CN108586584A - Functional polypeptide and application thereof - Google Patents

Functional polypeptide and application thereof Download PDF

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CN108586584A
CN108586584A CN201810414971.9A CN201810414971A CN108586584A CN 108586584 A CN108586584 A CN 108586584A CN 201810414971 A CN201810414971 A CN 201810414971A CN 108586584 A CN108586584 A CN 108586584A
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expression
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ala
wound
healing
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范汉东
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Hangzhou Shidimu Biological Technology Co Ltd
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Hangzhou Shidimu Biological Technology Co Ltd
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Abstract

It is the polypeptide with amino acid sequence shown in SEQ ID No.2 the invention discloses a kind of functional polypeptide；Either shown in SEQ ID No.2 on the basis of amino acid sequence after substitution, missing, the one or more amino acid of addition and the polypeptide with same or similar function.The invention also discloses above-mentioned functional polypeptides to prepare the purposes in promoting wound healing, the drug for inhibiting scar to generate.Functional polypeptide is dissolved in solvent, is smeared daily in wound surface, wound healing time can be shortened, and effectively inhibit scar incidence.

Description

Functional polypeptide and application thereof
Technical field
The present invention relates to biotechnologies, and in particular to a kind of functional polypeptide and application thereof.
Background technology
The healing of skin trauma is that a various kinds of cell and cell factor participate in jointly, and complicated, coordination, orderly tissue are repaiied Multiple regenerative process.It can be generally divided into following three processes：
1. the inflammatory reaction phase：Skin since wound generates a series of biochemical reaction, first the inflammatory factor in blood by Mononuclear macrophage is secreted, and the aggregation of a large amount of chemotactic neutrophil leucocytes occurs in the wound position of skin.Monokaryon granulocyte simultaneously Also the cell or tissue that phagocytosis injured areas damages is carried out to position aggregation, and secretes a variety of growth factors to promote pars affecta Position regeneration, and form new blood vessel.
2. proliferative phase：After inflammatory reaction, it is put into proliferative phase, proliferative phase is mainly associated restoration cell and substance Proliferation and increase.Skin wound wound relies primarily on granulation tissue to be repaired, and granulation tissue mainly rises for wound position To filling effect, position is created in completion.After granulation tissue filling process is completed, the keratinocyte near the surface of a wound starts to play and make With.New epithelium starts gradually to carry out covering formation to the surface of a wound by its proliferation and migration.
3. the phase of remodeling：The remodelling phase of wound repair is put into after proliferative phase, the stage, main event was scar The formation of tissue.Cicatricial tissue is mainly formed by the maturation of granulation tissue and conversion.Proteoglycans and each Collagen Type VI (I, II, III, VII, Ⅸ collagen etc.) by extracellular matrix degrading enzyme include-III point of metalloproteinases-I, metalloproteinases-II and metalloproteinases Solution.This process so that the arrangement of collagen and elastic fibers more rule, the prototype structure and characteristic of the surface of a wound are restored as far as possible. Capillary network also regenerates and changes to original state in the process.
In wound healing process, because of the influence of drug or other factors in individual constitution or agglutination, easy to produce more Sum velocity is slow, occurs the problems such as hyperplastic scar after healing.How wound or female body wound for face reduce scar Generation it is even more important.
Operation or contingency are the main reason for skin trauma generates, such as internal organ operation, beauty treatment, caesarean birth, vehicle How misfortune, burn etc., solve these speed of wound healing, reduces inflammation, while reducing generation of scar etc. all after healing It is present medicine and beauty culture problem to be solved.Therefore, healing factor is all widely used in these areas.Only to cut open For abdomen production, need the pregnant woman of caesarean birth 10,000,000 or so every year, healing factor presses daily 100 yuan, and each puerpera uses altogether It calculates within seven days, even if only 10% puerpera uses healing factor (1,000,000 people), can also realize that sales volume is about 1,000,000 * every year 700 yuan=700,000,000 yuan, net income is 300,000,000 or more.Therefore, it is effectively promoted wound healing, inhibits the healing factor of scar with considerable Economic benefit.
