CN107955075A - Truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide and preparation method thereof and encoding gene - Google Patents

Truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide and preparation method thereof and encoding gene Download PDF

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CN107955075A
CN107955075A CN201711260455.7A CN201711260455A CN107955075A CN 107955075 A CN107955075 A CN 107955075A CN 201711260455 A CN201711260455 A CN 201711260455A CN 107955075 A CN107955075 A CN 107955075A
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CN107955075B (en
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侯增淼
李晓颖
李敏
高恩
杨小琳
赵金礼
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Shaanxi HuiKang Bio Tech Co Ltd
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    • 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

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Abstract

The present invention provides a kind of truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide, including truncated-type human Agouti signal protein sequence and cell-penetrating peptide sequence；Wherein described truncated-type human Agouti signal protein sequence and the cell-penetrating peptide sequence are connected by connecting peptide.Present invention also offers the method for preparing fusion protein, including：Prepare plasmid, conversion, screening, fermentation and the purifying of multicopy insertion recon.Present invention also offers the encoding gene of fusion protein, expression vector, cell line comprising the encoding gene.

Description

Truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide and preparation method thereof with Encoding gene
Technical field
The invention belongs to bioengineering field, and in particular to a kind of truncated-type human Agouti signal protein is merged with cell-penetrating peptide Albumen and preparation method thereof, the invention further relates to the encoding gene of the fusion protein.
Background technology
Agouti signal protein (Agouti signaling protein, ASIP) is by Agouti gene codes, different plant species Agouti genes and Agouti signal protein have higher homology.The Agouti signal protein of Agouti gene codes is by 131 Amino acid profile, the functional areas of signal peptide and 109 amino acid residues comprising 22 amino acid residues of N-terminal.
Lu in 1994 etc. has found that agouti GAP-associated protein GAP (ASIP) and novel melanocortin receptor (MCR) have the affinity of height, ASIP Combined with α-rush melanocyte (MSH) competitiveness and Melanocortin-1 receptor (MC1R), the signal transduction in retardance α-MSH downstreams and then suppression Melanin genesis.External melanocyte culture experiment also confirms that, add ASIP can antagonism α-MSH, suppress the cAMP in melanocyte Level rise, the expression for suppressing tyrosinase TRP-1 and TRP-2, reduce melanocyte synthesis melanocyte.Also experiment shows The melanin genesis of ASIP energy competitive antagonism α-MSH in Skin autografts, makes the reduction of skin graft colorability, so as to prove skin The up-regulated expression of α-MSH is the major reason that skin graft is in hyperpigmentation in epidermal cell after piece transplanting.So as to draw Conclusion, synthesis of influences of the ASIP to skin color mainly by suppressing melanocyte are realized.
Agouti signal protein has the significant activity for suppressing melanin generation, has in cutaneous pigmentation good Application potential, but yet there are no the report of the expression of restructuring Agouti signal protein and application.
The content of the invention
The present invention goal of the invention be the fusion protein that a kind of truncated-type human Agouti signal protein and cell-penetrating peptide are provided and Its preparation method and encoding gene.
On the one hand, the present invention provides a kind of truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide, including cut Short people's Agouti signal protein sequence and cell-penetrating peptide sequence；Wherein described truncated-type human Agouti signal protein sequence and described wear film Peptide sequence is connected by connecting peptide.
On the other hand, the present invention provides the preparation method of foregoing fusion albumen, including：
(1) plasmid is prepared：SEQ ID No in artificial full genome composition sequence table:4 nucleotide sequence, to pPIC9K Carrier and SEQ ID No:4 nucleotide sequence carries out I double digestion of Xho I and Not, recycles digestion products, is connected with DNA ligase Connect, and convert Escherichia coli, extract plasmid；
(2) convert：Plasmid prepared by step (1) converts Pichia pastoris competent cell, bacterium colony after being converted；
(3) screening of multicopy insertion recon：Bacterium colony after step (2) is converted further is screened, and is turned Beggar；
(4) ferment：Fermented and cultured is carried out to the transformant that step (3) obtains and obtains zymotic fluid；
(5) purify：The zymotic fluid that step (4) obtains is subjected to separation of solid and liquid, filtering, concentration and ion-exchange chromatography successively Obtain product.
