CN105296579A - RhEGF (recombinant human Epidermal Growth Factor) liquid concentrate preparation technology - Google Patents
RhEGF (recombinant human Epidermal Growth Factor) liquid concentrate preparation technology Download PDFInfo
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Abstract
The invention discloses a rhEGF (recombinant human Epidermal Growth Factor) liquid concentrate preparation technology. A culture medium is prepared from the following ingredients in weight proportion: 5.0-7.0% of glycerin, 3.0-3.5% of YNB (Yeast Nitrogen Base), 1.8-2.2% of trace metal ions, 1.2-1.8% of an inducer, 0.5-1.0% of biotin, 0.5-1.0% of an accelerant and the balance of water, wherein the trace metal ions are one of ferric trichloride, calcium chloride, sodium molybdate, zinc chloride, copper sulfate, boric acid and aluminum sulfate; pH is adjusted to be 5.5-6.5 by phosphate buffer. According to the invention, plant raw material ingredients adopted in the invention are subjected to treatment of steam explosion, distillation, and treatment of a high-shear homogenizer, so that nanoemulsion plant oil ingredients with good compatibility to other formulas in the culture medium can be obtained; as the plant raw material ingredients are added in the culture medium as the accelerant, the yield of fermentation broth can be improved by 20-25% compared with that in the prior art.
Description
Technical field
The present invention relates to biological technical field, particularly relate to recombinant human epidermal growth factor stoste preparation technology.
Background technology
Urogastron (hEGF) is the polypeptide that body weight for humans is wanted, it has various biological function, such as can with special receptors bind on epidermic cell, information is transmitted into cell, change intracellular potential of hydrogen and free ca degree, thus promote glycolysis-and protein synthesis and increase some specific gene to transcribe, promote DNA replication dna and cell fission.Urogastron is a kind of growth regulator found from mouse submandibular gland in the sixties in last century by people such as Cohen, is made up of 53 amino acid, and research confirms that it has the effect stimulated cellular proliferation widely.1975, the people such as Starkey, Cohen were extracted human epidermal growth factor (humanEpidermalGrowthFactor, hEGF) from people's urine, have highly consistent structure, and have consistent biologic activity with M-EGF.Urogastron is extensively present in the various tissue of human body, various kinds of cell can be stimulated to breed, mainly the propagation of epithelial cell and endotheliocyte, and this is by working in conjunction with the EGF-R ELISA (EGFR) on cytolemma.EGF is partly combined outward by the film with cell receptor, activates its tyrosine kinase activity, inducible protein phosphorylation, starts DNA synthesis, activator RNA, protein synthesis, promotes hyperplasia, divides a word with a hyphen at the end of a line.Therefore, hEGF can promote epidermic cell, neurocyte and organ-tissue epithelial cell growth; The growth of newborn fetus tooth, bone and each organ can be promoted; Liver cell regeneration can be promoted; Gastrointestinal tract mucosa Growth of Cells can be promoted and protect gastrointestinal tract mucosa impaired.
Urogastron is extensively present in various tissue in human body, and content is atomic, has vital role for the normal physiological function maintaining the various histoorgan of human body (as angle conjunctiva, skin, various mucous membrane, body of gland etc.).With advancing age, body entocuticle growth factor levels declines gradually, show as aging and the degeneration of related tissue's organ, the particularly aging of epithelium, therefore, in time to human body supplementary table skin growth factor, can safeguard human normal function and delay senility, this is the application foundation of Urogastron in beauty and health care industry.In addition, when the impaired grade of human body, endogenous epidermal growth factor can not meet a large amount of needs of tissue repair, in time to damaged part supplementary table skin growth factor, the reparation of impaired when injected organism tissue can be promoted, apply widely clinically, as the corneal epithelial defect reparation caused for a variety of causes such as wound, inflammation, operation, chemical burn and xerophthalmia, the reparation of the various skins such as burn, wound, surgical operation, inflammation, ulcer and Mucosa Defect.