Invention content
It is an object of the present invention to provide a kind of functional polypeptides and application thereof, so as to solve the deficiencies in the prior art.
The present invention uses following technical scheme：
It is the polypeptide with amino acid sequence shown in SEQ ID No.2 the present invention provides a kind of functional polypeptide；Or After substitution, missing, the one or more amino acid of addition and have on the basis of being the amino acid sequence shown in SEQ ID No.2 There is the polypeptide of same or similar function.
Through the present invention also provides the encoding gene of functional polypeptide and on the basis of the base sequence and substitution, lacks, adding Add after one or more bases and the gene series with coding same or similar function.
It is the present invention also provides the carrier of the encoding gene and with protein expression function and can express same raw The active other protein expression vectors of object, including coli expression carrier, yeast expression system expression vector, baculoviral table It is carried up to system expression carrier, CHO expression systems expression vector, 293 expression system expression vectors, slow virus expression system expression Body, adenoviral expression systems expression vector.
The present invention also provides the host cell containing the carrier, including the Bacillus coli cells of protokaryon, eukaryon Yeast cells, baculovirus expression system host cell, CHO expression systems host cell, 293 expression system host cells, gland Virus expression systems host cell, slow virus expression system host cell.
The present invention also provides the functional polypeptides in preparing the drug for promoting wound healing, inhibition scar to generate Purposes.
The present invention also provides the drugs for promoting wound healing, scar being inhibited to generate, and contain the functional polypeptide conduct Active ingredient.
The drug for promoting wound healing, scar being inhibited to generate, in use, being applied after the drug and solvent are mixed Or it is sprayed on wound.
The present invention also provides the compounding ingredients containing the functional polypeptide.
Beneficial effects of the present invention：
The present invention is based on to pathology in wound healing process and Physiological Analysis, it was found that a kind of to promote wound healing simultaneously The biologically active polypeptide that scar can be inhibited to generate is dissolved in by vivoexpression and purifying in solvent, is applied daily in wound surface Smear, can effectively shorten wound healing time and effectively inhibit scar incidence.
Description of the drawings
Fig. 1 is that 1 48h of group observes result.
Fig. 2 is that 1 96h of group observes result.
Fig. 3 is that 1 192h of group observes result.
Fig. 4 is 1 wound location pathological section figure of group.
Fig. 5 is that group 2 the observes result for 24 hours.
Fig. 6 is that 2 120h of group observe result.
Fig. 7 is 2 wound location pathological section figures of group.
Fig. 8 is that group 3 the observes result for 24 hours.
Fig. 9 is that 3 72h of group observe result.
Figure 10 is that 3 120h of group observe result.
Figure 11 is that 3 168h of group observe result.
Figure 12 is 3 wound location pathological section figures of group.
Figure 13 is that group 4 the observes result for 24 hours.
Figure 14 is that 4 72h of group observe result.
Figure 15 is that 4 120h of group observe result.
Figure 16 is that 4 168h of group observe result.
Figure 17 is 4 wound location pathological section figures of group.
Specific implementation mode
The present invention is done with reference to embodiment and attached drawing and is further explained.The following example is merely to illustrate this hair It is bright, but be not used to limit the practical range of the present invention.
ELP-Intein systems are described below functional polypeptide is expressed and purified, it is big that other methods may also include protokaryon Enterobacteria expression system, the yeast expression system of eukaryon, baculovirus expression system, CHO expression systems, gland virus expression system Other protein expression systems such as system, 293 cell expression systems etc..