On the other hand, the present invention provides the encoding gene of foregoing fusion albumen.
On the other hand, the present invention provides the expression vector containing afore-mentioned code gene.
On the other hand, the present invention provides the cell line containing afore-mentioned code gene.
On the other hand, the application the present invention provides foregoing fusion albumen in medicine is prepared.
On the other hand, the application the present invention provides foregoing fusion albumen in cosmetics are prepared.
Truncated-type human agouti signal egg in the truncated-type human Agouti signal protein of the present invention and the fusion protein of cell-penetrating peptide It is the shearing to natural Agouti signal protein in vain, chooses 50 amino acid of C-terminal, but still there is suppression melanin generation to live for it Property；Cell-penetrating peptide is with the addition of in the N-terminal of truncated-type Agouti signal protein at the same time, in order to the application in pigmentation skin When pass through skin barrier.
Brief description of the drawings
Fig. 1 is pPIC9K-YARA-ASIP in embodiment 150Plasmid map.
Fig. 2 is that truncated-type human Agouti signal protein and the fusion protein zymotic fluid of cell-penetrating peptide are rear electric before purification in embodiment 1 Swimming figure.
Fig. 3 is the internal suppression tyrosinase activity measurement result column diagram carried out in embodiment 3.
Embodiment
In order to absolutely prove that the present invention solves the technical solution that technical problem is implemented to use.With reference to embodiment and attached Figure elaborates invention, but technical scheme, the embodiment of technical solution and protection domain are not merely It is limited to this.Unless otherwise stated, the science and technology and technical term that hereinafter occur are generally understood that with those skilled in the art Implication.
Agouti signal protein has the significant activity for suppressing melanin generation, has in cutaneous pigmentation good Application potential, but yet there are no the report of the expression of restructuring Agouti signal protein and application.The present inventor analyzes People's Agouti signal protein function region sequence, removes people's Agouti signal protein functional areas 109 amino acid N ends part and is unfavorable in table The region (being rich in Kex2 restriction enzyme sites) reached, retains 50 amino acid residues of C-terminal, and is merged with cell-penetrating peptide, realizes Expressed in Pichia pastoris, suppress B16 cell raw material as one kind, can be applied to medicine and cosmetics.
According to an aspect of the present invention, melting the present invention provides a kind of truncated-type human Agouti signal protein and cell-penetrating peptide Hop protein, including truncated-type human Agouti signal protein sequence and cell-penetrating peptide sequence；Wherein described truncated-type human Agouti signal protein Sequence and the cell-penetrating peptide sequence are connected by connecting peptide.
Wherein, truncated-type human Agouti signal protein sequence be 50 amino acid of people's Agouti signal protein C-terminal sequence or There is at least 85% homology, the amino acid sequence of the homology of preferably at least 90%, 93%, 95%, 97%, 98% or 99% with it Row.Wherein, the sequence of 50 amino acid of people's Agouti signal protein C-terminal is SEQ ID No in sequence table:1 amino acid sequence Row.Inventor is had found by studying, and is retained 50, end of people's Agouti signal protein sequence C amino acid residue, had both been eliminated people agouti It is unfavorable for the region of expression in signal protein sequence, and can be conducive to fusion protein wears film.
Wherein, cell-penetrating peptide is a kind of small peptide that can be carried macromolecular substances and enter cell, its wear film ability independent of Classical encytosis.In the present invention, cell-penetrating peptide sequence is SEQ ID No in sequence table:2 amino acid sequence, alternatively, with SEQ ID No:2 have at least 85% homology, the homology of preferably at least 90%, 93%, 95%, 97%, 98% or 99% Amino acid sequence.
Wherein, the sequence that connection peptide is made of glycine and serine；Common connection peptide is used equally in the present invention, Such as GGGS.
In a kind of preferred embodiment, fusion protein of the invention is SEQ ID No in sequence table:3 ammonia Base acid sequence.
