CN104829691A - Wrinkle-eliminating oligopeptide and preparation method thereof - Google Patents
Wrinkle-eliminating oligopeptide and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a recombinant protein in the field of genetic engineering and particularly relates to a novel wrinkle-eliminating oligopeptide and a preparation method thereof. The invention discloses the wrinkle-eliminating oligopeptide named as E14 composed of 14 amino acids and also discloses a process for fermenting by virtue of escherichia coil to industrially prepare recombinant E14 (short for rE14) on a large scale. Proven through detecting by using various biological means, the production process of rE14 is simple, low in cost, high in purity and stability, capable of bringing nutrient skin anti-aging products to the daily life of common people and wide in market application prospect.
Description
Technical field
The present invention relates to design and the preparation method of genetically engineered field recombinant protein, specifically a kind of novel smoothing wrinkle small peptide and preparation method thereof.
Background technology
The skin of people and higher animal is made up of epidermis, corium, subcutis.Epidermis does not have blood vessel, and the energy of cell and nutrition must be provided by corium microcirculation.Skin corium is the source of life of skin, is fine and close, the tough and tensile and resilient tissue of one deck, primarily of the composition such as fibre composition (collagen fabric, spandex and reticulin fiber), glycoprotein, glucosamine and Hyaluronic Acid.
Human research confirms, the generation of wrinkle is relevant with facial muscles fiber overstimulation.Therefore, from theory, can shrink by directly reducing muscular movement the generation alleviating wrinkle.The contraction of muscle is regulated and controled by neurotransmitter, and SNARE mixture is then the signal source that neurotransmitter is transmitted.Widely used novain is just by suppressing the activity of the function inhibitio neurocyte of SNARE mixture in the market, and then reaches the object removing wrinkle.But the side effect of novain is very huge, and the mankind are studying the functional analogue of novain always, make every effort to reduce its side effect under the prerequisite reaching identical object as far as possible.
The Argireline that Lipotec company finds is exactly a kind of functional analogue of novain.Argireline is made up of 6 amino acid, and amino acid consists of Ac-EEMQRR-NH2.Functional experiment confirms, Argireline has good percutaneous permeability, and effectively can suppress the function of SNARE, has very strong crease-resistant wrinkle of going active, is also referred to as Argireline.
At present, many series of the well-known makeup brand of a lot of country have been had to the addition of the effective constituent of Argireline Argireline as smoothing wrinkle.Also there are this production marketing in some biotechnology companies of China.But the preparation method of current Argireline is confined to chemosynthesis, and the cost of its great number limits widely using of this series products.Therefore, in order to the daily life that the skin-nourishing product of this kind of small peptide class can be made constantly to walk close to the common people, make the general population have safety, stable, practical high-quality makeup, be necessary design research and development smoothing wrinkle small peptide more efficiently, and explore the technology of extensive preparation smoothing wrinkle small peptide.
Summary of the invention
The present invention, in order to improve the biologic activity of existing smoothing wrinkle small peptide, reduces the production cost of this kind of beauty treatment peptide simultaneously, provides a kind of design of novel smoothing wrinkle small peptide, and provides its large-scale preparation method.
The present invention adopts following technical scheme to realize:
A kind of smoothing wrinkle small peptide, being made up of 14 amino acid, its aminoacid sequence is: Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln, is expressed as GPEEMQRRLEVLFQ with one-letter symbol, referred to as E14 small peptide, theoretical molecular is 1.732kD.
Above-mentioned E14 small peptide can be prepared by multiple production technique:
1, utilize the technique of Escherichia coli fermentation, the E14 of recombinant expressed preparation, be called for short rE14, production technique is simple, with low cost, is easy to promote.
2, utilize the technique synthesis E14 of chemosynthesis, be called for short sE14, cost is higher, but has the biological function similar with rE14.