ELP-Intein systems are developed by Princeton university WOOD professors and its partner, are situated between by Intein ELP systems and poly-β-hybroxybutyric acid (PHB) system led thoroughly has broken away from the constraint using chromatographic column.The bullet that Intein is mediated Property protein system is the Self cleavage characteristic of the self aggregation and Intein that make full use of ELP and builds a kind of very unique albumen Matter purification system.Its separation process is as follows：First, at room temperature (<Tt) soluble " the ELP-Intein- mesh that will be given expression to Mark albumen " triplet is detached by centrifugation with sundries such as cell cracking fragments；Then, temperature is elevated above Tt and be added A certain amount of salt such as NaCl promotes the generation of phase transformation, ELP labels that self aggregation occurs, and aggregation is collected by centrifugation or ultrafiltration, Temperature is reduced in Tt or less (such heating cooling can carry out two-wheeled)；Dissolving is resuspended with the buffer solution of low ph value (6.0-6.5) Self cleavage occurs for sediment fraction, induction Intein to discharge destination protein, using once phase-change process, equally centrifugation or ultrafiltration The target protein that the ELP label aggregations for being attached to Intein are removed and are purified.
SUMO expression systems can obtain soluble fusion protein, but most albumen are still in the form of inclusion body Expression, purification yield are low；And when the ELP-Intein system expression fusion proteins, fusion protein is expressed all in the form of soluble, Including albumen such as the cell factor in some inhuman sources, antibacterial peptide and alexins.And expressed albumen is without any mark Label, purifying process are simple.Basic step is：The method cloned using Standard PCR, fused polypeptide gene is subcloned to pET-EI In carrier, the plasmid of structure is converted into Escherichia coli BLR (DE3), high expression, and ELP are obtained under IPTG inductions Fusion protein is expressed in the form of soluble.One, pCF genes are artificial synthesized, and gene order is as shown in SEQ ID No.1.
Two, the structure of ELP-Intein fusion protein expression vectors, identification, conversion, expression and purification
According to molecular biology conventional method, by pCF gene clonings in the BsrG I and Hind of pET-EI-GFP plasmids Between III digestion site.PCR product is after electrophoretic separation, gel extraction target fragment, by the DNA amplification and pET-EI- of recycling GFP plasmids carry out digestion with BsrG I and Hind III respectively.DNA fragmentation is spare after cutting glue purification after digestion.Meanwhile it will be sweet Tablet, 37 DEG C of inversion overnight incubations are drawn in the E.coli DH5 α inoculations that oil freezes preservation；Picking monoclonal is to equipped with 3ml LB cultures In the test tube of liquid, 37 DEG C of 220rpm shake 12h；It draws in 1ml bacterium solutions to 1.5ml centrifuge tubes, 4 DEG C of 12000g centrifuge 3min, abandon Supernatant；With the CaCl of 400 μ l precoolings2Bacterial sediment is resuspended, 12000g centrifuges 3min, abandons supernatant；With the CaCl of 200 μ l precoolings2Again Secondary resuspension bacterial sediment is set on ice overnight；20 μ l of connection liquid are all added in 200 μ l competent bacterias, 1h on ice is set；42℃ Heat shock 90sec sets rapidly 5min in ice；The LB culture solutions of 600 μ l, 37 DEG C of preheatings are added；37 DEG C, 220rpm shakes 1h, centrifugation It is all coated on the LB tablets containing 50 μ g/ml Amp, 37 DEG C of inversion overnight incubations afterwards.4 monoclonals of random picking are to containing 50 μ g/ In the test tube of the 3mlLB culture solutions of ml Amp, 37 DEG C of 220rpm shake 4h, take 100 μ l centrifugations, bacterial sediment are taken, with 50 μ l ddH2O is resuspended, boiling water bath 5min, and 1 μ l supernatants of centrifuging and taking are cooked template, carries out PCR identifications.It will be accredited as the positive through bacterium colony PCR Cloning and sequencing, examining order are completed by Invitrogen biotech companies.It will identify that normal plasmid is named as PEI-CF.