According to another aspect of the present invention, the present invention provides the preparation method of foregoing fusion protein, including：
(1) plasmid is prepared：SEQ ID No in artificial full genome composition sequence table:4 nucleotide sequence, to pPIC9K Carrier and SEQ ID No:4 nucleotide sequence carries out I double digestion of Xho I and Not, recycles digestion products, is connected with DNA ligase Connect, and convert Escherichia coli, extract plasmid；
(2) convert：Plasmid prepared by step (1) converts Pichia pastoris competent cell, bacterium colony after being converted；
(3) screening of multicopy insertion recon：Bacterium colony after step (2) is converted further is screened, and is turned Beggar；
(4) ferment：Fermented and cultured is carried out to the transformant that step (3) obtains and obtains zymotic fluid；
(5) purify：The zymotic fluid that step (4) obtains is subjected to separation of solid and liquid, filtering, concentration and ion-exchange chromatography successively Obtain product.
Preferably, the purifying of step (5) concretely comprises the following steps：The zymotic fluid that step (4) obtains is subjected to solid-liquid through centrifugation Separation, takes supernatant, carries out micro-filtration to fermented supernatant fluid, collects filtrate, then carries out desalination and concentration by ultrafiltration, collects concentrate, so After carry out ion-exchange chromatography, you can obtain product.
In a kind of preferred embodiment, the preparation method of fusion protein of the invention, including：
(1) plasmid is prepared
SEQ ID No in artificial full genome composition sequence table:4 nucleotide sequence, then to pPIC9K carriers and people The SEQ ID No of work full genome synthesis:4 nucleotide sequence carries out I double digestion of Xho I and Not, recycles digestion products, uses DNA Ligase connects, and converts Escherichia coli, extracts plasmid, is named as pPIC9K-YARA-ASIP50；
(2) Pichia pastoris electricity conversion
The pPIC9K-YARA-ASIP that will be linearized through I restriction endonucleases of Sal50Plasmid, mixes with Pichia pastoris competent cell, Go in the electricity conversion cup of ice precooling, shock by electricity 4~10 milliseconds, the sorbitol solution for adding ice precooling mixes thalline, is coated with MD Culture medium flat plate, is inverted culture 3~4 days, bacterium colony is grown on MD culture medium flat plates；
(3) screening of multicopy insertion recon
It is respectively 1g/L, 2g/L, 3g/L, 4g/L that the bacterium colony grown on MD culture medium flat plates, which is corresponded to, and is inoculated into G418 concentration YPD tablets on, 30 DEG C culture, screening obtain transformant；
(4) ferment
The transformant screened is inoculated in BMGY culture mediums, when shaken cultivation 24 is small, as first order seed switching in In fermentation tank equipped with FBS culture mediums, pH value for 5.0 culture 16~20 it is small when, as secondary seed transfer into equipped with FBS cultivate The bulk fermentation of base, 30 DEG C of growth temperature, inducing temperature are less than growth temperature, pH value 5.5, and dissolved oxygen control exists>30%, induction When fermentation 36~42 is small；
(5) purify
The zymotic fluid that step (4) obtains is subjected to separation of solid and liquid through centrifugation, supernatant is taken, is 0.22 μm of hollow fibre with aperture Tie up microfiltration systems and micro-filtration is carried out to fermented supernatant fluid, collect filtrate, the Hollow Fiber Ultrafiltration system for being 1000D with molecular cut off Desalination and concentration by ultrafiltration is carried out, collects concentrate, ion-exchange chromatography is carried out using SP Sepharose FF, you can obtain product.
According to another aspect of the present invention, the present invention provides the encoding gene of foregoing fusion albumen.Preferably, it is described Encoding gene is SEQ ID No in sequence table:5 nucleotide sequence, alternatively, with SEQ ID No:5 is homologous with least 85% Property, the nucleotides sequence of the homology of preferably at least 90%, 93%, 95%, 97%, 98% or 99% and coding identical function albumen Row.
According to another aspect of the present invention, the present invention provides the expression vector containing afore-mentioned code gene.
According to another aspect of the present invention, the present invention provides the cell line containing afore-mentioned code gene.
