CN107955075B - Fusion protein of truncated human acanthogobius signal protein and cell-penetrating peptide, preparation method and coding gene thereof - Google Patents
Fusion protein of truncated human acanthogobius signal protein and cell-penetrating peptide, preparation method and coding gene thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Abstract
The invention provides a fusion protein of truncated human spiny mouse signal protein and cell-penetrating peptide, which comprises a truncated human spiny mouse signal protein sequence and a cell-penetrating peptide sequence; wherein the truncated human hamster signal protein sequence and the cell-penetrating peptide sequence are connected by a connecting peptide. The present invention also provides a method of preparing a fusion protein, comprising: preparing plasmid, transforming, screening multiple copies of inserted recombinant, fermenting and purifying. The invention also provides a coding gene of the fusion protein, an expression vector containing the coding gene and a cell line.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a fusion protein of truncated human hamster signaling protein and cell-penetrating peptide and a preparation method thereof, and also relates to a coding gene of the fusion protein.
Background
The Agouti signaling protein (ASIP) is coded by an Agouti gene, and the Agouti gene and the Agouti signaling protein of different species have higher homology. The Agouti gene coded rat signal protein consists of 131 amino acids and comprises a signal peptide with 22 amino acid residues at the N end and a functional region with 109 amino acid residues.
Lu et al, 1994, found that the acanthosis-related protein (ASIP) has high affinity with melanocortin receptor (MCR), and that ASIP competes with alpha-Melanocortin (MSH) and binds to melanocortin receptor-1 (MC1R), blocking signaling downstream of alpha-MSH and thereby inhibiting melanin synthesis. In vitro melanocyte culture experiments also prove that the addition of ASIP can antagonize alpha-MSH, inhibit the increase of cAMP level in melanocytes, inhibit the expression of tyrosinase TRP-1 and TRP-2, and reduce the synthesis of melanin by the melanocytes. Experiments also show that ASIP can competitively antagonize melanin synthesis of alpha-MSH in the autograft skin, so that the skin pigmentation capability is reduced, and the fact that the expression of the alpha-MSH in epidermal cells is up-regulated after the skin graft is transplanted is an important reason for hyperpigmentation of the skin graft. It can thus be concluded that the effect of ASIP on skin color is mainly achieved by inhibiting melanin synthesis.
The acanthus spinosus signal protein has obvious activity of inhibiting melanin generation and has good application potential in skin pigmentation, but the expression and application of the recombinant acanthus spinosus signal protein are not reported at present.
Disclosure of Invention
The invention aims to provide a fusion protein of truncated human acanthosis signal protein and cell-penetrating peptide, a preparation method and a coding gene thereof.
On one hand, the invention provides a fusion protein of a truncated human hamster signal protein and a cell-penetrating peptide, which comprises a truncated human rodent signal protein sequence and a cell-penetrating peptide sequence; wherein the truncated human hamster signal protein sequence and the cell-penetrating peptide sequence are connected by a connecting peptide.
In another aspect, the present invention provides a method for preparing the aforementioned fusion protein, comprising:
(1) plasmid preparation: artificially synthesizing a nucleotide sequence of SEQ ID No. 4 in a sequence table by a whole gene, carrying out Xho I and Not I double enzyme digestion on a pPIC9K vector and the nucleotide sequence of SEQ ID No. 4, recovering enzyme digestion products, connecting by using DNA ligase, converting escherichia coli, and extracting plasmids;
(2) and (3) transformation: transforming the plasmid prepared in the step (1) into a pichia pastoris competent cell to obtain a transformed bacterial colony;
(3) screening of multicopy insertion recombinants: further screening the bacterial colony transformed in the step (2) to obtain a transformant;
(4) fermentation: performing fermentation culture on the transformant obtained in the step (3) to obtain fermentation liquor;
(5) and (3) purification: and (4) sequentially carrying out solid-liquid separation, filtration, concentration and ion exchange chromatography on the fermentation liquor obtained in the step (4) to obtain a product.
In another aspect, the present invention provides a gene encoding the aforementioned fusion protein.
In another aspect, the present invention provides an expression vector comprising the aforementioned encoding gene.