1) with reference to routine CaCl2Method converts pEI-CF carriers to Escherichia coli BLR (DE3).Picking converts on tablet Monoclonal is inoculated in the test tube of the 3ml LB culture solutions containing 50 μ g/ml Amp, and 37 DEG C of 220rpm shakings are overnight；Next day presses 1： 100 are inoculated in the 30ml LB culture solutions of 50 μ g/ml Amp, and 37 DEG C of 220rpm shake to thalline OD600For 0.4 (about 2h).
2) IPTG to final concentration of 0.5mmol/l is added into culture, 37 DEG C of 220rpm shake 4h, induction ELP fusions Protein expression.
3) thalline after induction is centrifuged, collects the thalline of precipitation, ultrasonic disruption thalline (carries out) in ice bath：Setting For：Power 400W, work 4s, interval 8s.During ultrasonication, centrifuge opening is preheated to 35 DEG C.
4) ultrasonication finishes, and is crushed liquid with 16,000g, 4 DEG C of centrifugation 10min, stays supernatant, abandon precipitation.
5) supernatant is sub-packed in clean EP pipes, often the NaCl solution of a concentration of 3M is added to final concentration of in pipe 0.5M gently spins upside down mixing, and being placed in incubation 10min in 30 DEG C of thermostat water baths makes fusion protein undergo phase transition reaction.
6) it is incubated and finishes, be put into each pipe in preheated centrifuge immediately, 16,000g, 35 DEG C of centrifugation 10min；Centrifugation Terminate, rapid to take out EP pipes, the supernatant firmly thrown away in each pipe abandons it, since fusion protein undergoes phase transition aggregation, after centrifugation Precipitate, and foreign protein does not undergo phase transition therefore to appoint and be retained in supernatant.
7) PBS buffer solution of the pH 8.5 of the precipitation precooling containing fusion protein, which is gently resuspended, (reduces bubble to the greatest extent Generation) make fusion protein occur reversible transition solvable state is become again by precipitated form.Stay 10 μ l re-suspension liquids as electroresis appraisal Sample.During resuspension, opens an other centrifuge and be cooled to 4 DEG C in advance.
8) it is resuspended and finishes, each EP pipes are put and remove protein liquid with the centrifuge being pre-chilled with 16,000g, 4 DEG C of centrifugation 10min In remaining insoluble impurity.
9) centrifugation terminates, and collects in supernatant to new clean EP pipes, repeats another wheel phase transformation of step 5-8 progress and is melted with improving The purity of hop protein replaces PBS with the cutting buffer of pH 6.5.
10) collected purpose fusion protein is put in 25 DEG C of water-baths, automatic cutting reaction is carried out, to discharge purpose egg In vain；Automatic cutting process terminates, and the NaCl solution of final concentration of 1.5M is added into each EP pipes, and 30 DEG C of water-bath 10min induce phase Become.
11) ELP-Intein labels under cutting and complete fusion protein is not cut translate into precipitation, lose label Destination protein then keep solvable state to be present in supernatant.Freeze-drying is stored in -80 DEG C of refrigerators.Destination protein amino Acid sequence is as shown in SEQ ID No.2.
Three, promote wound healing, the preparation for the drug for inhibiting scar to generate
By the medical grade glycerine of sterile water and high-temperature sterilization by volume 8：2 ratio wiring solution-forming, adds 0.1w/ The AB serum of v% lysozymes and 50ul/ml, solvent is prepared into after being sufficiently mixed；By every sterile sealing point in ampere bottle of 2ml Dress.The pCF dry powder of freeze-drying is dissolved in sterile water, filtration sterilization, appropriate sterile mannitol is added, it is dense to be prepared into 1ug/ml Degree, then be divided in ampere bottle by the amount of 1ml every, it is freeze-dried in the workshop of ten thousand grades of standards, sealing.
One solvent and a pCF freeze-dried powder are sufficiently mixed when use, jet pipe made of one flexible rubber hose of housing, are directly squeezed Pressure is sprayed on wound.
