CN109553659A - A kind of cell-penetrating peptides and transdermal interferon - Google Patents
A kind of cell-penetrating peptides and transdermal interferon Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61P31/12—Antivirals
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
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Abstract
The invention belongs to biomedicine fields, disclose a kind of cell-penetrating peptides and transdermal interferon.Cell-penetrating peptides provided by the invention are the peptide that mediated delivery bioactive molecule enters cell, and the amino acid sequence of the cell-penetrating peptides includes sequence shown in SEQ ID NO:1；Wherein, the 3rd and/or the 8th and/or the 11st amino acid could alternatively be other amino acid by N-terminal.Cell-penetrating peptides of the invention transdermal activity with higher, and can be used as carrier delivery target substance (such as protein, DNA, siRNA) through skin epidermis, it has a wide range of applications；The transdermal interferon formed after cell-penetrating peptides and interferon fusion is easier Transdermal absorption, faster plays effect.
Description
Technical field
The invention belongs to biomedicine fields, specifically, being related to a kind of cell-penetrating peptides and transdermal interferon.
Background technique
Cutaneous penetration refer to drug by skin surface administration after, penetrate keratoderma enter capillary to
Into blood circulation, play the role of the drug delivery system of systemic therapy.As a kind of special administration route, prescription is given with traditional
Formula is oral compared with injection, has its unique advantage, advantage is: can to avoid liver first pass effect and drug in gastrointestinal tract
Inactivation, can achieve better drug effect, and reduce medication individual difference；Constant effective blood concentration is maintained, blood medicine is avoided
Concentration peak valley phenomenon reduces toxic side effect；Extend drug treating time, reduces administration number of times, most patient is made to be easy to receive；
No pain, make conveniently, patient can autonomous medication, medication can also be cancelled at any time.
During Drug Percutaneous Absorption, drug needs to diffuse into blood circulation across physical skin barrier, finally
Into target tissue.Many internal and external factors affect the transdermal process of drug during this.These are because being known as skin itself
Condition, physical and chemical properties of drugs, pharmaceutical formulation etc..The physicochemical property of drug will have a direct impact on drug in cutaneous penetration and be absorbed faster
Slowly.The dosage form and formula composition of preparation also have a direct impact cutaneous penetration efficiency.The condition of skin be also influence it is transdermal to
When medicine an important factor for Drug Percutaneous Absorption, the position of skin, damaged degree, thickness, pore distribution, temperature and humidity and clear
Clean degree etc. can all influence the efficiency of Drug Percutaneous Absorption.
Therefore, enhancing drug transdermal means of transportation, oneself is gradually applied in clinical treatment, and majority is concentrated on using chemistry
Dermal penetration enhancer and these two types of aspects of physics assist process.
Cell membrane is the semipermeable barrier between cell and extracellular environment, has selective permeation effect to make cell protect
Hold constant interior environment.Although this phospholipid bilayer is essential for the survival of cell and function, it is given into the cell
The exchange of outer cargo molecule proposes challenge.Since the drugs such as albumen, polypeptide, nucleotide macromolecular substances and developer have to
Reach cell interior competence exertion to act on accordingly, therefore realizes that the transmembrane transport of these substances becomes very necessary.
Currently, there is a kind of biological polypeptide, can quick penetration enter in mammalian cell, and still retain original structure and
Function, but they enter cell independent of encytosis, this kind of polypeptide is referred to as cell-penetrating peptides (cell
Penetrated peptide, CPP).Such polypeptide is rich in basic amino acid, therefore usually positively charged.CPP not only can be with
Itself penetrating cell can also load other substances and promote the Premeabilisation of cells of this substance, such as protein, DNA, siRNA, rouge
Plastid and nano material etc..Cell-penetrating peptides equally have penetration power to the epidermal cell of animal, therefore cell is worn in recent years
Saturating peptide is used as dermal penetration enhancer, with the cutaneous penetration for macromolecular drug.This substance is found to be cutaneous penetration technology
Provide convenience approach.
