CN100363500C - Preparation method of recombination human granular cell colony stimulating factor - Google Patents
Preparation method of recombination human granular cell colony stimulating factor Download PDFInfo
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- CN100363500C CN100363500C CNB200410027943XA CN200410027943A CN100363500C CN 100363500 C CN100363500 C CN 100363500C CN B200410027943X A CNB200410027943X A CN B200410027943XA CN 200410027943 A CN200410027943 A CN 200410027943A CN 100363500 C CN100363500 C CN 100363500C
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Abstract
The present invention relates to method for preparing a recombination human granular cell colony stimulating factor, which comprises the steps that: a. a DH5 alpha-PBV220-hGCSF bacterial strain of an engineering bacterium is adopted for fermentation cultivation; b. inclusion bodies of the mycelium obtained by fermentation cultivation are separated and washed to make the refined inclusion bodies; c. the refined inclusion bodies are denatured to obtain a denatured object; d. recoverability is carried out to the denatured object for the first time to obtain a first time recoverability object; e. the first time recoverability object is processed by ultrafiltration to obtain an ultrafiltration object; f. the recoverability is carried out to the ultrafiltration object for the second time to obtain the second time recoverability object; g. chromatography is carried out to the second time recoverability object to obtain the recombination human granular cell colony stimulating factor; the purity reaches more than 98%, and the specific activity is not lower than 4.0*10<8>IU/mg. The preparation method provides possibility to solve the problems of low expression rate, low mycelium output, low inclusion body recoverability rate, low activity, high cost, etc. for preparing the human granular cell colony stimulating factor.
Description
Technical field
The present invention relates to the preparation of pharmaceutical protein material, relate in particular to the method for utilizing the gene recombination engineering bacteria to carry out the target protein preparation, particularly the preparation method of recombinant methionyl human G-CSF.
Background technology
Welte.K in 1985 successfully from the culture supernatant of human bladder cancer cell's strain 5637 purifying and make with extra care out the Filgrastim (hereinafter to be referred as: hG-CSF), the Souza.L.M of the AMGEN company of the Welte.K and the U.S. further determines the N section sequence of amino acid of this hG-CSF again then, the hG-CSF gene cloning of 5637 cell strains will be derived from, adopt genetic engineering technique with this gene insert intestinal bacteria successfully make hG-CSF and develop recombinant methionyl human G-CSF (hereinafter to be referred as: rhG-CSF), commodity are called Filgratin (Hui Er blood).The drugs approved by FDA listing in 1991 of this medicine.The gene Granocyte (Ge Lanuosaite) that the rhG-CSF that hG-CSF is produced by Chinese hamster ovary celI (Chinese hamster ovary cell) derives from the human oral floor cells is a kind of 174 amino acid whose glycoprotein that contain, and its aminoacid sequence is identical with human body G-CSF with the sugar chain component.
But intracellular toxin, TNF-α and IFN-γ activated monocyte and scavenger cell produce G-CSF.In addition, inoblast, endotheliocyte, stellate cell and marrow stromal cell etc. also can be secreted G-CSF behind LPS, IL-1 or TNF-α thorn activating signal activation.But G-CSF is expressed on some leukemia cell and CHu-2 human oral cancer cells, 5637 human bladder cancer cells, MIAPa Ca-2 pancreatic cancer cell composition ground.
G-CSF cDNA cloned successfully in 1986, and G-CSF full length gene 2.5kb comprises 5 exons and 4 introns.The mankind have two kinds of different G-CSF cDNA, coding contains 207 and 204 amino acid whose precursor proteins respectively, 30 amino acid whose leaders are all arranged, the maturation protein molecule is respectively 177 and 174 amino acid, the former is except having inserted 3 amino acid at 35 places of ripe molecule N end, and remaining sequence is identical with 174 amino acid moleculars.The human G-CSF molecular weight is 19.6kDa, PI6.1, the 0-glycosylation is more stable relatively to soda acid (pH2～10), heat and denaturing agent etc.G-CSF has 5 halfcystines, Cys 36 and Cys42, and it is strong to form two pairs two sulphur between Cys74 and the Cys64, and Cys17 is unpaired halfcystine, and disulfide linkage is necessary factor for keeping the G-CSF biological function.