This product is directly applied on wound once a day suitable for wound, is placed in 4 DEG C of refrigerators after use, and every can only make With twice, that is, 48h is at most stored after mixing outside.
Four, effect experiment
Experimental rat：BALB/c mouse, female, weight 18-20g.
Experimental method：
1) mouse is anaesthetized, anaesthesia dosage is the 5w/v% chloraldurates of 0.6ml/100g, substantially may be used after about 5min It anaesthetizes successfully；
2) mouse back both sides stern is carried out at the same time with place and is shedded, expose the skin that about 5cm long is completely exposed；
3) about 3cm long together, the wound of 5mm depths are respectively scratched in both sides with scalpel；
4) experiment medication will be given after suture disinfection on the left of mouse, only iodine disinfection is handled after right side of mice suture；
5) all experiment mices are taken care of under same environment, raises, wound healing situation is observed in continuous two weeks；
6) wound skin biopsy is handled.
Experimental record：
It is grouped situation record：Each group is that administration is smeared in left back of the body external application, and once a day, the right back of the body only sterilizes, once a day, the 1 group compared with the 2nd group of healing factor (pCF) effect for various concentration, the 4th group have for document report promote Healing because The comparison of son, the 3rd group is healing factor and document report factor common results, specific as follows：
Group 1：500ng/ml healing factors are given,
Group 2：20ng/ml healing factors are given,
Group 3：500ng/ml healing factor+40ng/ml EGF+40ng/ml bFGF are given,
Group 4：Give 40ng/ml EGF+40ng/ml bFGF.
1 (giving 500ng/ml healing factors) record case of group：Group 1 48h observations are as a result, as shown in Figure 1, No. 1 mouse Left side healing factor side wound is intact, and right side control, which surveys to have, splits line, No. 2 mouse left and right sides no significant differences.Group 1 96h observations As a result, as shown in Fig. 2, No. 1 mouse is on the left of No. 2 mouse, to healing factor side, basic well-grown, right side control sides wound have bright Aobvious inflammation, phenomenon of bursting apart.1 192h of group observations as a result, as shown in figure 3, substantially completely healed on the left of No. 1 mouse and No. 2 mouse, wound Mouth has been taken out stitches, and right side control sides still have a small amount of non-healing areas.Fig. 4 is wound location pathological section figure (after 360h), No. 1 mouse Although significant difference is not seen from appearance after the 7th day with applying healing factor on the left of No. 2 mouse and being compareed with right side, from pathological section Observation, it is very much like to apply the structure that healing factor side pathologic structure and surrounding are not damaged, and control sides have been although wound has healed, But substantially fibrous connective tissue is embodied directly in scar almost without vascular distribution on the person at healing.From organizing 1 wound Recovery situation it could be assumed that：500ng/ml factor pair wounds are extremely helpful, can help wound healing acceleration, and at healing Institutional framework is similar to normal organization, not will produce apparent scar connective tissue, can be considered having for skin regeneration healing Imitate drug.
2 (giving 20ng/ml healing factors) record cases of group：The processing of low concentration healing factor, is observed, afterwards for 24 hours such as Fig. 5 institutes Show, side is administered in left side, and right side control sides, there is no notable differences for left and right sides upgrowth situation；120h is observed, as shown in fig. 6, left Side healing rate is obviously good compared with right side, and the left side starts to form a scab substantially, the relatively slow and chapped skin but the right skin is formed a scab.Fig. 7 is aobvious Show that the healing factor control sides of low concentration and the pathologic structure of administration side have no significant difference (after 360h), is finer and close knot Form tissue.