The invention provides the preparation method of above-mentioned smoothing wrinkle small peptide, comprise the steps:
(1), the structure of Recombinant organism
1.1, designing Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg(one-letter symbol and be expressed as LEVLFQGPEEMQRR) aminoacid sequence is that basic repeating unit L14 builds protein sequence pL14-n, wherein n is any number of 1-100;
1.2, the nucleotide sequence nL14 that protein sequence as implied above is corresponding is synthesized, (ctggaggtgctgtttcagggtccggaagagatgcagcgtcgc)
n;
1.3, nucleotide sequence nL14 clone is entered expression vector, then proceed in E. coli expression strains by expression vector, screening obtains Recombinant organism;
(2) fermentation culture of Recombinant organism
2.1, picking preferably after Recombinant organism list bacterium colony, as 35 ~ 38 DEG C of overnight incubation in LB substratum;
2.2, by bacterium liquid inoculation amplification culture, cultivate 2.5 ~ 3 hours, add IPTG and induce for 35 ~ 38 DEG C, 15 ~ 18 DEG C are continued cultivation 18 ~ 22 hours, and the albumen now given expression to is GST-pL14-n, collected by centrifugation thalline;
(3) purifying of restructuring smoothing wrinkle small peptide rE14
3.1, with the resuspended bacterium of Tris damping fluid, ultrasonication, collected by centrifugation supernatant liquor;
3.2, gsh affinity column purifying from supernatant liquor is utilized to obtain smoothing wrinkle small peptide rE14
It is as follows that rE14 small peptide of the present invention detects purity:
Smoothing wrinkle small peptide rE14 theoretical molecular is 1732Da, cannot be detected, need to utilize mass spectrometry method to identify by conventional SDS-PAGE.With Matrix Assisted Laser Desorption lonization-Time of Flight instrument MALDI-TOF AXIMA-CFR Plus(KRATOS Analytical, Shimadzu corporation, Japan) analyze under linear model.Analytical results as shown in Figure 3.
Compared with prior art, the smoothing wrinkle small peptide rE14 that prepared by the present invention has following characteristics:
1, novel smoothing wrinkle small peptide E14(GPEEMQRRLEVLFQ disclosed in this invention), be the sequence of long-term screening and optimizing, good water solubility, stability is strong.
2, the preparation method of novel smoothing wrinkle small peptide rE14 disclosed in this invention, adopt escherichia expression system, be suitable for extensive amplification, production cost is very low, and sequence in gene is carried out codon optimized for escherichia expression system, further increase output.
3, the preparation method of novel smoothing wrinkle small peptide E14 disclosed in this invention, product purity is high, utilizes mass spectrometry method to detect without obvious impurity component.
In sum, smoothing wrinkle small peptide E14 good water solubility disclosed by the invention, stability is strong, and purity is high, and production technique is simple, with low cost, smoothing wrinkle skin products can be allowed to walk close to the daily life of the common people, have wide market application foreground.
Accompanying drawing explanation
Fig. 1 represents the plasmid construction collection of illustrative plates of expression vector pGEX-6p-nL14-9.
Fig. 2 SDS-PAGE electrophoresis represents the purge process of rE14.Before enzyme is cut, gsh affinity column is the recombinant protein of GST-pL14-9, adds after PPase enzyme cuts, only surplus PPase enzyme and GST label protein on post material, rE14 is by wash-out results (because molecular weight is little, SDS-PAGE cannot be utilized to identify).
Fig. 3 represents the Mass Spectrometric Identification result of smoothing wrinkle small peptide rE14 in purified product.The theoretical molecular of rE14 is 1732Da, fits like a glove with mass spectrometric detection result.
After Fig. 4 represents that room temperature is placed 4 weeks, the Mass Spectrometric Identification result of smoothing wrinkle small peptide rE14 in purified product.RE14 demonstrates standard molecular weight 1732Da, proves do not have obvious degradation.
Embodiment
Below specific embodiments of the invention are described in detail.
A preparation method for smoothing wrinkle small peptide, comprises the steps:
(1), the structure of Recombinant organism
1.1, in order to enzyme cut after obtain smoothing wrinkle small peptide rE14, special design aminoacid sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg is basic repeating unit L14, builds protein sequence pL14-n.
