CN104829691B - Smoothing wrinkle small peptide and preparation method thereof - Google Patents
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Abstract
The present invention relates to the preparation method of genetic engineering field recombinant protein, specifically a kind of novel smoothing wrinkle small peptide and preparation method.The invention discloses a kind of smoothing wrinkle small peptides to be named as E14, is made of 14 amino acid；A kind of technique using Escherichia coli fermentation is also disclosed, may be implemented to industrialize large-scale Prepare restructuring E14（Abbreviation rE14）.The present invention is detected using various biological means, it was demonstrated that rE14 simple production process, of low cost, purity is good, and stability is high, and anti-aging skin nutrition product can be allowed to walk close to the daily life of the common people, have a vast market application prospect.
Description
Technical field
The present invention relates to the method that designs and prepares of genetic engineering field recombinant protein, specifically a kind of novel smoothing wrinkle small peptide
And preparation method thereof.
Background technology
The skin of people and higher mammal is made of epidermis, corium, subcutaneous tissue.Epidermis does not have a blood vessel, the energy of cell and
Nutrition must be provided by corium microcirculation.Skin corium is the source of life of skin, be one layer of densification, tough and tensile and flexible group
It knits, mainly by fibre composition（Collagen fabric, elastic fibers and reticular fibre）, glycoprotein, Glucosamine and sodium hyaluronate
Deng composition.
Human research confirms that the generation of wrinkle is related with facial muscles fiber overstimulation.Therefore, in theory,
The generation of wrinkle can be mitigated by directly reducing muscular movement contraction.The contraction of muscle be regulated and controled by neurotransmitter, and
SNARE compounds are then the signal sources that neurotransmitter is transmitted.Widely used creotoxin is exactly based on inhibition currently on the market
The activity of the function inhibitio nerve cell of SNARE compounds, and then achieve the purpose that wrinkle.But the secondary of creotoxin makees
With very huge, the mankind are studying always the functional analogue of creotoxin, make every effort to as possible subtract under the premise of reaching identical purpose
Its few side effect.
The Argireline that Lipotec companies find is exactly a kind of functional analogue of creotoxin.Argireline is by 6
Amino acid forms, and amino acid group becomes Ac-EEMQRR-NH2.Functional experiment confirms that Argireline has good Cutaneous permeation
Property, it and can effectively inhibit the function of SNARE, have and very strong crease-resistant go wrinkle activity, also referred to as Argireline.
Currently, having there is many series of the well-known cosmetics brand of many countries to be added to Argireline Argirelines work
For the active ingredient of smoothing wrinkle.Also there are product sale in some biotechnology companies of China.But the preparation method of Argireline at present
It is also all limited to chemical synthesis, the cost of great number limits being widely used for this kind of product.Therefore, this kind of short in order to make
The skin-nourishing product of peptides constantly walks close to the daily life of the common people so that ordinary people possesses safe and stable, practical high-quality
Cosmetics, it is necessary to which more efficient smoothing wrinkle small peptide is researched and developed in design, and explores the extensive technology for preparing smoothing wrinkle small peptide.
Invention content
The present invention reduces the production cost of this kind of beauty peptide to improve the biological activity of existing smoothing wrinkle small peptide,
A kind of design scheme of novel smoothing wrinkle small peptide is provided, and its large-scale preparation method is provided.
The present invention adopts the following technical scheme that realization：
A kind of smoothing wrinkle small peptide is made of 14 amino acid, and amino acid sequence is：Gly-Pro-Glu-Glu-Met-
Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln is expressed as GPEEMQRRLEVLFQ, referred to as E14 with one-letter symbol
Small peptide, theoretical molecular weight 1.732kD.
Above-mentioned E14 small peptides can be prepared by a variety of production technologies：
1, using the technique of Escherichia coli fermentation, the E14 of preparation, abbreviation rE14, simple production process, cost are recombinantly expressed
It is cheap, it is easy to spread.
2, E14 is synthesized using chemically synthesized technique, abbreviation sE14, cost is higher, but has the life similar with rE14
Object function.