Recombinant human epidermal growth factor (rhEGF) is by genetic engineering technique by intestinal bacteria or yeast secreted expression, and its goods purity can reach 98%.Recombinant human epidermal growth factor belongs to the macromolecular polypeptides class medicine of molecular weight more than 500 roads.At present, to Urogastron and the existing research of recombinant human epidermal growth factor, such as Chinese patent 97115284.5, disclose epdermal growth favtur (hEGF) engineering bacteria and use the method for this bacterium preparation table skin growth factor, researchist can obtain recombinant human epidermal growth factor (or being called recombinant human epidermal growth factor stoste) by Urogastron.Science and technology in continuous progress, for the research of Urogastron and recombinant human epidermal growth factor also in the middle of constantly carrying out.
Summary of the invention
The object of this invention is to provide recombinant human epidermal growth factor stoste preparation technology, comprise the following steps:
1) preparation of substratum: be made up of following part by weight composition:
Described trace metal ion is the one in iron trichloride, calcium chloride, Sodium orthomolybdate, zinc chloride, copper sulfate, boric acid, Tai-Ace S 150;
PH is adjusted to be 5.5-6.5 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, burstpressures 3.0-3.5Mpa, the dwell time maintains 150-200s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: the essential oil that 3. step is obtained by high-shear homogenizing machine process, linear velocity 30-40m/s, time 3-5min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming;
2) ferment, express: application 2L shaking flask and 20L in-situ sterilization fermentor cultivation genetic engineering bacterial strain, after 1O% culture medium inoculated, add inductor, 30 DEG C of cultivations, control pH7.0, oxygen saturation 10-50%, stir 150-170rpm, tank pressure 0.01378-0.03445MPa, stream glycerol adding to every L wet bacterium reach 100-300g after stream add methyl alcohol and keep its concentration to be 1% to carry out the abduction delivering of EGF;
3) be separated: express and terminate, centrifugation, collect supernatant and both obtained recombinant clone bacterial strain centrifugate;
4) purifying: by step 3) the recombinant clone bacterial strain centrifugate that obtains, adding ammonium sulfate makes concentration become 0.5M, be splined on the 20mM phosphoric acid buffer pH7.0 by 5 times of cylinder accumulated amounts, the drainage column that 0.5M amine sulfate solution equilibria is crossed, with 20mM phosphoric acid buffer pH7.0, sample introduction peak is washed till baseline by 0.5M amine sulfate, EGF peak is eluted with 20mM phosphoric acid buffer, this sample peak is splined on Q-sepharoseHighPerformance post after diluting 10 times with 20mMTris-HClpH7.6 damping fluid, with A liquid (20mMTris-HClpH7.6) to B liquid (20mMTris-HC1pH7.6, 0.5MNaCl) gradient elution method collects EGF peak, finally use molecular sieve Superdex30 column purification Urogastrone albumen, purity can reach more than 98%.
Further preferred, step 1) preparation of described substratum is according to following steps:
Substratum is made up of following part by weight composition:
Described trace metal ion is the one in iron trichloride, calcium chloride, Sodium orthomolybdate, zinc chloride, copper sulfate, boric acid, Tai-Ace S 150;
PH is adjusted to be 6.0 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, burstpressures 3.0-3.5Mpa, the dwell time maintains 150-200s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: essential oil step 3. obtained is by high-shear homogenizing machine process, and linear velocity 30-40m/s, time 3-5min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming.
Water of the present invention meets pharmaceutical industry standard.
The usage quantity of Japanese Honeysuckle of the present invention, the volume after preferably pulverizing is equivalent to the 1/4-1/3 of steam explosion tank volume.
Water of the present invention meets pharmaceutical industry standard, described genetic engineering bacterial strain reference prior art, preferred genetic engineering bacterial strain GS115-3EGF.
