand at low temperature
And lipid material, reduces cost, and the operability of technique is improved, it is adapted to industrialized production.
Brief description of the drawings
Fig. 1 is the HV provided in specific embodiment step 43Fermentation process cell concentration OD600 values and supernatant are anti-
Thrombin activity.
Specific embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to specific embodiment to this hair
The method that the lepirudin 023 ludon of the suitable industrialized production of bright offer is isolated and purified is described in further detail.
Hirudin (HV3) protein expression engineering bacteria, see the China Medicine University in ZhongYang Road, NanJing, Jiangsu Province Tong Jia lanes 24
" novel secretion expression vector and its application in recombinant hirudin expression technology " of Tan Shuhua, Wu Wutong application, patent Shen
Please number：01113526.3, authorized announcement date：20031119.
Step 1：Actication of culture
By hirudin (HV3) protein expression engineering bacteria strain one, in thaw at RT, is inoculated with, in LBA solids with collarium is connect
Rule on culture medium, be upside down in constant incubator, keep 37 DEG C of constant temperature static gas wave refrigerator 12 hours, obtain the circle of regular shape
Single bacterium colony；
Step 2：First order seed culture
From LBA solid mediums, selected shape rule, circular, single bacterium colony, picking single bacterium colony is inoculated in LBA liquid
37 DEG C of constant temperature is kept in body culture medium shaking table culture 14-16 hours, obtain primary seed solution；
Step 3：Secondary seed culture
5% primary seed solution is drawn, continuation is cultivated in being inoculated in LBA fluid nutrient mediums, keep 37 DEG C of shaking tables trainings of constant temperature
Support 14-16 hours, secondary seed solution is obtained, it is standby；
Step 4：Fermentation
The OD600nm values of above-mentioned secondary seed solution are monitored, when OD600nm values reach 4.2 or so, is started stream plus is mended carbon training
Support base, according to the change of dissolved oxygen, the speed of feed supplement is exponentially or linearly increasing, during feed supplement by dissolved oxygen control 20%~
40%, when OD600 no longer increases for continuous 5 hours or so, stop mending carbon, start slow stream plus mend nitrogen culture medium, treat anticoagulation
When enzymatic activity no longer increases, stop mending nitrogen, fermentation to thalline terminates fermentation when entering self-dissolving stage, the decline of Product formation ability,
Zymotic fluid is obtained, 20 hours or so are about during fermentation, as shown in Figure 1；
Step 5：Fermentation liquor pretreatment
Above-mentioned zymotic fluid is centrifuged, fermented liquid supernatant liquid is obtained, be adjusted to for pH with 12M hydrochloric acid by fermented liquid supernatant liquid
4.0, it is put into afterwards in fermentation tank and is heated to 80 DEG C, zymotic fluid is taken out into 2-8 DEG C of holding from tank after being kept for 60 minutes stands, and treats
Fermented liquid supernatant liquid is taken after layering, with 4 layers of filtered through gauze, and 0.45 μm of filter filtering；
Step 6：Zymotic fluid is purified
(1) cationic ion-exchange resin SP sepharose Fast Flow
Dress SP sepharose Fast Flow cation exchange resin columns, remove resin and preserve using deionized water rinsing
Liquid, is balanced with 50mM citric acids-NaOH (pH3.0) buffer solution, and the zymotic fluid after filtering is passed through with the speed loading of 5ml/min.
With 50mM citric acids-NaOH (pH3.0) buffer solutions and 50mM citric acids-NaOH (pH4.25) buffer solution wash away part foreign protein and
Pigment, wash-out destination protein is carried out using 50mM citric acids c-NaOH (pH5.5) buffer solution.Often step is collected through liquid, fibrin ferment
Titration is surveyed than living, and record data collects active part.
(2) fast protein liquid chromatography Fast protein liquid chromatography (FPLC)
SP sepharose Fast Flow cation-exchange chromatographies eluents are isolated and purified through C18 reversed-phase columns.
Used as mobile phase, A phases are 0.2%TFA/dd H to methanol-water2O, B phase are methyl alcohol, flow velocity 20ml/min, Detection wavelength 280nm.
Methanol-water gradient elution system：
Reversed-phase column 30mim is parallel to baseline for 15%B balances；0-30min 30%B are used successively；30-40min, 65%B；
40-50min, 60%-100%B.Collect active part.