In another aspect, the invention provides a cell line comprising the aforementioned coding gene.
In another aspect, the invention provides the use of the aforementioned fusion protein in the preparation of a medicament.
In another aspect, the invention provides the use of the aforementioned fusion protein in the preparation of a cosmetic.
The truncated human acanthis signal protein in the fusion protein of the truncated human acanthis signal protein and the cell-penetrating peptide is obtained by shearing natural acanthis signal protein, selecting 50 amino acids at the C end, and still having the activity of inhibiting melanin generation; meanwhile, cell-penetrating peptide is added at the N end of the truncated acanthosis signal protein, so that the cell-penetrating peptide can penetrate through skin barrier when being applied to the pigmented skin.
Drawings
FIG. 1 shows pPIC9K-YARA-ASIP in example 150And (3) plasmid mapping.
FIG. 2 is the electrophoresis chart before and after purification of the fermentation broth of the fusion protein of truncated human hamster signaling protein and cell-penetrating peptide of example 1.
FIG. 3 is a bar graph of the results of the in vivo tyrosinase inhibition assay performed in example 3.
Detailed Description
To fully explain the technical solution of the present invention to solve the technical problems. The present invention will be described in detail with reference to the following examples and drawings, but the technical solutions, embodiments of the technical solutions, and the scope of protection of the present invention are not limited thereto. Unless defined otherwise, technical and scientific terms appearing hereinafter have the meaning commonly understood by one of ordinary skill in the art.
The acanthus spinosus signal protein has obvious activity of inhibiting melanin generation and has good application potential in skin pigmentation, but the expression and application of the recombinant acanthus spinosus signal protein are not reported at present. The inventor analyzes the sequence of the functional region of the human spiny mouse signal protein, removes the 109 amino acid N-terminal part of the functional region of the human spiny mouse signal protein which is not beneficial to the expression region (rich in Kex2 enzyme cutting site), reserves the 50 amino acid residues at the C terminal, fuses with the cell penetrating peptide, realizes the expression in pichia pastoris, is used as a raw material for inhibiting the synthesis of melanin, and can be applied to medicines and cosmetics.
According to one aspect of the invention, the invention provides a fusion protein of a truncated human hamster signal protein and a cell-penetrating peptide, which comprises a truncated human rodent signal protein sequence and a cell-penetrating peptide sequence; wherein the truncated human hamster signal protein sequence and the cell-penetrating peptide sequence are connected by a connecting peptide.
Wherein the truncated human acanthus signal protein sequence is a sequence of 50 amino acids at C-terminal of the human acanthus signal protein or an amino acid sequence with at least 85% homology, preferably at least 90%, 93%, 95%, 97%, 98% or 99% homology. Wherein, the C-terminal 50 amino acid sequence of the human acanthopanax senticosus signal protein is the amino acid sequence of SEQ ID No. 1 in the sequence table. The inventor finds that 50 amino acid residues at the C end of the human spiny mouse signal protein sequence are reserved, so that the region which is not beneficial to expression on the human spiny mouse signal protein sequence is removed, and the membrane penetration of the fusion protein is facilitated.
Among them, the cell-penetrating peptide is a kind of short peptide capable of carrying macromolecular substance into cell, and its cell-penetrating ability is not dependent on classical endocytosis. In the present invention, the cell-penetrating peptide sequence is the amino acid sequence of SEQ ID No. 2 of the sequence Listing, or an amino acid sequence having at least 85% homology, preferably at least 90%, 93%, 95%, 97%, 98% or 99% homology with SEQ ID No. 2.
Wherein the connecting peptide is a sequence consisting of glycine and serine; commonly used linker peptides can be used in the present invention, such as GGGS.
In a preferred embodiment, the fusion protein of the invention is the amino acid sequence of SEQ ID No. 3 of the sequence Listing.