The repetition number of this unit and the harvest yield of final small peptide proportional.Usually 1-100 recomposition unit can be adopted to build, the target protein sequence (LEVLFQGPEEMQRR) built like this
nbe abbreviated as pL14-n, wherein n is any number of 1-100; Experiment proves as n=9, and the yield of smoothing wrinkle small peptide rE14 is higher.
The present embodiment is described for 9 repeating units, and the protein sequence pL14-9 of 9 repeating units is (LEVLFQGPEEMQRR)
9:
LEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRR。
1.2, synthesize the nucleotide sequence nL14 that protein sequence as implied above is corresponding, and be optimized according to the codon that intestinal bacteria are favourable, obtain nucleotide sequence nL14-9 as shown in SEQ ID NO.1:
ctggaagtgctgttccaaggtccggaagagatgcagcgtcgcctggaagtgctgttccagggtcctgaggaaatgcaacgtcgcctggaagttctgttccaaggcccggaggaaatgcaacgccgcctggaagttctgtttcaaggtccggaggagatgcagcgccgtctggaggtgctgttccagggtccggaagagatgcagcgccgcctggaggtgctgtttcagggtccggaagaaatgcaacgccgcctggaagtgctgttccaaggtccggaggaaatgcagcgccgcctggaggtgctgttccagggtccggaagaaatgcagcgccgcttagaagtgctgtttcagggcccggaggaaatgcagcgccgc。
1.3, nucleotide sequence nL14-9 clone is entered expression vector, then proceed in E. coli expression strains by expression vector, screening obtains Recombinant organism; Be specially:
Design upstream and downstream primer, primer is diluted to 50pmol/ μ l, primer and nucleotide sequence carry out PCR step, and agarose gel electrophoresis detects nL14-9 band and is positioned at about 350bp, reclaims goal gene.With
bami and
xhonL14-9 gene (above-mentioned nucleotide sequence) enzyme is cut into sticky end by I, same process pGEX-6p-1 carrier, and add T4 DNA ligase, 4 DEG C connect 12 hours, and transformation of E. coli DH5 α, for the amplification of plasmid; Hickie clone is chosen with penbritin-LB plate screening, the alkaline lysis of molecular cloning or boiling method is utilized to extract plasmid, enzyme is utilized to cut with the method qualification of PCR correct, then nucleotide sequence analysis contains correct gene order reading frame, successfully builds pGEX-6p-nL14-9 expression vector; By pGEX-6p-nL14-9 expression vector, transform BL21 bacterium (screening obtains Recombinant organism), for expressing protein.
(2) fermentation culture of Recombinant organism
2.1, picking preferably after Recombinant organism list bacterium colony, as 35 ~ 38 DEG C of overnight incubation in the LB substratum of 5ml, preferable temperature is 37 DEG C.
2.2, bacterium liquid is inoculated amplification culture according to 1:100, cultivate 2.5 ~ 3 hours, add IPTG and induce for 35 ~ 38 DEG C, 15 ~ 18 DEG C are continued cultivation 18 ~ 22 hours, be preferably 37 DEG C to cultivate 3 hours, add 0.5mM IPTG and induce, 16 DEG C are continued cultivation 20 hours; The albumen now given expression to is GST-pL14-9, collected by centrifugation thalline.
(3) purifying of restructuring smoothing wrinkle small peptide rE14
3.1, with the mixed bacterial sediment of PBS buffer solution; with Tris damping fluid 20-40ml volume resuspended about 500ml bacterium liquid precipitate; Triton X-100 is coordinated to help cracking bacterium with N,O-Diacetylmuramidase; under mixture of ice and water environment, (2s is ultrasonic, 5s interval, total length 45min for carrying out ultrasonic bacteria breaking; protection temperature 44 DEG C); the centrifugal 20min of 12000rpm/min, collects supernatant liquor, now namely contains a large amount of GST-pL14-9 recombinant proteins in solution.