Step 2) described inductor is methyl alcohol, consumption 1.2-1.8% (w/w).
Step 4) the preferred Phenylsepharose6FastFlow of described drainage column (highsub) post.
Compared with prior art, tool of the present invention has the following advantages:
1, the plant material composition that the present invention uses have passed through steam explosion, distillation, high-shear homogenizing machine process, obtains the plant oil components of nanometer emulsus, and in substratum, the consistency of other formulas is good.
The induction mode of 2, conventional in prior art engineering bacteria has temperature control or chemical substance, such as methyl alcohol, IPTG etc.The present invention is by adding plant material composition in the medium as promotor, and compared to the prior art, expressing output can improve 20-25%.
Embodiment
With embodiment, the invention will be further described below, but the present invention is not limited to these embodiments.
Embodiment 1:
Recombinant human epidermal growth factor stoste preparation technology, comprises the following steps:
1) preparation of substratum: be made up of following part by weight composition:
Described trace metal ion is iron trichloride;
PH is adjusted to be 5.5 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, volume is equivalent to 1/3 of steam explosion tank volume, burstpressures 3.0Mpa, and the dwell time maintains 200s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: the essential oil that 3. step is obtained by high-shear homogenizing machine process, linear velocity 30m/s, time 3min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming;
2) ferment, express: application 2L shaking flask and 20L in-situ sterilization fermentor cultivation genetic engineering bacterial strain, after 1O% culture medium inoculated, add inductor 1.2%, 30 DEG C of cultivations, control pH7.0, oxygen saturation 50%, stirs 150rpm, tank pressure 0.01378MPa, stream glycerol adding to every L wet bacterium reach 100g after stream add methyl alcohol and keep its concentration to be 1% to carry out the abduction delivering of EGF;
3) be separated: express and terminate, centrifugation, both obtained recombinant clone bacterial strain centrifugate;
4) purifying: by step 3) the recombinant clone bacterial strain centrifugate that obtains, adding ammonium sulfate makes concentration become 0.5M, be splined on the 20mM phosphoric acid buffer pH7.0 by 5 times of cylinder accumulated amounts, the drainage column (Phenylsepharose6FastFlow (highsub) post) that 0.5M amine sulfate solution equilibria is crossed, with 20mM phosphoric acid buffer pH7.0, sample introduction peak is washed till baseline by 0.5M amine sulfate, EGF peak is eluted with 20mM phosphoric acid buffer, this sample peak is splined on Q-sepharoseHighPerformance post after diluting 10 times with 20mMTris-HClpH7.6 damping fluid, with A liquid (20mMTris-HClpH7.6) to B liquid (20mMTris-HC1pH7.6, 0.5MNaCl) gradient elution method collects EGF peak, finally use molecular sieve Superdex30 column purification Urogastrone albumen, purity reaches 98.5%.
Embodiment 2:
Recombinant human epidermal growth factor stoste preparation technology, comprises the following steps:
1) preparation of substratum: be made up of following part by weight composition:
Described trace metal ion is calcium chloride;
PH is adjusted to be 6.5 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, volume is equivalent to 1/4 of steam explosion tank volume, burstpressures 3.5Mpa, and the dwell time maintains 180s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: the essential oil that 3. step is obtained by high-shear homogenizing machine process, linear velocity 35m/s, time 4min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming;
2) ferment, express: application 2L shaking flask and 20L in-situ sterilization fermentor cultivation genetic engineering bacterial strain, after 1O% culture medium inoculated, add inductor 1.5%, 30 DEG C of cultivations, control pH7.0, oxygen saturation 10%, stirs 170rpm, tank pressure 0.03445MPa, stream glycerol adding to every L wet bacterium reach 200g after stream add methyl alcohol and keep its concentration to be 1% to carry out the abduction delivering of EGF;
3) be separated: express and terminate, centrifugation, both obtained recombinant clone bacterial strain centrifugate;
4) purifying: by step 3) the recombinant clone bacterial strain centrifugate that obtains, adding ammonium sulfate makes concentration become 0.5M, be splined on the 20mM phosphoric acid buffer pH7.0 by 5 times of cylinder accumulated amounts, the drainage column (Phenylsepharose6FastFlow (highsub) post) that 0.5M amine sulfate solution equilibria is crossed, with 20mM phosphoric acid buffer pH7.0, sample introduction peak is washed till baseline by 0.5M amine sulfate, EGF peak is eluted with 20mM phosphoric acid buffer, this sample peak is splined on Q-sepharoseHighPerformance post after diluting 10 times with 20mMTris-HClpH7.6 damping fluid, with A liquid (20mMTris-HClpH7.6) to B liquid (20mMTris-HC1pH7.6, 0.5MNaCl) gradient elution method collects EGF peak, finally use molecular sieve Superdex30 column purification Urogastrone albumen, purity reaches 99.1%.