3 (giving 500ng/ml healing factor+40ng/ml EGF+40ng/ml bFGF) record cases of group：Group 3 the is seen for 24 hours Examine as a result, as shown in figure 8, high concentration healing factor and reported literature can promoting healing the factor it is common, side and right side is administered in left side There is no notable differences for control sides；72h is observed as a result, as shown in figure 9,3 whole recovery situations of group are bad, equal at wound incrustation There is the case where suspected infection, or even it is danger that water outlet, which has suppuration, and wound becomes No. 4 left back of the body infiltration of drying, especially, it is right Back of the body wound bursts apart again；120h is observed as a result, as shown in Figure 10, group 3 can be observed and start fast quick-recovery, and incrustation is more complete, But relatively group 1, group 2, the trace of wound incrustation are bigger and relatively slow；As shown in figure 11, that wound can be observed after 168h is bright Aobvious recovery, and left side back restores fast compared with right side, may be embodied in that scab print is apparent thin, and wound closure situation is more preferable.Figure 12 is aobvious Show high concentration healing factor and reported literature can the factor of promoting healing be used in conjunction with, administration side and right side control sides pathologic structure (after 360h) is finer and close fibrous connective tissue, and there is no notable differences.Conclusion：EGF and the b FGF of document report have suppression The function of healing factor processed cannot share.
4 (giving 40ng/ml EGF+40ng/ml bFGF) record cases of group：Reported literature can promoting healing the factor, such as scheme Shown in 13, there is no notable differences with right side control sides for the side of left side administration for 24 hours；As shown in figure 14, side is administered in whole left side after 72h There is no notable differences with right side control sides；As shown in figure 15, it is observed that mouse wound is serious dry and cracked nearby after 120h, it is whole There is no notable differences with right side control sides for administration side on the left of body；As shown in figure 16, the wound that group 4 can be observed after 168h does not have still There is too apparent healing.Concentration EGF, bFGF that can draw a conclusion substantially is bad for wound healing effect, substantially in vain.Figure 17 show, generate apparent fibrous connective tissue at administration (after 360h), and very fine and close, easily generate scar.
500ng/ml healing factors (polypeptide pCF) are maximally efficient for wound healing, and 20ng/ml healing factors take second place, this The kind factor is more fast and effective for wound healing, and institutional framework is similar to the structure of the non-injury region of surrounding at healing, without apparent Fine and close scar generates, and does not see there is side effect, and mouse restores very fast.Clinical wound healing promoting externally applied drug is can be developed into, not only It can promote wound healing, and be not likely to produce scar.
Sequence table
<110>The Hangzhou bio tech ltd Shi Dimu
<120>Functional polypeptide and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2063
<212> DNA
<213>PCF genes (artificial sequence)
<400> 1
aaactagcga caatggcact cttgtcagac ttctagcgct agcactagca ctcgctctgg 60
gccccgccgc cacccaggcg ggaccagcaa aaagtccata tcaactagta ctacaccaaa 120
gaagtctgag gggcaggcag caagacccaa aagtatgagc cgtgcagaag gtgatcggca 180
ccaacaggaa gtacttcacc aactgcaagc agtggtacca gaggaagatc tgcggcaaga 240
gcaccgtgat cagctacgag tgctgccccg gctacgagaa ggtgcccggc gagaagggct 300
gccccgccgc cctgcccctg agcaacctgt acgagaccct gggcgtggtg ggcagcacca 360