Interferon (Interferon, IFN) is that humans and animals cell is infected with the virus, or by nucleic acid, bacterium endogenous toxic material
After the induction of many factors such as element, cytokinin, by a type cytokines of recipient cell secretion.Antiviral activity is interference
One of basic function for plain family.Interferon mainly plays its antivirus action by body cell, itself is not
It can kill or inhibit virus.Cell can induce the generation and release of IFN through virus infection, is then infected cell death and collapses
Solution, IFN molecule are released and diffuse to whole body with blood circulation.IFN molecule in conjunction with the receptor-specific on peripheral cell film,
The activity for enhancing cell membrane adenyl cyclase, promotes the formation of adenosine cyclophosphate.Adenosine cyclophosphate increases the conjunction for being able to suppress DNA
At, division of cell etc., and then inhibit the duplication of virus.The cutaneous penetration for studying interferon, will be in diseases such as herpesviral, HPV
In terms of malicious treatment of infection, the compliance of patient is improved.
In view of this present invention is specifically proposed.
Summary of the invention
The technical problem to be solved in the present invention is that overcoming the deficiencies of the prior art and provide a kind of cell-penetrating peptides and transdermal
Interferon, cell-penetrating peptides of the invention transdermal activity with higher, and can be used as carrier delivery target substance (such as egg
White matter, DNA, siRNA etc.) skin epidermis is penetrated, it has a wide range of applications；It is formed after cell-penetrating peptides and interferon fusion
Transdermal interferon be easier Transdermal absorption, be conducive to faster play effect.
In order to solve the above technical problems, the present invention is using the basic conception of technical solution:
The first object of the present invention is to provide a kind of cell-penetrating peptides, and the cell-penetrating peptides are living for mediated delivery biology
Property molecule enters the peptide of cell, and the amino acid sequence of the cell-penetrating peptides includes sequence shown in SEQ ID NO:1；
Alternatively, the 3rd and/or the 8th and/or the 11st amino acid substitution is other amino acid by N-terminal.
The present invention screens the amino of the cell-penetrating peptides of acquisition by multi-turns screen using display technique in bacteriophage body
Acid sequence includes 15 amino acid, sequence, i.e. RKTWLRRLARRPQLK as shown in SEQ ID NO:1.
It has been found that sequence shown in SEQ ID NO:1 by N-terminal the 3rd, the 8th and the 11st can there are many
Selection when it is replaced respectively or replaces two of them position, or is replaced into other amino acid, the cell of acquisition simultaneously
Penetrating peptide also being capable of penetrability with higher.Therefore the amino acid sequence of the cell-penetrating peptides filtered out can be expressed as
RKX1WLRR X2AR X3PQLK, wherein X1、X2、X3It can also be replaced separately or together for shown in SEQ ID NO:1
At other amino acid.Applicant it is found through experiment that, the present invention screen obtain cell-penetrating peptides transdermal activity with higher, and
And can be used as carrier delivery target substance (such as protein, DNA, siRNA) through skin epidermis, have and widely answers
With.
Further embodiment, the sequence of the cell-penetrating peptides the 3rd amino acid substitution by N-terminal is S or A or R；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 3rd is S.
Further embodiment, the sequence of the cell-penetrating peptides the 8th amino acid substitution by N-terminal is I or V；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 3rd is I.
Further embodiment, the sequence of the cell-penetrating peptides by N-terminal the 11st amino acid substitution be K or H or
N；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 11st is K.
In above-described cell-penetrating peptides, the combination of amino acid sequence include it is a variety of, the 3rd, the 8th and
11 amino acid can be replaced respectively, or replacement two of them position, can also be replaced simultaneously.
The second object of the present invention is to provide a kind of nucleotide sequence for encoding cell-penetrating peptides as described above.
The nucleotides sequence of signified cell-penetrating peptides is classified as any one and can encode as described above arbitrarily in this programme
A kind of sequence of cell-penetrating peptide amino acid sequence.
The carrier, recombinant bacterium or recombination that the third object of the present invention is to provide a kind of nucleotide sequence as described above are thin
Born of the same parents.
Signified carrier can be expression vector, shuttle vector, integration vector etc. in this programme, and recombinant bacterium can be thin
Bacterium can be fungi, and recombinant cell, which can be virus, to be zooblast etc..
The fourth object of the present invention is to provide a kind of cell-penetrating peptides as described above as the intracellular use for transporting carrier
On the way；
Preferably, purposes of the cell-penetrating peptides as the carrier for transporting therapeutic agent in the cell.