According to another aspect of the present invention, the application the present invention provides foregoing fusion albumen in medicine is prepared.
According to another aspect of the present invention, the application the present invention provides foregoing fusion albumen in cosmetics are prepared.
In a kind of particularly preferred embodiment, the present invention is realized by following concrete scheme.
The fusion protein total length of the present invention is 65 amino acid, and carbon teminal is 50 amino acid sequences of people's Agouti signal protein C-terminal Arrange (SEQ ID No:1), nitrogen end is 11 amino acid sequences of cell-penetrating peptide (SEQ ID No:2), by being flexibly connected between two peptide fragments Peptide GGGS connections, the fusion protein have SEQ ID No:3 amino acid sequence, amino acid sequence are specifically：YARAAARQAR AGGGSVRPRT PLSAPCVATR NSCKPPAPAC CDPCASCQCR FFRSACSCRV LSLNC。
The preparation method of above-mentioned fusion protein includes the following steps：
1st, truncated-type human Agouti signal protein and the fusion protein expression vector of cell-penetrating peptide are built
According to the truncated-type human Agouti signal protein of design and the fusion protein amino acid sequence of cell-penetrating peptide, according to complete red ferment Female codon preference, designs simultaneously the manually core of full genome synthesis truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide Nucleotide sequence, while in 5 ' end addition I inscribe cleavage sites CTCGAG of Xho and Pichia pastoris kex2 cleavage sequences AAAAGA, 3 ' end addition I inscribe cleavage sites GCGGCCGC of Not, sequence is as follows, refers to SEQ ID No in sequence table:4：
The truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide synthesized to pPIC9K carriers and artificial full genome DNA sequence dna carry out I double digestion of Xho I and Not, recycle digestion products, connected with DNA ligase, and convert Escherichia coli, carried Plasmid is taken, is named as pPIC9K-YARA-ASIP50。
2nd, Pichia pastoris electricity conversion
The pPIC9K-YARA-ASIP that 10 μ g are linearized through I restriction endonucleases of Sal50Plasmid, with 80 μ L Pichia pastoris competence Cell mixes, and goes in the electricity conversion cup of 0.2cm ice precoolings, shocks by electricity 4~10 milliseconds, add the mountain of the 1mol/L of 1mL ice precoolings Pears alcoholic solution mixes thalline, is coated with MD culture medium flat plates, and 30 DEG C are inverted culture 3~4 days, and bacterium is grown on MD culture medium flat plates Fall；
3rd, the screening of multicopy insertion recon
It is respectively 1g/L, 2g/ that the bacterium colony sterile toothpick grown on MD culture medium flat plates, which is corresponded to, and is inoculated into G418 concentration L, on the YPD tablets of 3g/L, 4g/L, 30 DEG C of cultures, screening obtains transformant, and is identified through shaking flask；
4th, the fermentation of truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide
The transformant screened is inoculated in 400ml BMGY culture mediums, when 30 DEG C of shaken cultivations 24 are small, as level-one Seed is transferred in the 5L fermentation tanks equipped with 4L FBS culture mediums, and temperature is set as 30 DEG C, and pH value 5.0, it is small to cultivate 16~20 When, transfer as secondary seed into the 50L bulk fermentations equipped with FBS culture mediums, 30 DEG C of growth temperature, 29 DEG C of inducing temperature, pH It is worth for 5.5, dissolved oxygen control exists>30%, when induction fermentation 36~42 is small；
5th, the purifying of truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide
Zymotic fluid carries out separation of solid and liquid through centrifugation, takes supernatant, is 0.22 μm of doughnut to fermented supernatant fluid aperture Microfiltration systems carry out micro-filtration, collect filtered solution, then the Hollow Fiber Ultrafiltration system for being 1000D with molecular cut off carries out ultrafiltration and takes off Salt concentrates, and collects concentrate, then carries out ion-exchange chromatography with SP Sepharose FF, and it is more than 90% to obtain purity The fusion protein of truncated-type human Agouti signal protein and cell-penetrating peptide.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality Apply among a scope.The experimental method of actual conditions is not specified in the following example, according to conventional methods and conditions, or according to business Product specification selects.