According to another aspect of the present invention, there is provided a method for preparing the aforementioned fusion protein, comprising:
(1) plasmid preparation: artificially synthesizing a nucleotide sequence of SEQ ID No. 4 in a sequence table by a whole gene, carrying out Xho I and Not I double enzyme digestion on a pPIC9K vector and the nucleotide sequence of SEQ ID No. 4, recovering enzyme digestion products, connecting by using DNA ligase, converting escherichia coli, and extracting plasmids;
(2) and (3) transformation: transforming the plasmid prepared in the step (1) into a pichia pastoris competent cell to obtain a transformed bacterial colony;
(3) screening of multicopy insertion recombinants: further screening the bacterial colony transformed in the step (2) to obtain a transformant;
(4) fermentation: performing fermentation culture on the transformant obtained in the step (3) to obtain fermentation liquor;
(5) and (3) purification: and (4) sequentially carrying out solid-liquid separation, filtration, concentration and ion exchange chromatography on the fermentation liquor obtained in the step (4) to obtain a product.
Preferably, the specific steps of the purification of step (5) are: and (4) performing solid-liquid separation on the fermentation liquor obtained in the step (4) by centrifugation, taking supernatant, performing microfiltration on the fermentation supernatant, collecting filtrate, performing ultrafiltration, desalination and concentration, collecting concentrated solution, and performing ion exchange chromatography to obtain the product.
In a preferred embodiment, the fusion protein of the present invention is prepared by a method comprising:
(1) preparation of plasmids
Artificially synthesizing the nucleotide sequence of SEQ ID No. 4 in the sequence table by using whole gene, carrying out Xho I and Not I double enzyme digestion on the pPIC9K vector and the nucleotide sequence of the artificially synthesized SEQ ID No. 4 by using whole gene, recovering enzyme digestion products, connecting the enzyme digestion products by using DNA ligase, transforming escherichia coli, extracting a plasmid, and naming the plasmid as pPIC9K-YARA-ASIP50；
(2) Electrotransformation of Pichia pastoris
pPIC9K-YARA-ASIP linearized with SalI endonuclease50Uniformly mixing plasmids with pichia pastoris competent cells, transferring the mixture into an ice-precooled electric transformation cup, electrically shocking for 4-10 milliseconds, adding ice-precooled sorbitol solution to uniformly mix the thalli, coating an MD culture medium plate, performing inverted culture for 3-4 days, and growing colonies on the MD culture medium plate;
(3) screening for multicopy insertion recombinants
Correspondingly inoculating colonies growing on the MD culture medium plate to YPD plates with the G418 concentrations of 1G/L, 2G/L, 3G/L and 4G/L respectively, culturing at 30 ℃, and screening to obtain transformants;
(4) fermentation of
Inoculating the screened transformant into a BMGY culture medium, carrying out shake culture for 24 hours, transferring the transformant as a first-stage seed into a fermentation tank filled with the FBS culture medium, carrying out culture for 16-20 hours at a pH value of 5.0, transferring the transformant as a second-stage seed into a large tank filled with the FBS culture medium, carrying out fermentation at a growth temperature of 30 ℃, wherein the induction temperature is lower than the growth temperature, the pH value is 5.5, the dissolved oxygen content is controlled to be more than 30%, and carrying out induction fermentation for 36-42 hours;
(5) purification of
And (4) performing solid-liquid separation on the fermentation liquor obtained in the step (4) by centrifugation, taking supernatant, performing microfiltration on the fermentation supernatant by using a hollow fiber microfiltration system with the pore diameter of 0.22 mu m, collecting filtrate, performing ultrafiltration desalination concentration by using a hollow fiber ultrafiltration system with the molecular weight cutoff of 1000D, collecting concentrated solution, and performing ion exchange chromatography by using SP Sepharose FF to obtain the product.
According to another aspect of the present invention, the present invention provides a gene encoding the aforementioned fusion protein. Preferably, the coding gene is a nucleotide sequence of SEQ ID No. 5 in a sequence table, or a nucleotide sequence which has at least 85% homology, preferably at least 90%, 93%, 95%, 97%, 98% or 99% homology with SEQ ID No. 5 and encodes a protein with the same function.
According to another aspect of the present invention, there is provided an expression vector comprising the aforementioned encoding gene.
According to another aspect of the invention, there is provided a cell line comprising the aforementioned encoding gene.