Specifically refer to, carrier of the cell-penetrating peptides that the present invention filters out as drug delivery molecule, by the medicine of delivery
Object molecule is transferred to the cytoplasm and/or nucleus of aim cell.The drug may include viral infection resisting drug, resist swollen
Tumor medicine, cytotoxic drug, biofilm disruption drug, genomic medicine, neurotrophy molecule, photosensitive drug, stem cell are adjusted
Factor etc..
The fifth object of the present invention is to provide a kind of compound, the compound include cell-penetrating peptides as described above and
Cargo molecule；
Preferably, the cell-penetrating peptides and cargo molecule are mutually coupled；
Preferably, the cargo molecule is coupled at the end of cell-penetrating peptides.
In the present solution, the cargo molecule is the macromolecular for needing to enter by carrier of cell-penetrating peptides cell interior,
The macromolecular is preferably but not limited to the molecule with pharmaceutical activity, the molecule with label effect, point with targeting
At least one of son.
The coupling can be to be attached by way of covalent bond or non-covalent bond.
The sixth object of the present invention is to provide a kind of transdermal interferon, and the transdermal interferon includes as described above thin
The fusion protein that born of the same parents' penetrating peptide and interferon are formed, the cell-penetrating peptides are connected to the N-terminal of interferon.
The cell-penetrating peptides filtered out are connected to the N-terminal of interferon by this programme, are building up to expression cell on carrier and are penetrated
The fusion protein of peptide-interferon fusion protein, acquisition has the activity of interferon, while having the ability for penetrating skin, has
There is good patient compliance, the therapeutic effect in terms of virus infection can be improved.
Further embodiment, the interferon are selected from alpha interferon, beta interferon or interferon and homologous types of interference
Element；
Preferably, the interferon is Interferon Alpha-2b.
The seventh object of the present invention is to provide a kind of preparation method of transdermal interferon as described above, including following step
It is rapid:
(1) cell-penetrating peptides are connected to the N-terminal of interferon, building has cell-penetrating peptides-interferon fusion protein sequence
The recombinant vector of column；
(2) recombinant vector is transferred to aimed strain, screening obtains recombinant bacterium；
(3) engineering bacteria is subjected to fermented and cultured, collects thallus and cracked, purifying obtains transdermal interferon.
After adopting the above technical scheme, compared with the prior art, the invention has the following beneficial effects:
1, cell-penetrating peptides of the invention are easily prepared, molecular weight is small, be not likely to produce immunological rejection, penetration power is strong,
Transdermal activity with higher, and can be used as carrier delivery target substance (such as protein, DNA, siRNA) through skin
Epidermis has a wide range of applications.
2, the transdermal interferon formed after cell-penetrating peptides of the invention and interferon fusion has the activity of interferon, together
When with the ability for penetrating skin, it is easier to Transdermal absorption faster plays effect, have good patient compliance, can
To improve the therapeutic effect in terms of virus infection.
A specific embodiment of the invention is described in further detail with reference to the accompanying drawing.
Detailed description of the invention
Attached drawing is as a part of the invention, and for providing further understanding of the invention, of the invention is schematic
Examples and descriptions thereof are used to explain the present invention, but does not constitute an undue limitation on the present invention.Obviously, the accompanying drawings in the following description
Only some embodiments to those skilled in the art without creative efforts, can be with
Other accompanying drawings can also be obtained according to these attached drawings.In the accompanying drawings:
Fig. 1 is cell-penetrating peptides CPP secondary structure of the present invention and analysis of physical and chemical property；
Fig. 2 is CPP-IFN α -2b vivo transdermal experiment of the invention；
Fig. 3 is CPP-IFN α -2b of the invention real compared with PD-1-IFN α -2b and PEP-1-IFN α -2b vivo transdermal
It tests.
It should be noted that these attached drawings and verbal description are not intended to the design model limiting the invention in any way
It encloses, but illustrates idea of the invention by referring to specific embodiments for those skilled in the art.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical solution in embodiment is clearly and completely described, the following examples are intended to illustrate the invention, but
It is not intended to limit the scope of the invention.
Embodiment one
1, the screening of CPP
With display technique in bacteriophage body, screening can penetrate the CPP of skin.Specific method is,
1) phage peptide library is applied to nude mice skin surface by Transdermal delivery systems；
2) after transdermal after a period of time, blood is extracted out of mouse body；
3) Escherichia coli are added in blood, allow Phage Infection Escherichia coli, are cultivated later；
4) plaque is finally counted, phage titre is calculated.