ccacccagct gtacaccgac aggaccgaga agctgaggcc cgagatggag ggccccggca 420
gcttcaccat cttcgccccc agcaacgagg cctgggccag cctgcccgcc gaggtgctgg 480
acagcctggt gagcaacgtg aacatcagct gctgaacgcc ctgaggtacc acatggtggg 540
caggagggtg ctgaccgacg agctgaagca cggcatgacc ctgaccagca tgtaccagaa 600
cagcaacatc cagatccacc actaccccaa cggcatcgtg accgtgaact gcgccaggct 660
gctgaaggcc gaccaccacg ccaccaacgg cgtggtgcac ctgatcgaca aggtgatcag 720
caccatcacc aacaacatcc agcagatcat cgagatcgag gacaccttcg agaccctgag 780
ggccgccgtg gccgccagcg gcctgaacac catgctggag ggcaacggcc agtacaccct 840
gctggccccc accaacgagg ccttcgagaa gatccccagc gagaccctga acaggatcct 900
gggcgacccc gaggccctga gggacctgct gaacaaccac atcctgaaga gcgccatgtg 960
cgccgaggcc atcgtggccg gcctgagcgt ggagaccctg gagggcacca ccctggaggt 1020
gggctgcagc ggcgacatgc tgaccatcaa cggcaaggcc atcatcagca acaaggacat 1080
cctggccacc aacggcgtga tccactacat cgacgagctg ctgatccccg acagcgccaa 1140
gaccctgttc gagctggccg ccgagagcga cgtgagcacc gccatcgacc tgttcaggca 1200
ggccggcctg ggcaaccacc tgagcggcag cgaggctgac cctgctggcc cccctgaaca 1260
gcgtgttcaa ggacggcacc ccccccatcg acgcccacac caggaacctg ctgaggaacc 1320
acatcatcaa ggaccagctg gccagcaagt acctgtacca cggccagacc ctggagaccc 1380
tgggcggcaa gaagctgagg gtgttcgtgt acaggaacag cctgtgcatc gagaacagct 1440
gcatcgccgc ccacgacaag aggggcaggt acggcaccct gttcaccatg gacagggtgc 1500
tgaccccccc catgggcacc gtgatggacg tgctgaaggg cgacaacagg ttcagcatgc 1560
tggtggccgc catccagagc gccggcctga ccgagaccct gaacagggag ggcgtgtaca 1620
ccgtgttcgc ccccaccaac gaggccttca gggccctgcc ccccagggag aggagcaggc 1680
tgctgggcga cgccaaggag ctggccaaca tcctgaagta ccacatcggc gacgagatcc 1740
tggtgagcgg cggcatcggc gccctggtga ggctgaagag cctgcagggc gacaagctgg 1800
aggtgagcct gaagaacaac gtggtgagcg tgaacaagga gcccgtggcc gagcccgaca 1860
tcatggccac caacggcgtg gtgcacgtga tcaccaacgt gctgcagccc cccgcaacag 1920
gccccaggag aggggcgacg agctggccga cagcgccctg gagatcttca agcaggccag 1980
cgccttcagc agggccagcc agaggagcgt gaggctggcc cccgtgtacc agaagctgct 2040
ggagaggatg aagcactgaa ttc 2063
<210> 2
<211> 683
<212> PRT
<213>Polypeptide pCF (artificial sequence)
<400> 2
Met Ala Leu Phe Val Arg Leu Leu Ala Leu Ala Leu Ala Leu Ala Leu
1 5 10 15
Gly Pro Ala Ala Thr Leu Ala Gly Pro Ala Lys Ser Pro Tyr Gln Leu
20 25 30
Val Leu Gln His Ser Arg Leu Arg Gly Arg Gln His Gly Pro Asn Val
35 40 45
Cys Ala Val Gln Lys Val Ile Gly Thr Asn Arg Lys Tyr Phe Thr Asn
50 55 60
Cys Lys Gln Trp Tyr Gln Arg Lys Ile Cys Gly Lys Ser Thr Val Ile
65 70 75 80
Ser Tyr Glu Cys Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly
85 90 95
Cys Pro Ala Ala Leu Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val
100 105 110
Val Gly Ser Thr Thr Thr Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu
115 120 125
Arg Pro Glu Met Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser
130 135 140
Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Asp Ser Leu Val
145 150 155 160
Ser Asn Val Asn Ile Glu Leu Leu Asn Ala Leu Arg Tyr His Met Val
165 170 175
Gly Arg Arg Val Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr
180 185 190
Ser Met Tyr Gln Asn Ser Asn Ile Gln Ile His His Tyr Pro Asn Gly
195 200 205
Ile Val Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala
210 215 220
Thr Asn Gly Val Val His Leu Ile Asp Lys Val Ile Ser Thr Ile Thr
225 230 235 240
Asn Asn Ile Gln Gln Ile Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu
245 250 255
Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn
260 265 270
Gly Gln Tyr Thr Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu Lys Ile
275 280 285
Pro Ser Glu Thr Leu Asn Arg Ile Leu Gly Asp Pro Glu Ala Leu Arg
290 295 300
Asp Leu Leu Asn Asn His Ile Leu Lys Ser Ala Met Cys Ala Glu Ala
305 310 315 320
Ile Val Ala Gly Leu Ser Val Glu Thr Leu Glu Gly Thr Thr Leu Glu
325 330 335
Val Gly Cys Ser Gly Asp Met Leu Thr Ile Asn Gly Lys Ala Ile Ile