The present invention provides the preparation method of above-mentioned smoothing wrinkle small peptide, includes the following steps：
（1）, Recombinant organism structure
1.1, design Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg（With list
Character number is expressed as LEVLFQGPEEMQRR）Amino acid sequence is basic repetitive unit L14 structure protein sequence pL14-n, wherein
N is any number of 1-100；
1.2, the corresponding nucleotide sequence nL14 of protein sequence as shown above is synthesized,
（ctggaggtgctgtttcagggtccggaagagatgcagcgtcgc）n；
1.3, nucleotide sequence nL14 clones are entered into expression vector, expression vector is then transferred to Bacillus coli expression bacterium
In strain, screening obtains Recombinant organism；
（2）The fermented and cultured of Recombinant organism
2.1, the Recombinant organism single bacterium colony after picking is preferred was cultivated as 35~38 DEG C in LB culture mediums
Night；
2.2, bacterium solution is inoculated with amplification culture, 35~38 DEG C are cultivated 2.5~3 hours, and IPTG is added and is induced, and 15~18
DEG C continue culture 18~22 hours, the albumen given expression at this time is GST-pL14-n, and thalline were collected by centrifugation；
（3）Recombinate the purifying of smoothing wrinkle small peptide rE14
3.1, bacterium is resuspended with Tris buffer solutions, supernatant is collected by centrifugation in ultrasonication；
3.2, it is purified from supernatant using glutathione affinity column and obtains smoothing wrinkle small peptide rE14
RE14 small peptides detection purity of the present invention is as follows：
Smoothing wrinkle small peptide rE14 theoretical molecular weights are 1732Da, can not be detected by conventional SDS-PAGE, need profit
It is identified with mass spectrometry method.With Matrix Assisted Laser Desorption lonization-Time of Flight instrument MALDI-TOF AXIMA-CFR
Plus（KRATOS Analytical, Shimadzu corporation, Japan) it is analyzed under linear model.Analysis knot
Fruit is as shown in Figure 3.
Compared with prior art, the smoothing wrinkle small peptide rE14 that prepared by the present invention has the characteristics that：
1, novel smoothing wrinkle small peptide E14 disclosed in this invention（GPEEMQRRLEVLFQ）, it is the sequence of long-term screening and optimizing,
Good water solubility, stability are strong.
2, the preparation method of novel smoothing wrinkle small peptide rE14 disclosed in this invention is suitable for using escherichia expression system
Extensive amplification, production cost is very low, and for escherichia expression system to have carried out codon excellent for the sequence in gene
Change, further improves yield.
3, the preparation method of novel smoothing wrinkle small peptide E14 disclosed in this invention, product purity is high, is detected using mass spectrometry method
Without apparent impurity component.
In conclusion smoothing wrinkle small peptide E14 good water solubilities disclosed by the invention, stability is strong, and purity is high, production technology letter
It is single, it is of low cost, smoothing wrinkle skin products can be allowed to walk close to the daily life of the common people, have a vast market application prospect.
Description of the drawings
Fig. 1 shows the plasmid construction collection of illustrative plates of expression vector pGEX-6p-nL14-9.
Fig. 2 SDS-PAGE electrophoresis indicates the purification process of rE14.It is GST- before digestion, on glutathione affinity column
The recombinant protein of pL14-9 is added after PPase digestions, and only surplus PPase enzymes and GST label proteins, rE14 are eluted on column material
Harvest（Because molecular weight is small, SDS-PAGE can not be utilized to identify）.
Fig. 3 indicates the Mass Spectrometric Identification result of smoothing wrinkle small peptide rE14 in purified product.The theoretical molecular weight of rE14 is 1732Da,
It fits like a glove with Mass Spectrometer Method result.
Fig. 4 expressions were placed at room temperature for after 4 weeks, the Mass Spectrometric Identification result of smoothing wrinkle small peptide rE14 in purified product.RE14 is shown
Go out standard molecular weight 1732Da, it was demonstrated that there is no obvious degradation.
Specific implementation mode
Specific embodiments of the present invention are described in detail below.
A kind of preparation method of smoothing wrinkle small peptide, includes the following steps：
（1）, Recombinant organism structure
1.1, in order to obtain smoothing wrinkle small peptide rE14 after digestion, it is specifically designed amino acid sequence Leu-Glu-Val-Leu-
Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg is basic repetitive unit L14, structure protein sequence pL14-n.
The repetition number of this unit and the harvest yield of final small peptide are proportional.1-100 weight usually may be used
Group unit is built, the destination protein sequence built in this way（LEVLFQGPEEMQRR）nIt is abbreviated as pL14-n, wherein n is 1-
100 any number；Experiments have shown that as n=9, the yield of smoothing wrinkle small peptide rE14 is higher.