First round screening is carried out according to above step, usual first round screening has many nonspecific bacteriophages or saturating
The weaker bacteriophage of skin ability can also be screened to, and number is more.Therefore it needs by repeating to screen.It is above-mentioned by repeating
Method.The bacteriophage of certain purity is enriched to by 3-4 wheel screening.
Then by extract the bacteriophage, be sequenced its DNA sequence dna with polypeptide, to obtain polypeptide sequence.
By above step, CPP polypeptide sequence is filtered out, as follows:
RKTWLRRLARRPQLK
Or other schemes, wherein the 3rd amino acids T can be amino acid S or A or R；Wherein the 8th amino acids L can
For amino acid I or V；Wherein the 11st amino acids R can be amino acid K or H or N.
Wherein, the amino acid of 3 positions can have at one and be replaced, and can have at two and be replaced, and can also have at 3 simultaneously
It is replaced.
2, CPP analysis of physical and chemical property
Analysis of physical and chemical property is carried out to CPP sequence (RKTWLRRLARRPQLK) with tool ProtParam, it is known that its point
Son amount 2.0KD, iso-electric point=12.6 with higher, and there is stronger hydrophily (GRAVY:-1.59)；With
Protean software carries out secondary structure and analysis of physical and chemical property to CPP sequence (RKTWLRRLARRPQLK), as a result such as Fig. 1 institute
Show, which is in αhelix, and different degrees of hydrophily is presented in all amino acid.
Using same method, the CPP sequence of amino acid substitution has been carried out to the 3rd and/or the 8th and/or the 11st
Column carry out physico-chemical analysis, as a result similar with RKTWLRRLARRPQLK.
Embodiment two
Obtain fusion protein CPP-IFN α -2b
(1) engineering bacteria is constructed
The above-mentioned cpp screened and IFN α -2b gene order are stitched together (cpp-IFN α -2b), full genome is carried out
Synthesis.Wherein, the nucleotide sequence encoding amino acid sequence RKTWLRRLARRPQLK of Cpp is connected to the N section of IFN α -2b.
The nucleotide sequence of cpp-IFN α -2b is as shown in SEQ ID NO:2, and amino acid sequence is as shown in SEQ ID NO:3.
Cpp-IFN α -2b genetic fragment is building up on my company protokaryon soluble expression vector PHX53, transformed competence colibacillus is thin
Born of the same parents BL21 (DE3), by digestion, sequencing, screening obtains the genetic engineering bacterium containing PHX53-cpp-IFN α -2b.
(2) fermented and cultured engineering bacteria
The genetic engineering bacterium of above-mentioned acquisition is inoculated in 2 × YT culture medium, 37 DEG C of cultures in 15L fermentor；
Fermentation process: adjusting revolving speed control oxygen dissolving value between 50-65%, with ammonium hydroxide control pH between 6.0-7.5；When
The IPTG to working concentration 1mM of aseptic filtration is added in OD600 after reaching 0.8, continue 25 DEG C of Fiber differentiations 6 hours；Room temperature puts tank,
Thalline were collected by centrifugation for 12000rpm tubular type；Thallus pure water is centrifuged twice, removes the impurity in fermentation liquid.
Thallus press 1:20 (m/v), be suspended in lysate (20mMPB, 5mmol/L EDTA, 0.1%Triton-x100,
PH6.0 it in), is placed on mixture of ice and water, ultrasonication；12000rpm is centrifuged 30 minutes, takes supernatant.
(3) chromatographic purifying
By above-mentioned cell pyrolysis liquid, it is loaded to the CM column balanced with equilibrium liquid (50mM Tris-HCL, pH 8.0), then
5 column volumes are eluted with equilibrium liquid (50mM Tris-HCL, pH8.0), eluent (50mM Tris-HCL, pH 9.5) gradient is washed
It is de-, collect eluting peak.
The albumen that CM column is collected into, adjusts PH to 8.0, and loading to equilibrium liquid (50mM Tris-HCL, pH 8.0) balances
DEAE on, collection penetrates peak, and loading, which finishes, to be continued to be eluted with equilibrium liquid (50mM Tris-HCL, pH 8.0), continues to collect and wear
Saturating peak.Protein-20 DEG C will be collected into save backup.