Embodiment 1
1st, the structure of expression vector
According to the truncated-type human Agouti signal protein of design and the fusion protein amino acid sequence of cell-penetrating peptide, according to complete red ferment Female codon preference, designs simultaneously the manually core of full genome synthesis truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide Nucleotide sequence, while in 5 ' end addition I inscribe cleavage sites CTCGAG of Xho and Pichia pastoris kex2 cleavage sequences AAAAGA, 3 ' end addition I inscribe cleavage sites GCGGCCGC of Not, sequence is SEQ ID No in sequence table:4.
Xho I is carried out to the nucleotide sequence of pPIC9K carriers (being purchased from Invitrogen companies) and artificial full genome synthesis (being purchased from Thermo Fisher companies) and Not I (being purchased from Thermo Fisher companies) double digestion, recycles digestion products, uses DNA Ligase connects, and converts Escherichia coli, extracts plasmid, is named as pPIC9K-YARA-ASIP50(plasmid map such as Fig. 1 institutes Show).
2nd, Pichia pastoris electricity conversion
The pPIC9K-YARA-ASIP that 10 μ g are linearized through I restriction endonucleases of Sal50Plasmid, with 80 μ L Pichia pastoris competence Cell mixes, and goes in the electricity conversion cup of 0.2cm ice precoolings, shocks by electricity 4~10 milliseconds, add the mountain of the 1mol/L of 1mL ice precoolings Pears alcoholic solution mixes thalline, is coated with MD culture medium flat plates, and 30 DEG C are inverted culture 3~4 days, and bacterium is grown on MD culture medium flat plates Fall；
3rd, the screening of multicopy insertion recon
It is respectively 1g/L, 2g/ that the bacterium colony sterile toothpick grown on MD culture medium flat plates, which is corresponded to, and is inoculated into G418 concentration L, on the YPD tablets of 3g/L, 4g/L, 30 DEG C of cultures, screening obtains transformant；
4th, the fermentation of fusion protein
The transformant screened is inoculated in 400ml BMGY culture mediums, when 30 DEG C of shaken cultivations 24 are small, as level-one Seed switching is in equipped with 4L FBS (40g containing glycerine, K in 1L2SO4 18.2g、H3PO426.7ml、CaSO4.2H2O 0.93g、 MgSO414.9g, KOH 4.13g are mixed) in the 5L fermentation tanks of culture medium, temperature is set as 30 DEG C, pH value 5.0, training Support 16~20 it is small when, transfer as secondary seed into the 50L bulk fermentations equipped with 30L FBS culture mediums.In fermentation process, growth 30 DEG C of temperature, 29 DEG C of inducing temperature, pH value 5.5, dissolved oxygen is controlled 20%~30%, when dissolved oxygen suddenly rises, indicates base Sweet Fuel Exhausted in plinth salt culture medium, starts stream plus 18.15mL/h/L mass percentage concentrations as 50% glycerine, in quality hundred It is that the PTM1 of trace element containing 12mL/L (contains CuSO in 50% glycerine in 1L to divide concentration4.5H2O 6g、NaI 0.08g、MnSO4.H2O 3g、Na2MoO4.H2O 0.2g、H3BO3 0.02g、H2SO4 5ml、CoCl2.6H2O 0.5g、ZnCl2 20g、FeSO4.7H2O 75g, biotin 0.2g, are mixed), maintain dissolved oxygen to exist>30%.When the wet bacterium of zymotic fluid is weighed to 180mg/mL, stop mending Material, when starvation 1 is small, glycerol depletion, stream plus 20L mass percentage concentrations are 75% methanol induction fermentation, are in mass percentage concentration In 75% methanol PTM1 containing 12mL/L trace element, using speed for 7.5ml/L/h stream plus 2~3 it is small when, then raising speed extremely 10.9ml/L/h streams add capable fermentation.Dissolved oxygen is set to be more than 20% by adjusting rotating speed, tank pressure and throughput, induction fermentation 36~42 Hour.