Embodiment 3:
Recombinant human epidermal growth factor stoste preparation technology, comprises the following steps:
1) preparation of substratum: be made up of following part by weight composition:
Described trace metal ion is copper sulfate;
PH is adjusted to be 6.0 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, volume is equivalent to 1/3 of steam explosion tank volume, burstpressures 3.0Mpa, and the dwell time maintains 150s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: the essential oil that 3. step is obtained by high-shear homogenizing machine process, linear velocity 40m/s, time 5min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming;
2) ferment, express: application 2L shaking flask and 20L in-situ sterilization fermentor cultivation genetic engineering bacterial strain, after 1O% culture medium inoculated, add inductor 1.8%, 30 DEG C of cultivations, control pH7.0, oxygen saturation 30%, stirs 160rpm, tank pressure 0.02067MPa, stream glycerol adding to every L wet bacterium reach 300g after stream add methyl alcohol and keep its concentration to be 1% to carry out the abduction delivering of EGF;
3) be separated: express and terminate, centrifugation, both obtained recombinant clone bacterial strain centrifugate;
4) purifying: by step 3) the recombinant clone bacterial strain centrifugate that obtains, adding ammonium sulfate makes concentration become 0.5M, be splined on the 20mM phosphoric acid buffer pH7.0 by 5 times of cylinder accumulated amounts, the drainage column (Phenylsepharose6FastFlow (highsub) post) that 0.5M amine sulfate solution equilibria is crossed, with 20mM phosphoric acid buffer pH7.0, sample introduction peak is washed till baseline by 0.5M amine sulfate, EGF peak is eluted with 20mM phosphoric acid buffer, this sample peak is splined on Q-sepharoseHighPerformance post after diluting 10 times with 20mMTris-HClpH7.6 damping fluid, with A liquid (20mMTris-HClpH7.6) to B liquid (20mMTris-HC1pH7.6, 0.5MNaCl) gradient elution method collects EGF peak, finally use molecular sieve Superdex30 column purification Urogastrone albumen, purity reaches 98.8%.