The present embodiment is illustrated by taking 9 repetitive units as an example, and the protein sequence pL14-9 of 9 repetitive units is
（LEVLFQGPEEMQRR）9：
LEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQG
PEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRRLEVLFQGPEEMQRR。
1.2, the corresponding nucleotide sequence nL14 of protein sequence as shown above is synthesized, and advantageous close according to Escherichia coli
Numeral optimizes, and obtains nucleotide sequence nL14-9 as shown in SEQ ID NO.1：
ctggaagtgctgttccaaggtccggaagagatgcagcgtcgcctggaagtgctgttccagggtcctgaggaaatgca
acgtcgcctggaagttctgttccaaggcccggaggaaatgcaacgccgcctggaagttctgtttcaaggtccggagg
agatgcagcgccgtctggaggtgctgttccagggtccggaagagatgcagcgccgcctggaggtgctgtttcagggt
ccggaagaaatgcaacgccgcctggaagtgctgttccaaggtccggaggaaatgcagcgccgcctggaggtgctgtt
ccagggtccggaagaaatgcagcgccgcttagaagtgctgtttcagggcccggaggaaatgcagcgccgc。
1.3, nucleotide sequence nL14-9 clones are entered into expression vector, expression vector is then transferred to Bacillus coli expression
In bacterial strain, screening obtains Recombinant organism；Specially：
Upstream and downstream primer is designed, primer is diluted to 50pmol/ μ l, primer and nucleotide sequence carry out PCR step, agar
Sugared detected through gel electrophoresis nL14-9 bands are located at 350bp or so, recycle target gene.WithBamI andXhoI is by nL14-9 genes
（Above-mentioned nucleotide sequence）Digestion is same to handle pGEX-6p-1 carriers, addition T4 DNA ligases, 4 DEG C of connections at cohesive end
12 hours, bacillus coli DH 5 alpha is converted, is used for the amplification of plasmid；Choose hickie clone, profit with ampicillin-LB plate screenings
Plasmid is extracted with the alkaline lysis or boiling method of molecular cloning, is identified correctly using the method for digestion and PCR, then nucleotide
Sequence analysis contains correct gene order reading frame, successfully builds pGEX-6p-nL14-9 expression vectors；By pGEX-6p-
NL14-9 expression vectors convert BL21 bacterium（Screening obtains Recombinant organism）, for expressing albumen.
（2）The fermented and cultured of Recombinant organism
2.1, the Recombinant organism single bacterium colony after picking is preferred, as 35~38 DEG C in the LB culture mediums of 5ml
Overnight incubation, preferable temperature are 37 DEG C.
2.2, by bacterium solution according to 1：100 inoculation amplification cultures, 35~38 DEG C are cultivated 2.5~3 hours, and IPTG is added and is lured
It leads, 15~18 DEG C are continued culture 18~22 hours, and preferably 37 DEG C are cultivated 3 hours, and 0.5mM IPTG are added and are induced, 16 DEG C
Continue culture 20 hours；The albumen given expression at this time is GST-pL14-9, and thalline were collected by centrifugation.
（3）Recombinate the purifying of smoothing wrinkle small peptide rE14
3.1, mixed bacterial sediment is washed with PBS buffer solution, is resuspended about with Tris buffer solution 20-40ml volumes
500ml bacterium solutions precipitate, and crack bacterium with lysozyme cooperation Triton X-100 helps, the carrying out ultrasonic bacteria breaking under mixture of ice and water environment
（2s ultrasounds, 5s intervals, overall length 45min protect 44 DEG C of temperature）, 12000rpm/min centrifugation 20min, collection supernatant, at this time
Contain a large amount of GST-pL14-9 recombinant proteins in solution.
3.2, glutathione affinity column (Glutathione Sepharose beads) is cleaned with PBS buffer solution, then will
Column material and bacteria break supernatant mixing are educated altogether, and room temperature or on ice jog 30min, then upper prop, is washed with the PBS solution containing 1M NaCl
Foreign protein is washed, pure GST-pL14-9 recombinant proteins have been left on column.
Suitable Prescission Protease are added on column（Abbreviation PPase）Protease is in room temperature or light on ice
It shakes 2 hours, you can smoothing wrinkle small peptide rE14 is released from glutathione affinity column, smoothing wrinkle small peptide is eluted with PBS solution
RE14, products therefrom dialysed overnight, be lyophilized for dry powder it is for use.
Smoothing wrinkle small peptide rE14 prepared by the above method is made of 14 amino acid, and amino acid sequence is：Gly-Pro-
Glu-Glu-Met-Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln is expressed as with one-letter symbol
GPEEMQRRLEVLFQ.Using the technique of Escherichia coli fermentation, the rE14 of preparation is recombinantly expressed, simple production process is at low cost
It is honest and clean, it is easy to spread.
<110>Shanxi Jin Bo Biomedics Inc.