According to another aspect of the invention, the invention provides the use of the aforementioned fusion protein for the preparation of a medicament.
According to another aspect of the present invention, the present invention provides the use of the aforementioned fusion protein in the preparation of a cosmetic.
In a particularly preferred embodiment, the invention is achieved by the following embodiments.
The fusion protein has the total length of 65 amino acids, the carbon end is 50 amino acid sequences (SEQ ID No:1) at the C end of the human rat-stabbing signal protein, the nitrogen end is 11 amino acid sequences (SEQ ID No:2) of the cell-penetrating peptide, the two peptide segments are connected by the flexible connecting peptide GGGS, the fusion protein has the amino acid sequence of SEQ ID No:3, and the amino acid sequence is specifically as follows: YARAAARQAR AGGGSVRPRT PLSAPCVATR NSCKPPAPAC CDPCASCQCR FFRSACSCRV LSLNC。
The preparation method of the fusion protein comprises the following steps:
1. construction of fusion protein expression vector of truncated human acanthosis signal protein and cell-penetrating peptide
According to the designed amino acid sequence of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide, the nucleotide sequence of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide is designed and artificially synthesized in a whole gene according to the codon preference of pichia pastoris, meanwhile, an Xho I endonuclease cleavage site CTCGAG and a pichia pastoris kex2 cleavage site sequence AAAAGA are added at the 5 'end, an Not I endonuclease cleavage site GCGGCCGC is added at the 3' end, the sequence is as follows, and the sequence is detailed in SEQ ID No. 4 in the sequence table:
carrying out Xho I and Not I double enzyme digestion on the pPIC9K vector and the DNA sequence of the fusion protein of the truncated human hamster signaling protein and the cell-penetrating peptide synthesized by the artificial whole gene, recovering the enzyme digestion product, connecting the enzyme digestion product by using DNA ligase, transforming escherichia coli, extracting the plasmid, and naming the plasmid as pPIC 9-9K-YARA-ASIP50。
2. Electrotransformation of Pichia pastoris
Mu.g of SalI endonuclease linearized pPIC9K-YARA-ASIP50Mixing plasmids with 80 mu L of pichia pastoris competent cells uniformly, transferring the mixture into a 0.2cm ice-precooled electric transformation cup, electrically shocking for 4-10 milliseconds, adding 1mL ice-precooled 1mol/L sorbitol solution to mix the strains uniformly, coating an MD culture medium flat plate, performing inverted culture at 30 ℃ for 3-4 days, and growing colonies on the MD culture medium flat plate;
3. screening for multicopy insertion recombinants
Correspondingly inoculating colonies growing on the MD culture medium plate to YPD plates with the G418 concentrations of 1G/L, 2G/L, 3G/L and 4G/L by using sterile toothpicks, culturing at 30 ℃, screening to obtain transformants, and identifying by shaking bottles;
4. fermentation of fusion protein of truncated human acanthogobius signal protein and cell-penetrating peptide
Inoculating the screened transformant into 400ml of BMGY culture medium, carrying out shaking culture at 30 ℃ for 24 hours, transferring the transformant as a first-level seed into a 5L fermentation tank filled with 4L of FBS culture medium, setting the temperature at 30 ℃, controlling the pH value at 5.0, carrying out culture for 16-20 hours, transferring the transformant as a second-level seed into a 50L large tank filled with the FBS culture medium for fermentation, controlling the growth temperature at 30 ℃, the induction temperature at 29 ℃, the pH value at 5.5, controlling the dissolved oxygen at more than 30%, and carrying out induction fermentation for 36-42 hours;
5. purification of fusion protein of truncated human acanthosis signal protein and cell-penetrating peptide
And (2) performing solid-liquid separation on the fermentation liquor by centrifugation, taking supernatant, performing microfiltration on the fermentation supernatant by using a hollow fiber microfiltration system with the aperture of 0.22 mu m, collecting filtrate, performing ultrafiltration desalination concentration by using a hollow fiber ultrafiltration system with the molecular weight cutoff of 1000D, collecting concentrated solution, and performing ion exchange chromatography by using SP Sepharose FF to obtain the fusion protein of the truncated human spiny mouse signal protein and the cell-penetrating peptide, wherein the purity of the fusion protein is more than 90%.
