CN112625140A

CN112625140A - PEP-1-G4S-KGF2 fusion protein and coding gene and application thereof

Info

Publication number: CN112625140A
Application number: CN202011526321.7A
Authority: CN
Inventors: 甘向东; 龙民慧; 李飞; 张森
Original assignee: Beijing Gongzhi Shengke Technology Co ltd
Current assignee: Beijing Gongzhi Shengke Technology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-04-09

Abstract

The invention provides a PEP-1-G4S-KGF2 fusion protein, and an encoding gene and application thereof, belonging to the technical field of genetic engineering, wherein the amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No. 1. The PEP-1-G4S-KGF2 fusion protein provided by the invention improves the cell membrane penetrating capability of epithelial cells and improves the effective concentration of keratinocyte growth factor 2 reaching tissues of an injury part.

Description

PEP-1-G4S-KGF2 fusion protein and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a PEP-1-G4S-KGF2 fusion protein and an encoding gene and application thereof.

Background

Keratinocyte growth factor 2 (KGF 2), a member of the fibroblast growth factor family with heparin binding specificity, is also called FGF10, and consists of 208 amino acid residues, and the N-terminus thereof is a signal peptide sequence consisting of 39 hydrophobic residues. KGF2 is mainly secreted by fibroblasts and other mesenchymal cells, mainly acts on specific receptors FGFR 2-IIIb expressed in epithelial tissues in a mesenchymal-epithelial interaction mode, and effectively promotes proliferation, migration and differentiation of epithelial cells. The KGF2 is reported to play an important role in repairing various injuries such as acute and chronic inflammation, ulcer, physical and chemical burn and the like, and especially has an obvious effect on the injuries of the epithelial tissues which are difficult to treat, such as oral mucositis, venous ulcer, ulcerative colitis and the like caused by radiotherapy and chemotherapy. KGF2 is also involved in and regulates the formation and development of organs such as limbs, lung bronchi, teeth, pancreas, pituitary, eyelids, skin, and mandibular glands. However, the amount of KGF2 that penetrates deep into epithelial cells is limited, and no detailed study has been reported on how to increase the KGF2 penetration concentration.

Disclosure of Invention

In view of the above, the invention aims to provide the PEP-1-G4S-KGF2 fusion protein and the coding gene and the application thereof, and the PEP-1-G4S-KGF2 fusion protein provided by the invention improves the capability of penetrating the cell membrane of the epithelial cell and improves the effective concentration of the keratinocyte growth factor 2 reaching the tissue of the damaged part.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a PEP-1-G4S-KGF2 fusion protein, wherein the amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No. 1.

The invention also provides a gene for encoding the PEP-1-G4S-KGF2 fusion protein, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in the technical scheme in preparation of a drug for improving the cell membrane penetrating capability.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for repairing epithelial tissue injury.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for promoting cell proliferation.

Preferably, the cells comprise epithelial cells.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for promoting cell migration.

Preferably, the cells comprise epithelial cells.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of drugs for repairing acute and chronic inflammations, ulcers and physical and chemical burns.

The invention provides a PEP-1-G4S-KGF2 fusion protein, and a coding gene and application thereof, wherein the amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No. 1. The PEP-1-G4S-KGF2 fusion protein provided by the invention improves the capability of penetrating the cell membrane of epithelial cells, and improves the effective concentration of keratinocyte growth factor 2 reaching the tissue of the damaged part by utilizing the function that the cell-penetrating peptide PEP has the structure of carrying macromolecules to penetrate a biological membrane.

Drawings

FIG. 1 is a restriction enzyme identification map of the fusion sequence PEP-1-G4S-KGF 2;

FIG. 2 is a graph showing the growth of PEP-1-G4S-KGF2 fusion protein strain;

FIG. 3 is a diagram showing the expression of PEP-1-G4S-KGF2 fusion protein;

FIG. 4 shows a diagram of the purification of PEP-1-G4S-KGF2 fusion protein;

FIG. 5 is a diagram showing the cell migration promotion of PEP-1-G4S-KGF2 fusion protein;

FIG. 6 is a diagram showing the results of immunofluorescence assay of fusion proteins through membranes, in which PE is stained with KGF2 antibody and blue is stained with DAPI.

Detailed Description

The invention provides a PEP-1-G4S-KGF2 fusion protein, wherein the amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No.1, and is specifically shown as follows: KETWWETWWTEWSQPKKKKKVGGGGSLGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS are provided.

In the invention, the PEP-1 amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is KETWWETWWTEWSQPKKKKKV (SEQ ID No.12), and has an auxiliary membrane penetration effect; the G4S amino acid sequence is GGGGS, and the function of the GGGGS is linkage; the amino acid sequence of KGF2 is (SEQ ID No.13) LGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS, and the KGF2 has a repairing effect on epithelial tissue injury.