340 345 350
Ser Asn Lys Asp Ile Leu Ala Thr Asn Gly Val Ile His Tyr Ile Asp
355 360 365
Glu Leu Leu Ile Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala
370 375 380
Glu Ser Asp Val Ser Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly Leu
385 390 395 400
Gly Asn His Leu Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu
405 410 415
Asn Ser Val Phe Lys Asp Gly Thr Pro Pro Ile Asp Ala His Thr Arg
420 425 430
Asn Leu Leu Arg Asn His Ile Ile Lys Asp Gln Leu Ala Ser Lys Tyr
435 440 445
Leu Tyr His Gly Gln Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg
450 455 460
Val Phe Val Tyr Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala
465 470 475 480
Ala His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg
485 490 495
Val Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp
500 505 510
Asn Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr
515 520 525
Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn
530 535 540
Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly
545 550 555 560
Asp Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu
565 570 575
Ile Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu
580 585 590
Gln Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val
595 600 605
Asn Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val
610 615 620
Val His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Arg Pro Gln
625 630 635 640
Glu Arg Gly Asp Glu Leu Ala Asp Ser Ala Leu Glu Ile Phe Lys Gln
645 650 655
Ala Ser Ala Phe Ser Arg Ala Ser Gln Arg Ser Val Arg Leu Ala Pro
660 665 670
Val Tyr Gln Lys Leu Leu Glu Arg Met Lys His
675 680

Claims (8)

1. functional polypeptide, which is characterized in that be the polypeptide with amino acid sequence shown in SEQ ID No.2；Either in SEQ On the basis of amino acid sequence shown in ID No.2 after substitution, missing, the one or more amino acid of addition and with identical or The polypeptide of identity function.
2. the encoding gene of functional polypeptide described in claim 1 and on the basis of the base sequence by substitution, missing, addition After one or more bases and the gene with coding same or similar function is serial.
3. the carrier containing the encoding gene described in claim 2 and with protein expression function and to express same biology living Other protein expression vectors of property, including coli expression carrier, yeast expression system expression vector, baculovirus expression system System expression vector, CHO expression systems expression vector, 293 expression system expression vectors, slow virus expression system expression vector, gland Virus expression systems expression vector.
4. the host cell containing the carrier described in claim 3, including the yeast of the Bacillus coli cells of protokaryon, eukaryon are thin Born of the same parents, baculovirus expression system host cell, CHO expression systems host cell, 293 expression system host cells, adenovirus table Up to system host cell, slow virus expression system host cell.
5. functional polypeptide described in claim 1 is preparing the purposes in promoting wound healing, the drug for inhibiting scar to generate.
6. the drug for promoting wound healing, scar being inhibited to generate, which is characterized in that contain functional polypeptide described in claim 1 As active ingredient.
7. the drug according to claim 6 for promoting wound healing, scar being inhibited to generate, which is characterized in that in use, will Wound is applied or is sprayed on after the drug and solvent mixing.
8. the compounding ingredients containing functional polypeptide described in claim 1.
CN201810414971.9A 2018-05-03 2018-05-03 Functional polypeptide and application thereof Pending CN108586584A (en)

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