<120>Smoothing wrinkle small peptide and preparation method thereof
<160>1
<210>1
<211>378
<212>DNA
<400>
ctggaagtgctgttccaaggtccggaagagatgcagcgtcgcctggaagtgctgttccagggtcctgaggaaatgca
acgtcgcctggaagttctgttccaaggcccggaggaaatgcaacgccgcctggaagttctgtttcaaggtccggagg
agatgcagcgccgtctggaggtgctgttccagggtccggaagagatgcagcgccgcctggaggtgctgtttcagggt
ccggaagaaatgcaacgccgcctggaagtgctgttccaaggtccggaggaaatgcagcgccgcctggaggtgctgtt
ccagggtccggaagaaatgcagcgccgcttagaagtgctgtttcagggcccggaggaaatgcagcgccgc
Claims (5)
1. a kind of preparation method of smoothing wrinkle small peptide rE14, it is characterised in that：The amino acid sequence of the smoothing wrinkle small peptide rE14 is：
Gly-Pro-Glu-Glu- Met-Gln-Arg-Arg-Leu-Glu-Val-Leu-Phe-Gln indicate to utilize Escherichia coli fermentation
Technique recombinant expression smoothing wrinkle small peptide, preparation method includes the following steps：
（1）, Recombinant organism structure：
1.1, the amino acid sequence of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Glu-Glu-Met-Gln-Arg-Arg is designed
It is any number of 1-100 to be classified as basic repetitive unit pL14 structures protein sequence pL14-n, wherein n；
1.2, the corresponding nucleotide sequence L14-n of protein sequence as shown above is synthesized, i.e.,
（ctggaggtgctgtttcagggtccggaagagatgcagcgtcgc）n；
1.3, nucleotide sequence L14-n clones are entered into expression vector, expression vector is then transferred to E. coli expression strains
In, screening obtains Recombinant organism；
（2）The fermented and cultured of Recombinant organism：
2.1, the Recombinant organism single bacterium colony after picking is preferred, as 35~38 DEG C of overnight incubations in LB culture mediums；
2.2, by bacterium solution be inoculated with amplification culture, 35~38 DEG C cultivate 2.5~3 hours, be added IPTG induced, 15~18 DEG C after
Continuous culture 18~22 hours, the albumen given expression at this time is GST-pL14-n, and thalline were collected by centrifugation；
（3）Recombinate the purifying of smoothing wrinkle small peptide rE14：
3.1, bacterium is resuspended with Tris buffer solutions, supernatant is collected by centrifugation in ultrasonication；
3.2, on glutathione affinity column be added Prescission Protease protease, using glutathione affinity column from
Purifying obtains smoothing wrinkle small peptide rE14 in supernatant.
2. the preparation method of smoothing wrinkle small peptide rE14 according to claim 1, it is characterised in that：In step 1.1 and 1.2, n=
9。
3. the preparation method of smoothing wrinkle small peptide rE14 according to claim 1, it is characterised in that：Step 1.3 is specifically, design
Primer and nucleotide sequence are carried out PCR step, recycle target gene by upstream and downstream primer, with Bam I and Xho I by nucleotide
Sequence is cut into cohesive end, same to handle pGEX-6p-1 carriers, and T4 DNA ligases are added, and 4 DEG C connect 12 hours, and conversion is big
Enterobacteria DH5 α；With ampicillin-LB plate screenings choose hickie clone, using molecular cloning alkaline lysis or boil
Method extracts plasmid, identifies that correctly then nucleotide sequence analysis contains correct gene order and reads using the method for digestion and PCR
Frame frame successfully builds pGEX-6p-nL14-9 expression vectors；By pGEX-6p-nL14-9 expression vectors, BL21 bacterium are converted.
4. the preparation method of smoothing wrinkle small peptide rE14 according to claim 1, it is characterised in that：Step 2.2 is specifically, by bacterium
Liquid is according to 1：100 inoculation amplification cultures, 37 DEG C are cultivated 3 hours, and 0.5mM IPTG are added and are induced, and 16 DEG C to continue culture 20 small
When, thalline were collected by centrifugation.
5. the preparation method of smoothing wrinkle small peptide rE14 according to claim 1, it is characterised in that：Step 3.1 specifically, with
PBS buffer solution washs mixed bacterial sediment, is precipitated with Tris buffer solution resuspended bacterium solutions, with lysozyme cooperation Triton X-
100 help to crack bacterium, and the carrying out ultrasonic bacteria breaking under mixture of ice and water environment, 12000rpm/min centrifuges 20min, collects supernatant,
Contain a large amount of GST-pL14-9 recombinant proteins in solution at this time；
Then column material and bacteria break supernatant are mixed and are educated altogether specifically, clean glutathione affinity column with PBS buffer solution by step 3.2,
Room temperature or on ice jog 30min, then upper prop, washs foreign protein with the PBS solution containing 1M NaCl, has been left on column pure
GST-pL14-9 recombinant proteins；Prescission Protease protease is added on column, jog 2 is small in room temperature or on ice
When, you can smoothing wrinkle small peptide rE14 is released from glutathione affinity column.
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| 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 |
| 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 |
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 |
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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 |
|---|
| A型肉毒素面部祛皱常见副反应分析;王延金等;《河南科技大学学报》;20100615;第28卷(第2期);133-135 * |
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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. |