Hirudin (the HV after purification of table 13) the rate of recovery
When removing foreigh protein removing and lipid material in step 5, taken after large quqntitics of fermentation liquid tank external heat in conventional art
The processing mode of low-temperature centrifugation is inconvenient to operate, and the present invention takes fermented liquid supernatant to be adjusted by pH and 80 DEG C of holdings of upper tank heating
60 minutes afterwards stand at low temperature and filter processing mode, effectively except foreigh protein removing, substantially reduce the difficulty of subsequent purification technique
Degree, improves the operability of technique, and cost is relatively low, and is adapted to industrialized production.
The method that the lepirudin 023 ludon of suitable industrialized production provided by the present invention is isolated and purified has been carried out in detail above
It is thin to introduce.Specific case used herein is set forth to principle of the invention and implementation method, and above example is said
It is bright to be only intended to help and understand core concept of the invention.It should be pointed out that for those skilled in the art,
Under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention, these improve and modify
Fall into the protection domain of the claims in the present invention.
Claims (6)
1. a kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified, it is characterised in that step is as follows：
(1) hirudin (HV is taken3) protein expression engineering bacteria Spawn incubation fermentation obtain zymotic fluid, zymotic fluid is centrifuged, obtain zymotic fluid
Supernatant, it is standby；
(2) pH is adjusted to 3.8-4.2 by above-mentioned fermented liquid supernatant liquid hydrochloric acid, is put into afterwards in fermentation tank and is heated to 78-82 DEG C,
It is standby；
(3) gained zymotic fluid in step (2) is taken out into 2-8 DEG C of standing of holding from tank, fermented liquid supernatant liquid is taken after being layered, it is standby
With；
(4) fermented liquid supernatant liquid in step (3) is filtered with 0.45 μm of filter；Zymotic fluid after filtering is separated
Purifying obtains destination protein active part.
2. the method that the lepirudin 023 ludon of suitable industrialized production according to claim 1 is isolated and purified, it is characterised in that
The step of Spawn incubation ferments in step (1) is as follows：
A, the hirudin (HV that will be preserved3) protein expression engineering bacteria strain keeps constant temperature static gas wave refrigerator on LBA solid mediums
11-13 hours, single bacterium colony is obtained, it is standby；
B, above-mentioned single bacterium colony is chosen, be inoculated in LBA fluid nutrient mediums and keep constant-temperature table culture 14-16 hours, obtain one-level kind
Sub- liquid, it is standby；
C, the above-mentioned primary seed solution of absorption, continuation is cultivated in being inoculated in LBA fluid nutrient mediums, keeps constant-temperature table culture 14-16
Hour, secondary seed solution is obtained, it is standby；
D, the OD600nm values for monitoring above-mentioned secondary seed solution, when OD600nm values reach 4.0-4.4, start stream plus mend carbon culture
Base, by dissolved oxygen control in 20%-40% during feed supplement, when continuous 4.5-5.5 hour of OD600 no longer increases, stops benefit carbon,
Start slow stream plus mend nitrogen culture medium, treat hirudin (HV3) protein expression engineering bacteria bacterial activity when no longer increasing, stops mending
Nitrogen, fermentation to thalline terminates fermentation when entering self-dissolving stage, the decline of Product formation ability, and zymotic fluid is obtained, standby.
3. the method that the lepirudin 023 ludon of suitable industrialized production according to claim 2 is isolated and purified, it is characterised in that
In each step of Spawn incubation fermentation, thermostatic control is at 36.5-37.5 DEG C.
4. the method that the lepirudin 023 ludon of suitable industrialized production according to claim 3 is isolated and purified, it is characterised in that
In each step of Spawn incubation fermentation, thermostatic control is at 37 DEG C.
5. the method that the lepirudin 023 ludon of suitable industrialized production according to claim 1 is isolated and purified, it is characterised in that
In step (2), pH is adjusted to 4.0 by fermented supernatant fluid with 12M hydrochloric acid, zymotic fluid is put into 80 DEG C are heated in tank.