Test example one
Fusion protein CPP-IFN α -2b Activity determination
With the Pharmacopoeia of the People's Republic of China 2015 editions (three) " defined " interferon activity measuring method " measurement fusion
The biological activity of interferon.The result shows that fusion protein CPP-IFN α -2b, has 1.43 × 108Specific activity.
Test example two
The transdermal Activity determination of fusion protein CPP-IFN α -2b
It is internal using whether experiment in vivo verifying CPP-IFN α -2b can enter through rat skin.Steps are as follows:
1) taking 100ul concentration is the CPP-IFN α -2b of 0.5mg/ml, and being applied to rat abdomen baring skin surface (makes big
Mouse is in narcosis, prevents rat from rubbing drug), as experimental group.Taking 100ul concentration is the IFN α -2b of 0.5mg/ml,
It is applied to rat abdomen baring skin surface, as a control group.Respectively in 0min, 30min, 60min and 90min, from big
Caudal vein extracts blood 200ul, and serum is taken after being centrifuged, with CPP-IFN α -2b content in elisa technique detection serum.
(such as Fig. 2) as the result is shown, in 30 minutes, IFN α -2b can not almost be detected；And CPP-IFN α -2b is penetrated
The amount of 180pg, after sixty minutes, IFN α -2b only detect 5pg through skin；And CPP-IFN α -2b transit dose has been increased to
410pg.It is still very low also only in 8pg although the transdermal amount of IFN α -2b is increased after 90 minutes；CPP-IFNα-2b
Transdermal amount reached 420pg, drug has been likely to be breached infiltration-removing balance at this time.
By experiment in vivo, we have demonstrated that the transdermal capability of CPP-IFN α -2b.It further illustrates, institute of the present invention
The CPP of offer has the ability that protein penetrates skin that carries.As a result as shown in Figure 2.
Using same method, the CPP sequence of amino acid substitution has been carried out to the 3rd and/or the 8th and/or the 11st
Column have carried out the test with IFN α -2b fusion protein, as a result similar with RKTWLRRLARRPQLK, and there is carrying protein to penetrate
The ability of skin.
Test example three
For further prove cell-penetrating peptides CPP of the present invention transdermal capability advance, we are separately prepared for merging
Albumen PD-1-IFN α -2b and PEP-1-IFN α -2b, carries out transdermal Activity determination, for commonly using cell-penetrating peptides PD- with current
The comparison of 1 and PEP-1.Experimental method is referring to test example two.
(such as Fig. 3) as the result is shown, after sixty minutes, (CPP sequence is CPP-IFN α -2b transdermal experiment
RKTWLRRLARRPQLK), the transdermal amount of PD-1-IFN α -2b and PEP-1-IFN α -2b have reached maximum concentration, and test
The transdermal capability of group CPP-IFN α -2b is above other two groups within entire experimental period.
The above result shows that cell-penetrating peptides CPP of the invention, which carries IFN α -2b, not only has transdermal capability, Er Qiexiao
Fruit is better than common cell-penetrating peptides PD-1 and PEP-1 at present.
Using same method, the CPP sequence of amino acid substitution has been carried out to the 3rd and/or the 8th and/or the 11st
Column have carried out the test with IFN α -2b fusion protein, as a result similar with RKTWLRRLARRPQLK, and transdermal effect is normal better than at present
With cell-penetrating peptides PD-1 and PEP-1.
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though
So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this patent
Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little change or be modified to
The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention
Any simple modification, equivalent change and modification made by above embodiments, in the range of still falling within the present invention program.
Claims (10)
1. a kind of cell-penetrating peptides, which is characterized in that the cell-penetrating peptides are that mediated delivery bioactive molecule enters carefully
The peptide of born of the same parents, the amino acid sequence of the cell-penetrating peptides include sequence shown in SEQ ID NO:1；
Alternatively, the 3rd and/or the 8th and/or the 11st amino acid substitution is other amino acid by N-terminal.
2. a kind of cell-penetrating peptides according to claim 1, which is characterized in that the sequence of the cell-penetrating peptides is by N
The amino acid substitution for having held the 3rd is S or A or R；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 3rd is S.
3. a kind of cell-penetrating peptides according to claim 1 or 2, which is characterized in that the sequence of the cell-penetrating peptides
The 8th amino acid substitution is I or V by N-terminal；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 3rd is I.