G-CSF mainly acts on propagation, differentiation and the activation of neutrophil series (lineage) hematopoietic cell.Stimulate the formation of neutrophil leucocyte colony in the marrow hemopoiesis progenitor cell at external G-CSF, prolong the survival time of ripe neutrophil leucocyte, the activation neutrophil leucocyte promotes its ADCC, the generation of superoxide anion and alkaline phosphatase synthetic.Studies show that recently, separately G-CSF or form with collaborative propagation, the stem cell parent cell colony that can promote pluripotential hemopoietic stem cell of SCF and body in the formation of CFU-S.G-CSF also has the chemotaxis to human granulocyte, monocyte, inoblast, smooth muscle cell and myofibroblast.Can improve the level of neutrophil leucocyte in the circulation of blood behind the tumour patient injection G-CSF, this effect may enter the time of S phase and increase the granulocytic progenitor cell quantity of generation relevant with some medullary cell of shortening.
The rhG-CSF that applicant Xinpeng Biotech Co., Ltd., Shenzhen produces, commodity auspicious blood by name is new, adopt engineering bacteria DH5 α-PBV220-hGCSF to be prepared from, contain 175 amino acid, molecular weight is 18.8kDa, iso-electric point 6.2 ± 0.4, except first amino acid, the G-CSF sequence of other 174 sequence of amino acid and natural human body is identical, in China's listing that gets the Green Light, applicable to the leukopenia that cancer is put, reason such as chemotherapy causes, be that tumour is put, important adjuvant drug in the chemotherapy process.
At present, the domestic and international various rhG-CSF commodity of producing all have been applied to clinical, and have obtained good effect.People are also seeking to simplify rhG-CSF preparation technology, increase the activity of goods, the various methods that reduce cost.These methods can be divided into three classes: first method is to start with from the recombination engineering bacterial strain that can secrete rhG-CSF, in the hope of to obtain output high, be convenient to the later stage and extract the bacterial classification of handling, each discloses the coli strain of a kind of rhG-CSF of secretion as Chinese patent ZL98103011.4 and Chinese patent application 01800753.8; Second method is can be from the genetic engineering bacterium bacterial strain carries out high-density, quickly breeding is started with to recombinating, though lot of documents is mentioned the cultivation of bacterial strain, but touch on lightly mostly, this kind method is not furtherd investigate and analyzed, in other words, be exactly not have good breakthrough in this field all the time; The third method then is that extraction, the purifying from rhG-CSF started with, as disclosing a kind of employing external-compression type Hollow Fiber Ultrafiltration dialysis renaturation among the Chinese patent ZL 96106418.8, with ion-exchange chromatography, hydrophobic chromatography and molecular sieve column chromatography sequential combination purifying with preparation high purity medical rhG-CSF protein process.
How prior art solves exists problem aspects such as renaturing inclusion bodies rate on the low side and cost on the low side, active is higher all the time in the preparation of rhG-CSF, not have the solution of getting well all the time.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, from reorganization genetic engineering bacterium bacterial strain is carried out high-density, quickly breeding is started with, problem such as solve that the expression rate that exists in the preparation of rhG-CSF is on the low side, thalline output is on the low side, the renaturing inclusion bodies rate is on the low side, active on the low side and cost is higher.