Examples
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
1. Construction of expression vectors
According to the designed amino acid sequence of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide, the nucleotide sequence of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide is designed and artificially synthesized in a whole gene according to the codon preference of pichia pastoris, and meanwhile, an Xho I incision enzyme cutting site CTCGAG and a pichia pastoris kex2 cutting site sequence AAAAGA are added at the 5 'end, an Not I incision enzyme cutting site GCGGCCGC is added at the 3' end, and the sequence is SEQ ID No:4 in the sequence table.
The pPIC9K vector (purchased from Invitrogen) and the artificially synthesized nucleotide sequence of the whole gene were subjected to double digestion with Xho I (purchased from Thermo Fisher) and Not I (purchased from Thermo Fisher), the digested products were recovered, ligated with DNA ligase, transformed into E.coli, and the plasmid was extracted and named pPIC9K-YARA-ASIP50(the plasmid map is shown in FIG. 1).
2. Electrotransformation of Pichia pastoris
Mu.g of SalI endonuclease linearized pPIC9K-YARA-ASIP50Mixing plasmid with 80 μ L of Pichia pastoris competent cells, transferring to a 0.2cm ice-precooled electrotransformation cup, shocking for 4-10 ms, adding 1mL ice-precooled 1mol/L sorbitol solution to mix the thallus, coating MD medium plate, performing inverted culture at 30 deg.C for 3-4 days, and culturing on the MD medium plateGrowing bacterial colonies;
3. screening for multicopy insertion recombinants
Correspondingly inoculating colonies growing on the MD culture medium plate to YPD plates with the G418 concentrations of 1G/L, 2G/L, 3G/L and 4G/L by using sterile toothpicks, culturing at 30 ℃, and screening to obtain transformants;
4. fermentation of fusion proteins
The selected transformant was inoculated into 400ml of BMGY medium, cultured with shaking at 30 ℃ for 24 hours, and transferred as first-order seed to a medium containing 4L FBS (1L of 40 g/K glycerol)2SO4 18.2g、H3PO426.7ml、CaSO4.2H2O 0.93g、MgSO414.9g and 4.13g of KOH) was cultured at 30 ℃ and a pH of 5.0 for 16 to 20 hours in a 5-L fermenter containing 30L of FBS medium, and transferred to a 50-L jar containing 30L of FBS medium as a secondary seed for fermentation. In the fermentation process, the growth temperature is 30 ℃, the induction temperature is 29 ℃, the pH value is 5.5, the dissolved oxygen is controlled to be 20-30%, when the dissolved oxygen is suddenly increased, the glycerol in the basic salt culture medium is indicated to be exhausted, 18.15mL/h/L of glycerol with the mass percentage concentration of 50% is fed, and 12mL/L of trace element PTM1(1L of glycerol containing CuSO) is added in the glycerol with the mass percentage concentration of 50%4.5H2O 6g、NaI 0.08g、MnSO4.H2O 3g、Na2MoO4.H2O 0.2g、H3BO3 0.02g、H2SO4 5ml、CoCl2.6H2O 0.5g、ZnCl2 20g、FeSO4.7H2O75 g and biotin 0.2g, mixed to prepare the medicament) for maintaining dissolved oxygen in>30 percent. Stopping feeding when the wet bacteria weight of the fermentation liquor reaches 180mg/mL, starving for 1 hour, exhausting glycerol, feeding 20L of methanol with the mass percentage concentration of 75% in a fed-batch manner for induction fermentation, feeding 12mL/L of PTM1 trace elements in the methanol with the mass percentage concentration of 75% for 2-3 hours at the speed of 7.5mL/L/h, and then increasing the speed to 10.9mL/L/h for feeding and fermentation. And (3) adjusting the rotating speed, the tank pressure and the ventilation quantity to enable the dissolved oxygen to be more than 20%, and performing induced fermentation for 36-42 hours.