3.2, with PBS buffer solution for cleaning gsh affinity column (Glutathione Sepharose beads), then post material and bacteria break supernatant mixing are educated altogether, room temperature or on ice jog 30min, then upper prop, with the PBS solution washing foreign protein containing 1M NaCl, post just leaves pure GST-pL14-9 recombinant protein.
Post adds appropriate Prescission Protease(and is called for short PPase) proteolytic enzyme, in room temperature or jog 2 hours on ice, smoothing wrinkle small peptide rE14 can be discharged from gsh affinity column, with PBS solution wash-out smoothing wrinkle small peptide rE14, products therefrom dialysed overnight, freeze-drying is that dry powder is stand-by.
Smoothing wrinkle small peptide rE14 prepared by aforesaid method is made up of 14 amino acid, and its aminoacid sequence is: Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln, is expressed as GPEEMQRRLEVLFQ with one-letter symbol.Utilize the technique of Escherichia coli fermentation, the rE14 of recombinant expressed preparation, production technique is simple, with low cost, is easy to promote.
<110> Shanxi Jin Bo Biomedics Inc.
<120> smoothing wrinkle small peptide and preparation method thereof
<160>1
<210>1
<211>378
<212>DNA
<400>
ctggaagtgctgttccaaggtccggaagagatgcagcgtcgcctggaagtgctgttccagggtcctgaggaaatgcaacgtcgcctggaagttctgttccaaggcccggaggaaatgcaacgccgcctggaagttctgtttcaaggtccggaggagatgcagcgccgtctggaggtgctgttccagggtccggaagagatgcagcgccgcctggaggtgctgtttcagggtccggaagaaatgcaacgccgcctggaagtgctgttccaaggtccggaggaaatgcagcgccgcctggaggtgctgttccagggtccggaagaaatgcagcgccgcttagaagtgctgtttcagggcccggaggaaatgcagcgccgc
Claims (6)
1. a smoothing wrinkle small peptide, its aminoacid sequence is: Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln.
2. prepare a preparation method for smoothing wrinkle small peptide according to claim 1, it is characterized in that: comprise the steps:
(1), the structure of Recombinant organism
1.1, the aminoacid sequence designing Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg – Arg is that basic repeating unit L14 builds protein sequence pL14-n, and wherein n is any number of 1-100;
1.2, the nucleotide sequence nL14 that protein sequence as implied above is corresponding is synthesized, (ctggaggtgctgtttcagggtccggaagagatgcagcgtcgc)
n;
1.3, nucleotide sequence nL14 clone is entered expression vector, then proceed in E. coli expression strains by expression vector, screening obtains Recombinant organism;
(2) fermentation culture of Recombinant organism
2.1, picking preferably after Recombinant organism list bacterium colony, as 35 ~ 38 DEG C of overnight incubation in LB substratum;
2.2, by bacterium liquid inoculation amplification culture, cultivate 2.5 ~ 3 hours, add IPTG and induce for 35 ~ 38 DEG C, 15 ~ 18 DEG C are continued cultivation 18 ~ 22 hours, and the albumen now given expression to is GST-pL14-n, collected by centrifugation thalline;
(3) purifying of restructuring smoothing wrinkle small peptide rE14
3.1, with the resuspended bacterium of Tris damping fluid, ultrasonication, collected by centrifugation supernatant liquor;
3.2, gsh affinity column purifying from supernatant liquor is utilized to obtain smoothing wrinkle small peptide rE14.
3. the preparation method of smoothing wrinkle small peptide according to claim 2, is characterized in that: in step 1.1 and 1.2, n=9.
4. the preparation method of smoothing wrinkle small peptide according to claim 3, is characterized in that: step 1.3 is specially, and design upstream and downstream primer, carries out PCR step by primer and nucleotide sequence, reclaim goal gene, use
bami and
xhonucleotide sequence is cut into sticky end by I, same process pGEX-6p-1 carrier, and add T4 DNA ligase, 4 DEG C connect 12 hours, transformation of E. coli DH5 α; Hickie clone is chosen with penbritin-LB plate screening, the alkaline lysis of molecular cloning or boiling method is utilized to extract plasmid, enzyme is utilized to cut with the method qualification of PCR correct, then nucleotide sequence analysis contains correct gene order reading frame, successfully builds pGEX-6p-nL14-9 expression vector; By pGEX-6p-nL14-9 expression vector, transform BL21 bacterium.