The present invention solves the problems of the technologies described above the technical scheme that is adopted, and proposes a kind of preparation method of recombinant methionyl human G-CSF, comprises step, and: a. selects for use engineering bacteria DH5 α-PBV220-hGCSF to do bacterial strain to carry out fermentation culture; B. fermentation culture gained thalline is carried out the separation and the washing of inclusion body, obtain refining inclusion body; C. refining inclusion body is carried out denaturing treatment, obtain the sex change gains; D. the sex change gains are carried out the renaturation processing first time, obtain renaturation gains for the first time; E. to the first time renaturation gains carry out uf processing, obtain the ultrafiltration gains; F. the ultrafiltration gains are carried out the renaturation processing second time, obtain renaturation gains for the second time; G. to the second time renaturation gains carry out chromatography and handle, obtain recombinant methionyl human G-CSF.
Compare with prior art, adopt the preparation method of recombinant methionyl human G-CSF of the present invention, problem such as on the low side and cost on the low side for the renaturing inclusion bodies rate that exists in the preparation that solves rhG-CSF, active is higher provides may.
Embodiment
Below the present invention is further elaborated.
The preparation method of recombinant methionyl human G-CSF of the present invention comprises step: a. selects for use engineering bacteria DH5 α-PBV220-hGCSF bacterial strain to carry out fermentation culture; B. fermentation culture gained thalline is carried out the separation and the washing of inclusion body, obtain refining inclusion body; C. refining inclusion body is carried out denaturing treatment, obtain the sex change gains; D. the sex change gains are carried out the renaturation processing first time, obtain renaturation gains for the first time; E. to the first time renaturation gains carry out uf processing, obtain the ultrafiltration gains; F. the ultrafiltration gains are carried out the renaturation processing second time, obtain renaturation gains for the second time; G. to the second time renaturation gains carry out chromatography and handle, obtain recombinant methionyl human G-CSF.
The benefit of selecting for use engineering bacteria DH5 α-PBV220-hGCSF to do bacterial strain is: its rhG-CSF albumen accounts for more than 40% of bacterial protein.
Wherein, step a comprises cultivation of step seed liquor and fed-batch fermentation again, and described seed liquor is cultivated and comprised following substep:
1.. the screening of bacterial classification: preserve the described engineering bacteria of taking-up the storehouse from bacterial classification, draw the dull and stereotyped 30 ℃ of overnight incubation of LB, picking list colony inoculation is cultivated in the 5ml LB test tube that contains 100 μ g/ml penbritins, to OD
600Value is 0.4-0.6, again in 42 ℃ of cultivations 4 hours, and centrifugal collection thalline, SDS-PAGE analyzes, and uses the highest clone of recombinant methionyl human G-CSF expression amount as the fermentation seed, and packing glycerine is preserved, and every batch fermentation is taken one;
2.. primary seed solution is cultivated: seed is inoculated in the 50ml triangular flask that contains the 2YT seed culture medium by 2% volume ratio, and 30 ℃, the 200r/min shaking table is cultivated, to OD
600Value is 0.6-1.0;
3.. secondary seed solution is cultivated: primary seed solution is inoculated in the 500ml triangular flask that contains 200ml 2YT seed culture medium with 1% volume ratio, 30 ℃, 250r/min, shaking table was cultivated 12-16 hour, to OD
600Value is 3.5-6.5, and microscopy does not have assorted bacterium, and the bacterial classification form is normal, is the fermentation seed;
Described fed-batch fermentation comprises following substep:
1.. the preparation before the fermentation: adopt NBS 20L fermentor tank, pH electrode, dissolved oxygen electrode are proofreaied and correct and installed; The 8L fermention medium is inserted fermentor tank, set 115 ℃ of sterilising temps, 30min, the sterilization of fermentor tank automatic on-line;
2.. basic fermentation: after the sterilization, when treating that the substratum temperature drops to 30 ℃, insert secondary seed solution, 30 ℃, cultivated the beginning abduction delivering 8 hours by inoculum size 10% volume ratio;
3.. induce fermentation: inducing temperature is 42 ℃, induces 4 hours, finishes fermentation;
Above-mentioned fed-batch fermentation substep is 7.0-7.1 by adding 4mol/L NaOH (sodium hydroxide) control pH 2. and 3., and by regulating the air velocity and the rotating speed of fermentor tank, the control oxygen dissolving value is 50%; And work as OD
600Value is 2 o'clock, the beginning feed supplement, and the feed supplement mode is for per hour adding each 100ml of feed supplement I and II.