4. a kind of cell-penetrating peptides according to claim 1 to 3, which is characterized in that the cell-penetrating peptides
Sequence by N-terminal the 11st amino acid substitution be K or H or N；
Preferably, in amino acid sequence, the amino acid substitution that N-terminal plays the 11st is K.
5. a kind of nucleotide sequence of cell-penetrating peptides described in coding claim 1-4 any one.
6. a kind of carrier including the nucleotide sequence described in claim 5, recombinant bacterium or recombinant cell.
7. a kind of cell-penetrating peptides as described in claim 1-4 any one are as the intracellular purposes for transporting carrier；
Preferably, purposes of the cell-penetrating peptides as the carrier for transporting therapeutic agent in the cell.
8. a kind of compound, which is characterized in that the compound includes cell-penetrating peptides described in claim 1-4 any one
And cargo molecule；
Preferably, the cell-penetrating peptides and cargo molecule are mutually coupled；
Preferably, the cargo molecule is coupled at the end of cell-penetrating peptides.
9. a kind of transdermal interferon, which is characterized in that the transdermal interferon includes cell-penetrating described in claim 1-4
The fusion protein that peptide and interferon are formed, the cell-penetrating peptides are connected to the N-terminal of interferon；
Preferably, the interferon is selected from alpha interferon, beta interferon or interferon and homologous types of interference element；
Preferably, the interferon is Interferon Alpha-2b.
10. a kind of preparation method of transdermal interferon as claimed in claim 9, which comprises the following steps:
(1) cell-penetrating peptides are connected to the N-terminal of interferon, building has cell-penetrating peptides-interferon fusion protein sequence
Recombinant vector；
(2) recombinant vector is transferred to aimed strain, screening obtains recombinant bacterium；
(3) engineering bacteria is subjected to fermented and cultured, collects thallus and cracked, purifying obtains transdermal interferon.
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| CN1240830A (en) * | 1999-05-25 | 2000-01-12 | 上海华新生物高技术有限公司 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
| WO2008063113A1 (en) * | 2006-11-20 | 2008-05-29 | Cepep Iii Ab | Cell -penetrating peptides and constructs containing them consisting 15-25 amino acids of tumor supressor protein p14arf or p19arf |
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| US20160151512A1 (en) * | 2012-07-11 | 2016-06-02 | Gemvax & Kael Co., Ltd. | Cell-Penetrating Peptide, Conjugate Comprising Same and Composition Comprising Same |
| CN107556376A (en) * | 2017-09-08 | 2018-01-09 | 上海华新生物高技术有限公司 | A kind of interferon alpha 2 b mutant and its preparation method and application |
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| CN1240830A (en) * | 1999-05-25 | 2000-01-12 | 上海华新生物高技术有限公司 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
| WO2008063113A1 (en) * | 2006-11-20 | 2008-05-29 | Cepep Iii Ab | Cell -penetrating peptides and constructs containing them consisting 15-25 amino acids of tumor supressor protein p14arf or p19arf |
| US20120065124A1 (en) * | 2009-05-20 | 2012-03-15 | Toray Industries, Inc. | Cell-penetrating peptides |
| CN102428096A (en) * | 2009-05-20 | 2012-04-25 | 东丽株式会社 | Cell membrane-permeable peptides |
| US20140141452A1 (en) * | 2011-05-23 | 2014-05-22 | Phylogica Limited | Method of Determining, Identifying or Isolating Cell-Penetrating Peptides |
| US20160151512A1 (en) * | 2012-07-11 | 2016-06-02 | Gemvax & Kael Co., Ltd. | Cell-Penetrating Peptide, Conjugate Comprising Same and Composition Comprising Same |
| WO2016036060A1 (en) * | 2014-09-04 | 2016-03-10 | 디오셀 주식회사 | Novel cell membrane-penetrating peptide, and use thereof as bioactive substance carrier |
| CN106795205A (en) * | 2014-09-04 | 2017-05-31 | 迪沃势股份有限公司 | Neoblast membrane penetrating peptide and its purposes as bioactivator carrier |
| CN107556376A (en) * | 2017-09-08 | 2018-01-09 | 上海华新生物高技术有限公司 | A kind of interferon alpha 2 b mutant and its preparation method and application |
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