5. Purification of fusion proteins
5.1 solid-liquid separation and decolorization of fermentation broth
After fermentation is finished, centrifugally separating fermentation liquor, taking 35L of supernatant, carrying out microfiltration by using a hollow fiber microfiltration system with the aperture of 0.22 mu m, collecting filtrate, carrying out ultrafiltration desalination concentration by using a hollow fiber ultrafiltration system with the molecular weight cutoff of 1000D, collecting concentrated solution when the conductivity is less than 1ms/cm, and removing a large amount of yellow-green substances generated by fermentation of pichia pastoris by using the two-step ultrafiltration method.
5.2 ion exchange chromatography
Performing anion exchange chromatography on the concentrated solution, selecting SP Sepharose FF (purchased from GE company in America) as a filler, using A phase as 10mmol/L citric acid buffer solution with pH of 5.8, using B phase as A phase +1M NaCl, balancing 3 times of column volume of A phase, loading, using A phase + 5% B phase to flush the column, then using 10 times of column volume of A phase + 10% B phase to elute, collecting eluent, using roll-type ultrafiltration membrane with molecular weight of 1000D, desalting, collecting concentrated solution, freeze-drying by a freeze-drying machine to obtain the fusion protein of truncated type human hamster signal protein and cell-penetrating peptide with purity of more than 90%, wherein the purification result is shown in figure 2.
Example 2
The melanin synthesis inhibition rate of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide is detected:
the purified fusion protein of truncated human acanthis serratus signal protein and cell-penetrating peptide, acanthis serratus signal protein (containing 109 amino acid functional region, purchased from Wuhan Huamei organism) and whitening component arbutin (purchased from Xianyuan Sen organism) are diluted with culture solution to 10 concentrations-1、10-2、10-3、10-4、10-5mol/L. The blank control group was added with only the corresponding vehicle used to dilute the drug.
The density of mouse melanoma cells B16 was adjusted to 5X 105Inoculating each well in 6-well plate, changing liquid after 6 hr, adding 1ml of medicinal solution into each well, setting 3 multiple wells for each concentration, adding fresh culture solution instead of medicinal solution into control group, and adding 5% CO2Incubating at 37 deg.C for 3d (replacing medium once), discarding supernatant, adding 0.25% pancreatin 1ml per well, digesting at room temperature for 5min, adding 4ml culture medium to stop digestionBlowing and beating the mixture into single cell suspension, taking 20 mu l of the single cell suspension as cell count, centrifuging the rest cell suspension for 5min at 1500r/min, discarding the solution, adding 1ml of 1N NaOH solution, shaking for 5min, heating in 80 ℃ water bath for 1h, transferring to a 96-well plate, adding 100 mu l of the solution into each well, selecting a wavelength of 405nm, adjusting zero by using a blank hole, measuring the absorbance value on an enzyme-linked immunosorbent assay detector, and repeating each experiment for 3 times. Melanin synthesis inhibition (%) [1- (drug well absorbance value ÷ drug well cell density) ÷ (control well absorbance value ÷ control well cell density)]X 100%. The results are shown in the following table, and it can be seen from the table that the fusion protein of the present invention has a significant inhibitory effect on melanogenesis, has a dose-dependent relationship, i.e., the inhibitory rate on melanogenesis increases with the increase of dose, and has a similar inhibitory activity to agouti signaling protein, and in addition, the inhibitory rate of melanin synthesis at the same dose is higher than that of arbutin, which is a natural melanin inhibitor.