The invention also provides a gene for encoding the PEP-1-G4S-KGF2 fusion protein, the nucleotide sequence of the gene is shown as SEQ ID No.2, and the gene is specifically as follows:

in the invention, the nucleotide sequence of the PEP-1 amino acid sequence is shown as SEQ ID No.3, and specifically comprises the following steps:

aag gag acc tgg tgg gag acc tgg tgg acc gag tgg agc cag ccc aag aag aag aag aag gtg。

in the invention, the nucleotide sequence of the amino acid sequence of the G4S is shown as SEQ ID No.4, and specifically comprises the following steps:

ggc ggc ggc ggc agc。

in the present invention, the nucleotide sequence encoding the amino acid sequence of KGF2 is shown in SEQ ID No.5, and specifically as follows:

the method for obtaining the PEP-1-G4S-KGF2 fusion protein and the gene for coding the PEP-1-G4S-KGF2 fusion protein is not particularly limited, and the fusion protein can be obtained by conventional obtaining or synthesis by entrusting by adopting a method well known by the technicians in the field.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in the technical scheme in preparation of a drug for improving the cell membrane penetrating capability. In the invention, the PEP-1-G4S-KGF2 fusion protein is preferably used as the only active substance in the medicament. The invention has no special limitation on the dosage form of the medicament, and the PEP-1-G4S-KGF2 fusion protein is adopted to be a medically acceptable dosage form.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for repairing epithelial tissue injury. In the invention, the PEP-1-G4S-KGF2 fusion protein is preferably used as the only active substance in the medicament. The invention has no special limitation on the dosage form of the medicament, and the PEP-1-G4S-KGF2 fusion protein is adopted to be a medically acceptable dosage form.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for promoting cell proliferation. In the invention, the PEP-1-G4S-KGF2 fusion protein is preferably used as the only active substance in the medicament. The invention has no special limitation on the dosage form of the medicament, and the PEP-1-G4S-KGF2 fusion protein is adopted to be a medically acceptable dosage form. In the present invention, the cells preferably comprise epithelial cells.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of a drug for promoting cell migration. In the invention, the PEP-1-G4S-KGF2 fusion protein is preferably used as the only active substance in the medicament. The invention has no special limitation on the dosage form of the medicament, and the PEP-1-G4S-KGF2 fusion protein is adopted to be a medically acceptable dosage form. In the present invention, the cells preferably comprise epithelial cells.

The invention also provides application of the PEP-1-G4S-KGF2 fusion protein in preparation of drugs for repairing acute and chronic inflammations, ulcers and physical and chemical burns. In the invention, the PEP-1-G4S-KGF2 fusion protein is preferably used as the only active substance in the medicament. The invention has no special limitation on the dosage form of the medicament, and the PEP-1-G4S-KGF2 fusion protein is adopted to be a medically acceptable dosage form.

In order to further illustrate the present invention, the following detailed description of the invention is given in conjunction with examples, which should not be construed to limit the scope of the invention.

Example 1

PEP-1-G4S-KGF2 fusion protein sequence design process:

designing a primer:

p1 (upstream primer) (SEQ ID No. 6): 5'-atgga tcc aaa gaa acc tgg tgg gaa acc tgg tgg ac-3', respectively;

p1' (downstream primer) (SEQ ID No. 7): 5'-cgc cac cca ctt tac gtt ttt ttt tcg gct gag acc att cgg tcc acc agg ttt ccc ac-3', respectively;

ligation primer L (SEQ ID No. 8): 5'-act acc gcc gcc acc tga gcc acc acc gcc act gcc acc gcc acc cat gtc cag gcc cag-3', respectively;

KGF2 amplification upstream primer (SEQ ID No. 9): 5'-ctg ggc cag gac atg gtg ag-3', respectively;

KGF2 amplification downstream primer (SEQ ID No. 10): 5'-aaa gtc gac tca gct gtg gac-3' are provided.

The nucleic acid sequence with ID No. 2255 (SEQ ID No.11) retrieved in genebank was specified as follows, and based on this sequence, SEQ ID No.5 was synthesized:

atgtggaaatggatactgacacattgtgcctcagcctttccccacctgcccggctgctgctgctgctgctttttgttgctgttcttggtgtcttccgtccctgtcacctgccaagcccttggtcaggtcatggtgtcaccagaggccaccaactcttcttcctcctccttctcctctccttccagcgcgggaaggcatgtgcggagctacaatcaccttcaaggagatgtccgctggagaaagctattctctttcaccaagtactttctcaagattgagaagaacgggaaggtcagcgggaccaagaaggagaactgcccgtacagcatcctggagataacatcagtagaaatcggagttgttgccgtcaaagccattaacagcaactattacttagccatgaacaagaaggggaaactctatggctcaaaagaatttaacaatgactgtaagctgaaggagaggatagaggaaaatggatacaatacctatgcatcatttaactggcagcataatgggaggcaaatgtatgtggcattgaatggaaaaggagctccaaggagaggacagaaaacacgaaggaaaaacacctctgctcactttcttccaatggtggtacactca。

based on the codon recognition bias of Escherichia coli, the codon was changed without changing the amino acid sequence, and some unnecessary 5' amino acid sequence was deleted according to the active domain, and a novel KGF2 sequence (SEQ ID No.5) was synthesized by an artificial synthesis method.