6. the method that the lepirudin 023 ludon of suitable industrialized production according to claim 5 is isolated and purified, it is characterised in that
Zymotic fluid is put into tank be heated to 80 DEG C keep 50-70 minutes, effectively except foreigh protein removing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710244487.1A CN106834395A (en) | 2017-04-14 | 2017-04-14 | A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710244487.1A CN106834395A (en) | 2017-04-14 | 2017-04-14 | A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106834395A true CN106834395A (en) | 2017-06-13 |
Family
ID=59146993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710244487.1A Pending CN106834395A (en) | 2017-04-14 | 2017-04-14 | A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106834395A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107353338A (en) * | 2017-07-27 | 2017-11-17 | 宁波博睿修存生物科技有限公司 | A kind of method for separating pigment molecular in hirudin zymotic fluid |
| CN107459572A (en) * | 2017-07-27 | 2017-12-12 | 宁波博睿修存生物科技有限公司 | A kind of method of hirudin in concentrated broth |
| CN109957008A (en) * | 2019-04-16 | 2019-07-02 | 广东双骏生物科技有限公司 | The method for extraction and purification of hirudin mutant and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373216A (en) * | 2011-10-28 | 2012-03-14 | 元昊 | Construction of recombinant eukaryotic hansenula engineering bacterial strains containing medical hirudin genes and production process of recombinant hirudin |
| CN102965419A (en) * | 2011-08-28 | 2013-03-13 | 杭州路富生物科技有限公司 | Fermentation optimization technology of recombinant human growth hormone engineering bacteria |
-
2017
- 2017-04-14 CN CN201710244487.1A patent/CN106834395A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965419A (en) * | 2011-08-28 | 2013-03-13 | 杭州路富生物科技有限公司 | Fermentation optimization technology of recombinant human growth hormone engineering bacteria |
| CN102373216A (en) * | 2011-10-28 | 2012-03-14 | 元昊 | Construction of recombinant eukaryotic hansenula engineering bacterial strains containing medical hirudin genes and production process of recombinant hirudin |
Non-Patent Citations (2)
| Title |
|---|
| 周卫斌等: ""毕赤酵母发酵液的脱色和重组水蛭素的分离"", 《生物工程学报》 * |
| 崔莉等: ""重组水蛭素Ⅲ的工程菌的培养及高密度发酵"", 《药物生物技术》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107353338A (en) * | 2017-07-27 | 2017-11-17 | 宁波博睿修存生物科技有限公司 | A kind of method for separating pigment molecular in hirudin zymotic fluid |
| CN107459572A (en) * | 2017-07-27 | 2017-12-12 | 宁波博睿修存生物科技有限公司 | A kind of method of hirudin in concentrated broth |
| CN107353338B (en) * | 2017-07-27 | 2020-11-17 | 宁波博睿修存生物科技有限公司 | Method for separating pigment molecules in hirudin fermentation liquor |
| CN109957008A (en) * | 2019-04-16 | 2019-07-02 | 广东双骏生物科技有限公司 | The method for extraction and purification of hirudin mutant and its application |
| CN109957008B (en) * | 2019-04-16 | 2021-12-24 | 广东双骏生物科技有限公司 | Extraction and purification method of hirudin mutant and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11518986B2 (en) | Process and system for obtaining botulinum neurotoxin | |
| US10414795B2 (en) | Techniques of preparing collagen active peptides | |
| CN106834395A (en) | A kind of method that lepirudin 023 ludon of suitable industrialized production is isolated and purified | |
| JP2012532932A5 (en) | ||
| NO169497B (en) | PROCEDURE FOR MANUFACTURING A SERUM-BASED HUMAN CELL LINE | |
| CN102190722A (en) | Purifying method for recombinant human serum albumin | |
| CN103739704B (en) | Testudinate collagen and extracting method thereof | |
| CN100392078C (en) | Acid trehalosease and preparation process thereof | |
| CN108840928B (en) | Radix pseudostellariae trypsin inhibitor and preparation method thereof | |
| CN105622623A (en) | Chlorophyll extraction process | |
| CN103627683A (en) | Production method for recombinant humanized superoxide dismutase | |
| CN104387459A (en) | Industrial separation and purification method of bacterial source antibacterial peptide | |
| CN106148308A (en) | A kind of isolation and purification method of recombination bacillus subtilis fibrinolysin | |
| CN110042139A (en) | A kind of preparation method and applications of animal blood activity complex peptides | |
| CN1286850C (en) | Process for preparing thymosin | |
| CN105061575B (en) | Tuna liver antibacterial peptide and preparation method and application thereof | |
| CN105483201B (en) | Fermentation preparation method of whey antioxidant peptide | |
| CN101717430A (en) | Purification method of recombinant human tissue type profibrinolysin activator modified body | |
| CN102372777A (en) | Method for purifying natural serine protease inhibitors | |
| CN101696241A (en) | Method for inducing and extracting metallothionein from housefly larva | |
| CN109370996A (en) | A kind of catalase sublimate method of purification | |
| CN203295493U (en) | Liquid enzyme preparation ultrafiltration concentration system | |
| CN113121638B (en) | Method for purifying recombinant protein | |
| CN102304502A (en) | Preparation method for bacterial-originated defibrase | |
| Zhou et al. | Purification, properties, and characterization of amylase: a review |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170613 |
|
| RJ01 | Rejection of invention patent application after publication |