TABLE 1 Effect of fusion proteins on melanin synthesis
Example 3
The in vivo inhibition tyrosinase activity of the fusion protein of the truncated human acanthosis signal protein and the cell-penetrating peptide is determined:
18 KM mice, each half of male and female, were randomly divided into three groups (6 mice per group), a blank group, a spiny mouse signal protein group, and a fusion protein group, and after removing back hair with 10% sodium sulfide, and 24 hours of breeding, the mice were treated with physiological saline and 10 cotton swabs according to the groups-1mol/L acanthous signal protein (containing 109 amino acid functional region, purchased from Wuhan Huamei organism), 10-1mol/L fusion protein (the fusion protein of truncated human spiny mouse signal protein and cell-penetrating peptide prepared in example 1) was smeared twice a day, 5 days later, the mice were sacrificed, 1.5cm × 1.5cm of back skin was taken, physiological saline was added to prepare 10% homogenate, the supernatant was centrifuged to take, tyrosinase activity was measured by a mouse Tyrosinase (TYR) elisa kit, and statistical analysis was performed by sps statistical software. The results show (see FIG. 3), that the rodent signaling protein group compared to the blank group,the tyrosinase activity is obviously inhibited, and the difference is obvious (P)<0.05); compared with the blank group, the fusion protein group has obvious inhibition and obvious difference (P)<0.01); the fusion protein group has more obvious inhibition of tyrosinase activity than the acanthomys signal protein group, and the two have very obvious difference (P)<0.01)。
The study in example 2 shows that the fusion protein of the acanthopanax senticosus signal protein, the truncated human acanthopanax senticosus signal protein and the cell-penetrating peptide has similar melanin generation inhibiting activity in vitro cell experiments. The in vivo experimental study of example 3 shows that the fusion protein of truncated human acanthosis signal protein and cell-penetrating peptide has more significant tyrosinase activity inhibiting effect than the acanthosis signal protein. Comprehensive analysis shows that the fusion protein of truncated human hamster signaling protein and cell-penetrating peptide can effectively and competitively antagonize the combination of alpha-MSH and melanin receptor, thus inhibiting the expression of tyrosinase and reducing the synthesis of melanin by melanocyte.
The present invention has been disclosed in the foregoing in terms of preferred embodiments, but it will be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to those of the embodiments are intended to be included within the scope of the claims of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined in the claims.
Sequence listing
<110> Shanxi Huikang Biotechnology Limited liability company
<120> truncated human acanthomys signal protein and cell-penetrating peptide fusion protein, preparation method and coding gene thereof
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Gln Cys Arg Phe Phe Arg Ser Ala Cys Ser Cys Arg Val Leu Ser Leu
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<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala
1 5 10
<210> 3
<211> 65
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala Gly Gly Gly Ser Val
1 5 10 15
Arg Pro Arg Thr Pro Leu Ser Ala Pro Cys Val Ala Thr Arg Asn Ser
20 25 30
Cys Lys Pro Pro Ala Pro Ala Cys Cys Asp Pro Cys Ala Ser Cys Gln
35 40 45
Cys Arg Phe Phe Arg Ser Ala Cys Ser Cys Arg Val Leu Ser Leu Asn
50 55 60
Cys
65
<210> 4
<211> 218
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctcgagaaaa gatacgctag agctgctgct agacaagcta gagctggtgg tggtagtgtt 60
agaccaagaa ccccattgtc cgctccatgt gttgctacca gaaactcctg taagccacca 120
gctccagctt gttgtgaccc atgtgcttcc tgtcaatgta gattcttcag atccgcttgt 180
tcctgtagag ttttgtcctt gaactgttaa gcggccgc 218
<210> 5
<211> 195
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tacgctagag ctgctgctag acaagctaga gctggtggtg gtagtgttag accaagaacc 60
ccattgtccg ctccatgtgt tgctaccaga aactcctgta agccaccagc tccagcttgt 120
tgtgacccat gtgcttcctg tcaatgtaga ttcttcagat ccgcttgttc ctgtagagtt 180
ttgtccttga actgt 195
Claims (10)
1. A fusion protein of truncated human acanthosis signal protein and cell-penetrating peptide is characterized in that the fusion protein is an amino acid sequence of SEQ ID No. 3 in a sequence table.
2. The method of producing the fusion protein according to claim 1, comprising:
(1) plasmid preparation: artificially synthesizing a nucleotide sequence of SEQ ID No. 4 in a sequence table by a whole gene, carrying out Xho I and Not I double enzyme digestion on a pPIC9K vector and the nucleotide sequence of SEQ ID No. 4, recovering enzyme digestion products, connecting by using DNA ligase, converting escherichia coli, and extracting plasmids;
(2) and (3) transformation: transforming the plasmid prepared in the step (1) into a pichia pastoris competent cell to obtain a transformed bacterial colony;
(3) screening of multicopy insertion recombinants: further screening the bacterial colony transformed in the step (2) to obtain a transformant;
(4) fermentation: performing fermentation culture on the transformant obtained in the step (3) to obtain fermentation liquor;
(5) and (3) purification: and (4) sequentially carrying out solid-liquid separation, filtration, concentration and ion exchange chromatography on the fermentation liquor obtained in the step (4) to obtain a product.