Amplifying the P1 and the P1' to obtain a fragment containing PEP-1; then using the fragment as a template 1, and amplifying G4S into a template 2 by using a primer P1 and a connecting primer; the synthesized SEQ ID No.5 which is changed into the preferred codon sequence of the escherichia coli but does not change the amino acid sequence is used as a template, a KGF2 fragment is synthesized by using an upstream primer and a downstream primer of a primer KGF2 and is used as a template 3, finally, the template 2 and the template 3 are used as final templates, and the nucleotide sequences of target gene fragments PEP-1-G4S-KGF2 and PEP-1-G4S-KGF2 genes are synthesized by using P1 and downstream primers of KGF-2 and are shown as SEQ ID No. 2.

The amino acid of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No. 1.

Example 2

1. Enzyme digestion identification process of fusion protein

The fusion protein carrier plasmid PET28a-PEP-1-G4S-KGF2 is cut by restriction enzymes BamHI and SalI at 37 ℃ for 12h, and 10 mu l of cut products are used for agarose gel electrophoresis after the cut products are cut.

The sequence restriction identification of the fusion protein is shown in figure 1, and the success of the construction of the fusion protein plasmid can be proved from figure 1.

2. Growth curve diagram of fusion protein strain

Inoculating the fusion protein strain PET28a-PEP-1-G4S-KGF2 monoclonal colony into 5ml fresh KANLB culture solution, performing shake culture at 37 ℃ and 200r/min for 72 hours, and determining OD respectively at 0h, 24h, 48h and 72h₆₀₀The value is obtained. The results are shown in FIG. 2, from which FIG. 2To see that the fusion protein strain grew well within this 72 h.

3. Preparation, expression and purification of fusion protein

Construction of fusion protein plasmid: the PCR products of the vector PET28a and PEP-1-G4S-KGF2 were digested with the same restriction enzymes BamH I and Sal I at 37 ℃ for 12h, as shown in Table 1:

TABLE 1 reaction System

After digestion at 37 ℃, agarose gel electrophoresis detection is carried out, and digestion products are recovered.

The DNA recovery and purification kit recovers the target DNA fragment, and the operation steps are as follows:

(1) and cutting off the gel block containing the target DNA fragment and transferring the gel block into a centrifuge tube.

(2) According to the kit instructions (see Bomaide agarose recovery kit)

And (3) connection reaction:

a connection system: mu.l of vector, 6. mu.l of target gene, 5U/. mu.l of T4 ligase, 1.5. mu.l of buffer, and 10. mu.l in total.

And (3) transformation:

the DH5a and BL21(de3) allelochemicals frozen from-80 ℃ were thawed on ice, and 1. mu.l of the ligation product was added separately and ice-cooled for 30 min. The mixture was heated at 42 ℃ for 90s and then cooled in ice bath for 2 min. Adding 1ml LB culture medium, culturing at 37 deg.C for 2h, centrifuging at 5000rpm for 1min, and collecting bacteria. Suck 900 μ l of the supernatant and discard it, leave 100 μ lLB to blow off the bacteria solution. The culture solution is smeared on LB agar medium containing KAN respectively and cultured overnight.

The constructed and sequenced recombinant plasmid PET28a-PEP-1-G4S-KGF2 and the empty vector PET28a were transferred to BL21(de3) according to the transformation method above, and plated for culture. From the resistant plate, 1 single clone each was inoculated in 5ml of LB liquid containing KAN to OD₆₀₀0.5, inoculating in 20ml of fresh KAN LB culture solution according to 1% inoculum size, shake culturing for 3 hr, OD₆₀₀When the temperature reaches 0.5-0.7, IPTG (final product) is addedConcentration 1mM), at 37 ℃ and 200r/min, and culturing is continued for 4 h. Samples were taken every 2 hours from 0 to 4 hours for 0, 2, and 4 hours, 1ml each. The cell was resuspended in 48. mu.l of PBS, 12. mu.l of 5 Xloading buffer was added and mixed, boiled in boiling water for 10min, and the expression of the protein was detected by SDS-PAGE (FIG. 3), which shows that the fusion protein was highly expressed in FIG. 3.