5. the preparation method of smoothing wrinkle small peptide according to claim 4, is characterized in that: step 2.2 is specially, and bacterium liquid is inoculated amplification culture according to 1:100, cultivate 3 hours for 37 DEG C, add 0.5mM IPTG to induce, 16 DEG C are continued cultivation 20 hours, collected by centrifugation thalline.
6. the preparation method of smoothing wrinkle small peptide according to claim 5, it is characterized in that: step 3.1 is specially, with the mixed bacterial sediment of PBS buffer solution, with the resuspended bacterium liquid precipitate of Tris damping fluid, Triton X-100 is coordinated to help cracking bacterium, carrying out ultrasonic bacteria breaking under mixture of ice and water environment, the centrifugal 20min of 12000rpm/min with N,O-Diacetylmuramidase, collect supernatant liquor, now namely contain a large amount of GST-pL14-9 recombinant proteins in solution;
Step 3.2 is specially, and with PBS buffer solution for cleaning gsh affinity column, then post material and bacteria break supernatant mixing is educated altogether, room temperature or on ice jog 30min, then upper prop, with the PBS solution washing foreign protein containing 1M NaCl, post just leaves pure GST-pL14-9 recombinant protein; Post adds Prescission Protease proteolytic enzyme, in room temperature or jog 2 hours on ice, smoothing wrinkle small peptide rE14 can be discharged from gsh affinity column.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109021071A (en) * | 2018-08-21 | 2018-12-18 | 山西锦波生物医药股份有限公司 | Peptide and its preparation method and application |
| CN116162133A (en) * | 2022-06-15 | 2023-05-26 | 山西锦波生物医药股份有限公司 | Wrinkle-removing short peptide and preparation method thereof |
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|---|---|---|---|---|
| US7015192B1 (en) * | 1999-04-23 | 2006-03-21 | Lipotec, S.A. | Neuronal exocytosis inhibiting peptides and cosmetic and pharmaceutical compositions containing said peptides |
| CN104402975A (en) * | 2014-12-11 | 2015-03-11 | 太原锦波生物医药科技有限公司 | Anti-aging short peptide and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7015192B1 (en) * | 1999-04-23 | 2006-03-21 | Lipotec, S.A. | Neuronal exocytosis inhibiting peptides and cosmetic and pharmaceutical compositions containing said peptides |
| CN104402975A (en) * | 2014-12-11 | 2015-03-11 | 太原锦波生物医药科技有限公司 | Anti-aging short peptide and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 王延金等: "A型肉毒素面部祛皱常见副反应分析", 《河南科技大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109021071A (en) * | 2018-08-21 | 2018-12-18 | 山西锦波生物医药股份有限公司 | Peptide and its preparation method and application |
| CN109021071B (en) * | 2018-08-21 | 2020-08-04 | 山西锦波生物医药股份有限公司 | Peptides, their preparation and use |
| CN116162133A (en) * | 2022-06-15 | 2023-05-26 | 山西锦波生物医药股份有限公司 | Wrinkle-removing short peptide and preparation method thereof |
| CN116162133B (en) * | 2022-06-15 | 2023-10-20 | 山西锦波生物医药股份有限公司 | Wrinkle-removing short peptide and preparation method thereof |
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Address after: 030032 No.18, Jinbo street, Tanghuai Park, Taiyuan comprehensive reform demonstration zone, Taiyuan City, Shanxi Province Patentee after: SHANXI JINBO BIOMEDICAL Co.,Ltd. Address before: 030032 Shanxi city of Taiyuan province by the economic and Technological Development Zone No. 18 North Street Patentee before: SHANXI JINBO BIOMEDICAL Co.,Ltd. |