5th, the purifying of fusion protein
The separation of solid and liquid of 5.1 zymotic fluids and decoloration
After fermentation, zymotic fluid centrifuges, and takes supernatant 35L, is 0.22 μm of doughnut microfiltration systems with aperture Micro-filtration is carried out, collects filtered solution, then the Hollow Fiber Ultrafiltration system for being 1000D with molecular cut off carries out desalination and concentration by ultrafiltration, When electrical conductivity is less than 1ms/cm, concentrate is collected, is the big of removable Pichia pastoris fermentation generation by above-mentioned two steps ultrafiltration Measure yellowish green color substance.
5.2 ion-exchange chromatography
Above-mentioned concentrate is subjected to anion-exchange chromatography, filler selects SP Sepharose FF (to be purchased from U.S. GE public affairs Department), it is the 10mmol/L citrate buffer solutions that pH is 5.8 with A phases, B phases balance each other for A phase+1M NaCl, the A of 3 times of column volumes, Loading, then mutually rushes column with A phases+5%B, then is mutually eluted with the A phases+10%B of 10 times of column volumes, collects eluent, then uses and divides Son amount is the rolling ultrafiltration membrane ultrafiltration of 1000D, and desalination, collects concentrate, and freeze drier freezes, and it is more than 90% to obtain purity The fusion protein of truncated-type human Agouti signal protein and cell-penetrating peptide, purification result are shown in Fig. 2.
Embodiment 2
The melanin genesis inhibiting rate of truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide detects:
Purified truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide, Agouti signal protein (are included 109 Aminoacid functional area, purchased from the magnificent biology in Wuhan) and whitening composition ursin (being purchased from the gloomy biology in Xi'an source) utilize nutrient solution Diluted concentration is respectively that drug dilution concentration is respectively 10-1、10-2、10-3、10-4、10-5mol/L.Blank control group only adds dilute The corresponding solvent to release the drug used in thing.
Mouse black-in tumor cell B16 density is adjusted, with 5 × 105A/hole is inoculated in 6 orifice plates, and liquid is changed after 6h, is added per hole The drug solution of 1ml, each concentration set 3 multiple holes, and control group adds fresh medium alternatives to medication solution, 5%CO2, 37 DEG C be incubated 3d after (liquid is changed once in centre), abandoning supernatant, per hole add 0.25% pancreatin 1ml digest 5min at room temperature, add Enter 4ml nutrient solutions stop digestion, blow and beat into single cell suspension, take 20 μ l to make cell count, remaining cell suspension 1500r/min from Heart 5min, abandons upper liquid, adds the NaOH solution of 1ml 1N, shakes 5min, after 80 DEG C of heating water bath 1h, is transferred to 96 orifice plates, often Hole adds 100 μ l, selects 405nm wavelength, is returned to zero with blank well, and absorbance, each experiment weight are surveyed on enzyme-linked immunosorbent assay instrument It is 3 times multiple.Melanin genesis inhibiting rate (%)=[1- (medicine hole absorbance ÷ medicines hole cell density) ÷ (control wells absorbances Value ÷ control wells cell density)] × 100%.As a result see the table below, as can be seen from the table, fusion protein of the invention gives birth to melanocyte Into with obvious inhibitory action, having dose-dependence, i.e., the inhibiting rate of melanogenesis is improved with the increase of dosage, and There is similar inhibitory activity to Agouti signal protein, in addition, compared with natural black pigment inhibitor ursin, Isodose is black Element synthesis inhibiting rate is higher than ursin.