Claims (4)
1. recombinant human epidermal growth factor stoste preparation technology, is characterized in that, comprises the following steps:
1) preparation of substratum: be made up of following part by weight composition:
Described trace metal ion is the one in iron trichloride, calcium chloride, Sodium orthomolybdate, zinc chloride, copper sulfate, boric acid, Tai-Ace S 150;
PH is adjusted to be 5.5-6.5 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, burstpressures 3.0-3.5Mpa, the dwell time maintains 150-200s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: the essential oil that 3. step is obtained by high-shear homogenizing machine process, linear velocity 30-40m/s, time 3-5min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming;
2) ferment, express: application 2L shaking flask and 20L in-situ sterilization fermentor cultivation genetic engineering bacterial strain, after 10% culture medium inoculated, add inductor, 30 DEG C of cultivations, control pH7.0, oxygen saturation 10-50%, stir 150-170rpm, tank pressure 0.01378-0.03445MPa, stream glycerol adding to every L wet bacterium reach 100-300g after stream add methyl alcohol and keep its concentration to be 1% to carry out the abduction delivering of EGF;
3) be separated: express and terminate, centrifugation, both obtained recombinant clone bacterial strain centrifugate;
4) purifying: by step 3) the recombinant clone bacterial strain centrifugate that obtains, adding ammonium sulfate makes concentration become 0.5M, be splined on the 20mM phosphoric acid buffer pH7.0 by 5 times of cylinder accumulated amounts, the drainage column that 0.5M amine sulfate solution equilibria is crossed, with 20mM phosphoric acid buffer pH7.0, sample introduction peak is washed till baseline by 0.5M amine sulfate, EGF peak is eluted with 20mM phosphoric acid buffer, this sample peak is splined on Q-sepharoseHighPerformance post after diluting 10 times with 20mMTris-HClpH7.6 damping fluid, with A liquid (20mMTris-HClpH7.6) to B liquid (20mMTris-HC1pH7.6, 0.5MNaCl) gradient elution method collects EGF peak, finally use molecular sieve Superdex30 column purification Urogastrone albumen, purity can reach more than 98%.
2. recombinant human epidermal growth factor stoste preparation technology according to claim 1, is characterized in that, step 1) described substratum is made up of following part by weight composition:
Described trace metal ion is the one in iron trichloride, calcium chloride, Sodium orthomolybdate, zinc chloride, copper sulfate, boric acid, Tai-Ace S 150;
PH is adjusted to be 6.0 with phosphate buffered saline buffer;
Described promotor adopts following methods preparation:
1. Feedstock treating: Japanese Honeysuckle is clean, drying, pulverizing;
2. Steam explosion treatment: steam explosion tank put into by material step 1. obtained, burstpressures 3.0-3.5Mpa, the dwell time maintains 150-200s;
3. process is distilled: distiller put into by material step 2. obtained, bottom distiller, heating flame or pass into steam, when hot steam is filled in distiller, water vapour, by prolong, is introduced in condenser, then by water-and-oil separator, collects essential oil;
4. high-shear homogenizing machine process: essential oil step 3. obtained is by high-shear homogenizing machine process, and linear velocity 30-40m/s, time 3-5min, makes nano-emulsion, and obtains with filtering with microporous membrane is degerming.
3. recombinant human epidermal growth factor stoste preparation technology according to claim 1 and 2, it is characterized in that: the usage quantity of described Japanese Honeysuckle, the volume after pulverizing is equivalent to the 1/4-1/3 of steam explosion tank volume.
4. recombinant human epidermal growth factor stoste preparation technology according to claim 1, is characterized in that: step 2) described inductor is methyl alcohol, consumption 1.2-1.8%.
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Citations (2)
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|---|---|---|---|---|
| CN1210145A (en) * | 1997-08-29 | 1999-03-10 | 中国医学科学院基础医学研究所 | Engineering bacteria for epidermal growth factor and preparation of epidermal growth factor by using this bacteria |
| CN101492675A (en) * | 2008-01-21 | 2009-07-29 | 吉林圣元科技有限责任公司 | Method for producing recombinant human epidermal growth factor acceptor 2 cytoplasmic region with methylotrophy yeast |
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2015
- 2015-11-26 CN CN201510843354.7A patent/CN105296579A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1210145A (en) * | 1997-08-29 | 1999-03-10 | 中国医学科学院基础医学研究所 | Engineering bacteria for epidermal growth factor and preparation of epidermal growth factor by using this bacteria |
| CN101492675A (en) * | 2008-01-21 | 2009-07-29 | 吉林圣元科技有限责任公司 | Method for producing recombinant human epidermal growth factor acceptor 2 cytoplasmic region with methylotrophy yeast |
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