The prescription of described LB is: peptone 10g, and yeast powder 5g, sodium-chlor 10g adds purified water and is settled to 1L.
The prescription of described 2YT substratum is: peptone 16g, and yeast powder 10g, sodium-chlor 5g adds purified water and is settled to 1L.
Above-mentioned after inducing fermentation in 4 hours, through centrifugal collection, on average wet bacterium yield can reach 80-100g/L.
The difficult point of fermenting process of the present invention is the growth characteristics according to selected engineering bacteria, and provide corresponding fermentation condition, specifically comprise temperature, pH value control, dissolved oxygen amount control, such as: why dissolved oxygen amount control adopts 50% in the present invention, rather than adopt about 30% in the general zymotechnique, exactly through analyzing, test the preference data that draws repeatedly.
The difficult point of fermenting process of the present invention also is to be fit to the selection of culture medium prescription of the growth characteristics of engineering bacteria, and the selection that feed supplement itself is filled a prescription during the timesharing batch feeding.
The fermentative medium formula that the present invention selects for use is: peptone 20g, and yeast powder 120g, glycerine 40ml, ammonium sulfate 50g, glucose 100g, sodium-chlor 5g, dipotassium hydrogen phosphate 100g, potassium primary phosphate 20g adds purified water and is settled to 8L.
The prescription of the feed supplement I that the present invention selects for use is: glucose 300g, ammonium sulfate 50g adds purified water and is settled to 1L.
The prescription of the feed supplement II that the present invention selects for use is: yeast powder 300g, and casein hydrolysate 20g, VITMAIN B1 0.1g adds purified water and is settled to 1L.
Adopt fermentation process of the present invention, can from reorganization genetic engineering bacterium bacterial strain is carried out high-density, quickly breeding is started with, problem such as solve that the expression rate that exists in the preparation of rhG-CSF is on the low side, thalline output is on the low side, active on the low side and cost is higher.
Thalline can carry out preservation, transhipment behind the fermenting process of collecting in-20 ℃ refrigerating plant, also can carry out the separation of inclusion body and extractions such as washing, renaturation, ultrafiltration and chromatography, purifying process process immediately with preparation high purity medical rhG-CSF albumen.
Step b of the present invention comprises: with the g/ml ratio of described engineering bacteria thalline by 1: 5, add in the bacterial cell disruption damping fluid, ultrasonication to bacterial cell disruption rate is more than 98%, 10000r/min, and centrifugal 40min keeps precipitation; To precipitate with washings washing three times; Add 1% sodium deoxycholate the 3rd time, use deionized water wash again 2 times, 10000r/min, centrifugal 40min, collecting precipitation is refining inclusion body;
The prescription of wherein said broken damping fluid is: 1mmol/L EDTANa
2(disodium ethylene diamine tetraacetate), 80mmol/L NaCl (sodium-chlor), 50mmol/L Tris-HCl (Tutofusin tris-hydrochloric acid), pH8.0;
The prescription of described washings is: 5mmol/L EDTANa
2, 80mmol/L NaCl, 3mmol/L Urea, 50mmol/LTris-HCl, pH8.0.
Step c of the present invention comprises: with 1: 20 g/ml ratio, will make with extra care inclusion body with sex change liquid mixing, and stir more than 2 hours, and be the sex change gains; The prescription of described sex change liquid is: 1mmol/L EDTANa
2, 10mmol/LDTT (DTT), 8mol/L Urea (urea), 50mmol/L Tris-HCl, pH8.0.