Influence of 1 fusion protein of table to B16 cell
Embodiment 3
Suppress tyrosinase activity measure in truncated-type human Agouti signal protein and the fusion protein body of cell-penetrating peptide：
KM mouse 18, half male and half female, is randomly divided into three groups (every group 6), blank group, Agouti signal protein group, fusion Protein groups, with 10% vulcanized sodium remove back hair, recover raising 24 it is small when after, by different groups with cotton swab smear physiology salt Water, 10-1Mol/L Agouti signal proteins (109 aminoacid functional areas being included, purchased from the magnificent biology in Wuhan), 10-1Mol/L merges egg (truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide prepared by embodiment 1) in vain, smears twice, smears 5 days daily Afterwards, mouse is put to death, skin of back 1.5cm × 1.5cm is taken, adds physiological saline, be prepared into 10% homogenate, centrifuging and taking supernatant, by small Mouse tyrosinase (TYR) elisa kit measurement tyrosinase activities, statistical analysis is carried out by spss statistical softwares.As a result Display is (see Fig. 3), and compared with blank group, tyrosinase activity substantially suppresses Agouti signal protein group, significant difference (P<0.05)； Compared with blank group, tyrosinase activity substantially suppresses fusion protein group, difference extremely significantly (P<0.01)；Fusion protein group is relatively pierced Mouse signal protein group tyrosinase activity suppresses to become apparent, the two difference extremely significantly (P<0.01).
Research shows in embodiment 2, the fusion protein of Agouti signal protein, truncated-type human Agouti signal protein and cell-penetrating peptide There is similar suppression melanin generation activity in cell experiment in vitro.The experiment in vivo of embodiment 3, which is studied, to be shown, truncated-type People's Agouti signal protein and the fusion protein of cell-penetrating peptide have suppression tyrosinase activity more significantly more than Agouti signal protein Effect.Comprehensive analysis illustrates that the fusion protein of truncated-type human Agouti signal protein and cell-penetrating peptide can be with more efficiently competitiveness Antagonism α-MSH are combined with melanocyte acceptor, so as to suppress the expression of tyrosinase, reduce melanocyte synthesis melanocyte, at this During Transdermal absorption, the cell-penetrating peptide in fusion protein plays an important role.
The present invention is hereinbefore disclosed with preferred embodiment, but it should be understood by those skilled in the art that, these Embodiment is only used for describing the present invention, and should not be construed as limiting the scope of the invention.It should be noted that every implement with these Example equivalent change and displacement, should all be set to be covered by scope of the presently claimed invention.Therefore, protection scope of the present invention The scope that should be subject to defined in claims.
Sequence table
<110>Shaanxi Hui Kang biotechnologies Co., Ltd
<120>Truncated-type human Agouti signal protein and the fusion protein of cell-penetrating peptide and preparation method thereof and encoding gene
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Val Arg Pro Arg Thr Pro Leu Ser Ala Pro Cys Val Ala Thr Arg Asn
1 5 10 15
Ser Cys Lys Pro Pro Ala Pro Ala Cys Cys Asp Pro Cys Ala Ser Cys
20 25 30
Gln Cys Arg Phe Phe Arg Ser Ala Cys Ser Cys Arg Val Leu Ser Leu
35 40 45
Asn Cys
50
<210> 2
<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 (14)

1. the fusion protein of a kind of truncated-type human Agouti signal protein and cell-penetrating peptide, it is characterised in that including truncated-type human agouti Signal protein sequence and cell-penetrating peptide sequence；Wherein described truncated-type human Agouti signal protein sequence and the cell-penetrating peptide sequence pass through Connect peptide connection.
2. fusion protein according to claim 1, it is characterised in that the truncated-type human Agouti signal protein sequence is people The sequence of 50 amino acid of Agouti signal protein C-terminal has at least 85% homology with it, preferably at least 90%, 93%, 95%th, the amino acid sequence of 97%, 98% or 99% homology；
The sequence of wherein 50 amino acid of people's Agouti signal protein C-terminal is SEQ ID No in sequence table:1 amino acid sequence Row.
3. fusion protein according to claim 1, it is characterised in that the cell-penetrating peptide sequence is SEQ ID in sequence table No:2 amino acid sequence, alternatively, with SEQ ID No:2 have at least 85% homology, preferably at least 90%, 93%, 95%, 97%th, the amino acid sequence of 98% or 99% homology.
4. fusion protein according to claim 1, it is characterised in that the connection peptide is made of glycine and serine Sequence.
5. according to claim 1-4 any one of them fusion proteins, it is characterised in that the fusion protein is in sequence table SEQ ID No:3 amino acid sequence.