3. The preparation method according to claim 2, wherein the purification of step (5) comprises the following specific steps: and (4) performing solid-liquid separation on the fermentation liquor obtained in the step (4) by centrifugation, taking supernatant, performing microfiltration on the fermentation supernatant, collecting filtrate, performing ultrafiltration, desalination and concentration, collecting concentrated solution, and performing ion exchange chromatography to obtain the product.
4. The method of claim 2 or 3, comprising:
(1) preparation of plasmids
Artificially synthesizing the nucleotide sequence of SEQ ID No. 4 in the sequence table by using whole gene, carrying out Xho I and Not I double enzyme digestion on the pPIC9K vector and the nucleotide sequence of the artificially synthesized SEQ ID No. 4 by using whole gene, recovering enzyme digestion products, connecting the enzyme digestion products by using DNA ligase, transforming escherichia coli, extracting a plasmid, and naming the plasmid as pPIC9K-YARA-ASIP50；
(2) Electrotransformation of Pichia pastoris
pPIC9K-YARA-ASIP linearized with SalI endonuclease50Uniformly mixing plasmids with pichia pastoris competent cells, transferring the mixture into an ice-precooled electric transformation cup, electrically shocking for 4-10 milliseconds, adding ice-precooled sorbitol solution to uniformly mix the thalli, coating an MD culture medium plate, performing inverted culture for 3-4 days, and growing colonies on the MD culture medium plate;
(3) screening for multicopy insertion recombinants
Correspondingly inoculating colonies growing on the MD culture medium plate to YPD plates with the G418 concentrations of 1G/L, 2G/L, 3G/L and 4G/L respectively, culturing at 30 ℃, and screening to obtain transformants;
(4) fermentation of
Inoculating the screened transformant into a BMGY culture medium, carrying out shake culture for 24 hours, transferring the transformant as a first-stage seed into a fermentation tank filled with the FBS culture medium, carrying out culture for 16-20 hours at a pH value of 5.0, transferring the transformant as a second-stage seed into a large tank filled with the FBS culture medium, carrying out fermentation at a growth temperature of 30 ℃, wherein the induction temperature is lower than the growth temperature, the pH value is 5.5, the dissolved oxygen content is controlled to be more than 30%, and carrying out induction fermentation for 36-42 hours;
(5) purification of
And (4) performing solid-liquid separation on the fermentation liquor obtained in the step (4) by centrifugation, taking supernatant, performing microfiltration on the fermentation supernatant by using a hollow fiber microfiltration system with the pore diameter of 0.22 mu m, collecting filtrate, performing ultrafiltration desalination concentration by using a hollow fiber ultrafiltration system with the molecular weight cutoff of 1000D, collecting concentrated solution, and performing ion exchange chromatography by using SP Sepharose FF to obtain the product.
5. A gene encoding the fusion protein according to claim 1.
6. The encoding gene of claim 5, wherein the encoding gene is the nucleotide sequence of SEQ ID No. 5 of the sequence Listing.
7. An expression vector comprising the coding gene of claim 5 or 6.
8. A cell line comprising the gene of claim 5 or 6.
9. Use of the fusion protein of claim 1 for the preparation of a medicament.
10. Use of the fusion protein according to claim 1 for the preparation of a cosmetic product.
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| CN106632690A (en) * | 2016-12-29 | 2017-05-10 | 陕西慧康生物科技有限责任公司 | Recombinant human heat shock protein 10 as well as encoding gene and preparation method thereof |
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| CN106589137A (en) * | 2016-12-12 | 2017-04-26 | 陕西慧康生物科技有限责任公司 | Cell-penetrating peptide and human Beta-defensin 3 fusion protein and preparation method and application thereof |
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