Steps d of the present invention comprises: the sex change gains with the centrifugal 40min of 13000r/min, are discarded precipitation, supernatant is slowly poured in the renaturation solution, the control protein concentration stirs more than 20 hours at 0.05mg/ml, is renaturation gains for the first time; The prescription of described renaturation solution is 1mol/L Urea, 0.1mmol/L GSH (reduced glutathion), 0.01mmol/LGSSG (Sleep-promoting factor B), 30mmol/LTris-HCl, pH8.5.
Step e of the present invention comprises: with Millipore ultrafiltration instrument, ultra-filtration membrane molecular weight cut-off 10KDa uses purified water and the abundant cleaning system of distilled water earlier, for the first time renaturation gains ultrafiltration and concentration before using, final volume is 1/15 of an original volume, is the ultrafiltration gains.
Step f of the present invention comprises: the ultrafiltration gains are positioned over 2-8 ℃ of environment, continue renaturation more than 96 hours, renaturation yield reaches more than 92%, is renaturation gains for the second time.
Step g of the present invention comprises: the Glacial acetic acid adjusting with 10% renaturation gains pH for the second time is 5.4, centrifugal 10000r/min, and 30min keeps supernatant liquor; It with SP Sepharose High Performance chromatography column pH 5.4 30mmol/mol NaAC (sodium-acetate) balance, with the supernatant liquor upper prop, with pH is 5.4 the 30mmol/LNaAC wash-out that contains 120mmol/L NaCl, collects the main peak material of SP Sepharose High Performance chromatography column; Is 4.0 10mmol/L NaAC balance with Sephadex G25 post with pH, with SephadexG25 post on the main peak material of the SP Sepharose High Performance chromatography column collected, collects main peak, is recombinant methionyl human G-CSF.
Adopt the preparation method's of a recombinant methionyl human G-CSF of the present invention concrete result of implementation to be exactly: auspicious blood is new, and it is through the scanning of SDS-PAGE electrophoresis, and purity reaches more than 99%, and HPLC shows unimodal, and the rhG-CSF specific activity is not less than 4.0 * 10
8IU/mg.
Claims (6)
1. the preparation method of a recombinant methionyl human G-CSF may further comprise the steps:
A. select for use engineering bacteria DH5 α-PBV220-hGCSF bacterial strain to carry out fermentation culture;
B. fermentation culture gained thalline is carried out the separation and the washing of inclusion body, obtain refining inclusion body;
C. refining inclusion body is carried out denaturing treatment, obtain the sex change gains;
D. with the sex change gains with the centrifugal 40min of 13000r/min, discard precipitation, supernatant is slowly poured in the renaturation solution, the control protein concentration stirred 20 hours at 0.05mg/ml at least, was the renaturation gains first time; The prescription of described renaturation solution is: 1mol/L urea, 0.1mmol/l reduced glutathion, 0.01mmol/l Sleep-promoting factor B, 30mmol/l Tutofusin tris-hydrochloric acid, pH8.5;
E. select ultra-filtration membrane molecular weight cut-off 10KDa, with the renaturation gains ultrafiltration and concentration first time, final volume is 1/15 of an original volume, is the ultrafiltration gains;
F. the ultrafiltration gains are positioned over 2-8 ℃ of environment, at least 96 hours, promptly get renaturation gains for the second time;