6. the preparation method of claim 1-5 any one of them fusion proteins, it is characterised in that including：
(1) plasmid is prepared：SEQ ID No in artificial full genome composition sequence table:4 nucleotide sequence, to pPIC9K carriers And SEQ ID No:4 nucleotide sequence carries out I double digestion of Xho I and Not, recycles digestion products, is connected with DNA ligase, and Escherichia coli are converted, extract plasmid；
(2) convert：Plasmid prepared by step (1) converts Pichia pastoris competent cell, bacterium colony after being converted；
(3) screening of multicopy insertion recon：Bacterium colony after step (2) is converted further is screened, and obtains transformant；
(4) ferment：Fermented and cultured is carried out to the transformant that step (3) obtains and obtains zymotic fluid；
(5) purify：The zymotic fluid that step (4) is obtained carries out separation of solid and liquid, filtering, concentration and ion-exchange chromatography and obtains successively Product.
7. preparation method according to claim 6, it is characterised in that the purifying of the step (5) concretely comprises the following steps：Will The zymotic fluid that step (4) obtains carries out separation of solid and liquid through centrifugation, takes supernatant, carries out micro-filtration to fermented supernatant fluid, collects filtrate, Desalination and concentration by ultrafiltration is carried out again, is collected concentrate, is then carried out ion-exchange chromatography, you can obtain product.
8. the preparation method according to claim 6 or 7, it is characterised in that including：
(1) plasmid is prepared
SEQ ID No in artificial full genome composition sequence table:4 nucleotide sequence is then complete to pPIC9K carriers and manually The SEQ ID No of gene chemical synthesis:4 nucleotide sequence carries out I double digestion of Xho I and Not, recycles digestion products, is connected with DNA Enzyme connects, and converts Escherichia coli, extracts plasmid, is named as pPIC9K-YARA-ASIP50；
(2) Pichia pastoris electricity conversion
The pPIC9K-YARA-ASIP that will be linearized through I restriction endonucleases of Sal50Plasmid, mixes with Pichia pastoris competent cell, goes to In the electricity conversion cup of ice precooling, shock by electricity 4~10 milliseconds, the sorbitol solution for adding ice precooling mixes thalline, coating MD cultures Base tablet, is inverted culture 3~4 days, bacterium colony is grown on MD culture medium flat plates；
(3) screening of multicopy insertion recon
It is respectively 1g/L, 2g/L, 3g/L, 4g/L that the bacterium colony grown on MD culture medium flat plates, which is corresponded to, and is inoculated into G418 concentration On YPD tablets, 30 DEG C of cultures, screening obtains transformant；
(4) ferment
The transformant screened is inoculated in BMGY culture mediums, when shaken cultivation 24 is small, as first order seed switching in equipped with In the fermentation tank of FBS culture mediums, pH value for 5.0 culture 16~20 it is small when, transfer as secondary seed into equipped with FBS culture mediums Bulk fermentation, 30 DEG C of growth temperature, inducing temperature are less than growth temperature, pH value 5.5, and dissolved oxygen control exists>30%, induction fermentation 36~42 it is small when；
(5) purify
The zymotic fluid that step (4) obtains is subjected to separation of solid and liquid through centrifugation, takes supernatant, it is micro- for 0.22 μm of doughnut with aperture Filter system carries out micro-filtration to fermented supernatant fluid, collects filtrate, and the Hollow Fiber Ultrafiltration system for being 1000D with molecular cut off carries out Desalination and concentration by ultrafiltration, collects concentrate, carries out ion-exchange chromatography using SP Sepharose FF, you can obtain product.
9. the encoding gene of claim 1-5 any one of them fusion proteins.
10. encoding gene according to claim 9, it is characterised in that the encoding gene is SEQ ID in sequence table No:5 nucleotide sequence, alternatively, with SEQ ID No:5 have at least 85% homology, preferably at least 90%, 93%, 95%, 97%th, the nucleotide sequence of 98% or 99% homology and coding identical function albumen.
11. the expression vector containing the encoding gene described in claim 9 or 10.
12. the cell line containing the encoding gene described in claim 9 or 10.
13. application of the claim 1-5 any one of them fusion proteins in medicine is prepared.
14. application of the claim 1-5 any one of them fusion proteins in cosmetics are prepared.
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