G. to the second time renaturation gains carry out chromatography and handle, obtain recombinant methionyl human G-CSF.
2. the preparation method of recombinant methionyl human G-CSF as claimed in claim 1 is characterized in that described step a comprises seed liquor cultivation and fed-batch fermentation,
Described seed liquor is cultivated and is comprised following substep:
1) screening of bacterial classification: preserve the described engineering bacteria of taking-up the storehouse from bacterial classification, draw the dull and stereotyped 30 ℃ of overnight incubation of LB, picking list colony inoculation is cultivated in the 5ml LB test tube that contains 100 μ g/ml penbritins, to OD
600Value is 0.4-0.6, again in 42 ℃ of cultivations 4 hours, and centrifugal collection thalline, SDS-PAGE analyzes, and uses the highest clone of recombinant methionyl human G-CSF expression amount as the fermentation seed, and packing glycerine is preserved, and every batch fermentation is taken one;
2) primary seed solution is cultivated: seed is inoculated in the 50ml triangular flask that contains the 2YT seed culture medium by 2% volume ratio, and 30 ℃, the 200r/min shaking table is cultivated, to OD
600Value is 0.6-1.0;
3) secondary seed solution is cultivated: primary seed solution is inoculated in the 500ml triangular flask that contains the 200ml2YT seed culture medium with 1% volume ratio, and 30 ℃, 250r/min, shaking table was cultivated 12-16 hour, to OD
600Value is 3.5-6.5, and microscopy does not have assorted bacterium, and the bacterial classification form is normal, is the fermentation seed;
Described fed-batch fermentation comprises following substep:
1) preparation before the fermentation: the 8L fermention medium is inserted in the fermentor tank of 20L, sterilized 30 minutes for 115 ℃;
2) basic fermentation: after the sterilization, when treating that the substratum temperature drops to 30 ℃, insert secondary seed solution, 30 ℃, cultivated the beginning abduction delivering 8 hours by inoculum size 10% volume ratio;
3) induce fermentation: inducing temperature is 42 ℃, induces 4 hours, finishes fermentation;
Described fed-batch fermentation substep 2) and 3) in, be 7.0-7.1 by adding 4mol/L sodium hydroxide control pH, by regulating the air velocity and the rotating speed of fermentor tank, the control oxygen dissolving value is 50%; And work as OD
600Value is 2 o'clock, the beginning feed supplement, and the feed supplement mode is for per hour adding each 100ml of feed supplement I and II;
The prescription of described fermention medium is: peptone 20g, and yeast powder 120g, glycerine 40ml, ammonium sulfate 50g, glucose 100g, sodium-chlor 5g, dipotassium hydrogen phosphate 100g, potassium primary phosphate 20g adds purified water and is settled to 8L;
The prescription of described feed supplement I is: glucose 300g, and ammonium sulfate 50g adds purified water and is settled to 1L;
The prescription of described feed supplement II is: yeast powder 300g, and casein hydrolysate 20g, VITMAIN B1 0.1g adds purified water and is settled to 1L.
3. the preparation method of recombinant methionyl human G-CSF as claimed in claim 1, it is characterized in that described step b comprises: with the g/ml ratio of described engineering bacteria thalline in 1: 5, add in the bacterial cell disruption damping fluid, ultrasonication to bacterial cell disruption rate is more than 98%, 10000r/min, centrifugal 40min keeps precipitation; To precipitate with washings washing three times; Add 1% sodium deoxycholate the 3rd time, use deionized water wash again 2 times, 10000r/min, centrifugal 40min, collecting precipitation is refining inclusion body; The prescription of wherein said broken damping fluid is: 1mmol/L disodium ethylene diamine tetraacetate, 80mmol/L sodium-chlor, 50mmol/L Tutofusin tris-hydrochloric acid, pH8.0; The prescription of described washings is: 5mmol/L disodium ethylene diamine tetraacetate, 80mmol/L sodium-chlor, 3mmol/L urea, 50mmol/L Tutofusin tris-hydrochloric acid, pH8.0.
4. the preparation method of recombinant methionyl human G-CSF as claimed in claim 1 is characterized in that, described step c comprises: with 1: 20 g/ml ratio, exquisite inclusion body with sex change liquid mixing, was stirred 2 hours at least, be the sex change gains.
5. the preparation method of recombinant methionyl human G-CSF as claimed in claim 4 is characterized in that, the prescription of described sex change liquid is: the 1mmol/L disodium ethylene diamine tetraacetate, the 10mmol/L DTT, 8mol/L urea, 50mmol/L Tutofusin tris-hydrochloric acid, pH8.0.
6. the preparation method of recombinant methionyl human G-CSF as claimed in claim 1 is characterized in that, described step g comprises: the Glacial acetic acid adjusting with 10% renaturation gains pH for the second time is 5.4, centrifugal 10000r/min, and 30min keeps supernatant liquor; It with SP Sepharose High Performance chromatography column pH 5.4 30mmol/L sodium-acetate balance, with the supernatant liquor upper prop, with pH is 5.4 the 30mmol/L sodium-acetate wash-out that contains 120mmol/L sodium-chlor, collects the main peak material of SP Sepharose HighPerformance chromatography column; Is 4.0 10mmol/L sodium-acetate balance with Sephadex G25 post with pH, with Sephadex G25 post on the main peak material of the SP Sepharose High Performance chromatography column collected, collects main peak, is recombinant methionyl human G-CSF.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101045742B (en) * | 2006-03-27 | 2011-12-21 | 华北制药集团新药研究开发有限责任公司 | Dipurification process of recombinant humangranulocyte colony stimulating factor |
| CN101830977B (en) * | 2010-05-07 | 2012-01-25 | 厦门特宝生物工程股份有限公司 | Method for purifying recombined human granulocyte stimulating factors |
| CN102154189B (en) * | 2010-12-31 | 2015-11-11 | 鲁南制药集团股份有限公司 | A kind of fermentation culture method of rhG-CSF recombinant bacterial strain |
| CN102344931A (en) * | 2011-09-16 | 2012-02-08 | 江苏普罗赛生物技术有限公司 | Simple and convenient chemical industrial technology for prokaryotic expression and purification of recombinant human granulocyte-colony stimulating factor (G-CSF) |
| CN104846002B (en) * | 2015-06-06 | 2019-02-19 | 山东新时代药业有限公司 | Recombinate the building and expression of G-CSF (15-75) peptide carrier |
| CN110066331B (en) * | 2018-01-23 | 2022-05-24 | 江苏恒瑞医药股份有限公司 | Preparation method of recombinant human granulocyte colony stimulating factor |
| CN110567949B (en) * | 2019-09-10 | 2021-12-14 | 广东天地和实业控股集团有限公司 | Bacterial toxin detection method and treatment instrument applied to same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1129255A (en) * | 1995-02-16 | 1996-08-21 | 中国人民解放军军事医学科学院基础医学研究所 | Method for making recombined granular macrophage colony stimulating factor of human body |
| CN1228476A (en) * | 1998-03-05 | 1999-09-15 | 上海华晨生物技术研究所 | Method for preparing human granulocyte macrophage colony stimulating factor and its expressing carrier and engineering bacteria |
| CN1487086A (en) * | 2003-05-26 | 2004-04-07 | 海南国栋药物研究所 | Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor |
-
2004
- 2004-07-07 CN CNB200410027943XA patent/CN100363500C/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1129255A (en) * | 1995-02-16 | 1996-08-21 | 中国人民解放军军事医学科学院基础医学研究所 | Method for making recombined granular macrophage colony stimulating factor of human body |
| CN1228476A (en) * | 1998-03-05 | 1999-09-15 | 上海华晨生物技术研究所 | Method for preparing human granulocyte macrophage colony stimulating factor and its expressing carrier and engineering bacteria |
| CN1487086A (en) * | 2003-05-26 | 2004-04-07 | 海南国栋药物研究所 | Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor |
Non-Patent Citations (3)
| Title |
|---|
| 大肠杆菌表达重组人粒细胞一巨唑细胞集落刺激因子的中试纯化. 李智华，姬秋彦等.云南医药，第22卷第1期. 2001 * |
| 重组GM-CSF/IL-3融合蛋白的纯化. 张毅等.生物化学与生物物理学报，第32卷第3期. 2000 * |
| 重组人粒细胞集落刺激因子的纯化. 储淳等.微生物免疫学进展，第26卷第2期. 1998 * |
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