Collecting strains

Inoculating the cultured strain PET28a-PEP-1-G4S-KGF2 seed solution at a ratio of 1%, culturing at 37 deg.C under stirring speed of 200rpm, controlling pH to 7.0, and allowing the thallus to grow to OD₆₀₀Induction was started at 1mM IPTG concentration 0.5, samples were collected after 5 hours, the fermentation broth was centrifuged (5000rpm, 15min, 4 ℃) to remove the supernatant, and the precipitate was collected.

Ultrasonic bacteria breaking:

resuspending the cells with PBS at a ratio of 1:30 (w/v); and then carrying out ultrasonic bacteria breaking with the power of 700w, wherein the bacteria breaking cycle times are 10-15 min, ultrasonic treatment is carried out for 4s, and the bacteria breaking rate needs to reach 99% by microscopic examination after ultrasonic treatment is stopped for 11 s. Performing ice bath in the whole bacteria breaking process, and controlling the temperature at 15 ℃; after the disruption, the supernatant was centrifuged (12000rpm, 15min, 4 ℃ C.) and collected.

Affinity chromatography purification

The target protein is fused and expressed together with the his label, so that a nickel column is selected to collect the required his fusion protein. The steps of target protein purification are as follows:

(1) the sample and PBS buffer were filtered through a 0.45um microfiltration membrane.

(2) Filling Ni into a column, wherein the volume of the column body is 10 ml;

(3) 5 column volumes were equilibrated with buffer 1 at a flow rate of 1 ml/min;

(4) filtering the crushed solution with a 0.45um filter membrane, and loading the sample at the flow rate of 1 ml/min;

(5) washing 5 column volumes with buffer 2 at a flow rate of 0.5 ml/min;

(6) performing stage elution with buffer solution 3 containing 10 mM, 20 mM, 50mM, 100 mM, 200 mM, 300 mM and 400mM of imidazole respectively at a flow rate of 1ml/min, collecting elution peaks at each stage, and detecting the molecular weight and purity of the fusion protein by SDS-PAGE; washing 5 column volumes with pure water, washing 3 column volumes with 20% ethanol at a flow rate of 2ml/min, and storing the column in low temperature environment.

The buffer composition is as follows:

buffer 1:

preparation of 50mM PBS buffer (pH7.4): 0.5M NaH₂PO₄ 19ml，0.5M Na₂HPO₄81ml of NaCl, 29.3g of NaCl and a proper amount of water are added for dissolving, and then the volume is adjusted to 1000 ml.

Buffer 2:

50mM phosphate buffer, pH7.4, i.e., PBS solution at pH7.4, to prepare: 0.5M NaH₂PO₄19ml，0.5M Na₂HPO₄81ml, NaCl 29.3g and imidazole 34g, adding a proper amount of water to adjust the pH value and then fixing the volume to 1000 ml.

Buffer solution 3

Buffer B was prepared at different imidazole concentrations as shown in table 2:

TABLE 2 buffer configuration

Concentration of imidazole	Buffer	1 amount (ml)	Buffer 2 volume (ml)
				10mM	98	2
50mM	90	10
			100mM	80	20
200mM	60	40
			300mM	40	60
400mM	20	80

The fusion protein is further separated and purified.

Purifying by ion exchange chromatography

The strong anion exchange column comprises the following specific operation steps:

(1) carrying out ultrafiltration concentration on the protein collected liquid after nickel column purification by using an ultrafiltration tube, wherein a concentrated sample is reserved;

(2) preparing 1mM PBS buffer solution and 1mM NaCl solution, and ultrasonically defoaming for later use;

(3) 2ml of the concentrated protein mixture was added to an ion exchange column and the flow-through was collected.

(4) Washing with PBS and collecting the rinsing liquid.

(5) Elution was then carried out with varying concentration gradients of Nacl (5%, 10%, 20%, 30%), again requiring collection of eluents at each concentration.

(6) And detecting the distribution of the target protein by SDS-PAGE of the collected flow-through liquid, rinsing liquid and eluent.

The expression pattern of the fusion protein is shown in FIG. 3 and the purification pattern of the fusion protein is shown in FIG. 4, from which FIG. 3 it can be concluded that the fusion protein is highly expressed; from FIG. 4, it can be seen that the fusion protein with higher purity can be collected.

Example 3

Fusion proteins promote cell migration

(1) Firstly, marking lines are evenly drawn on the back of the 6-hole plate by using a marking pen compared with a ruler, and the marking lines cross through the holes at intervals of about 0.5-1 cm.

(2) Adding about 5X 10 to 6-well plate⁵Fibroblasts, 5% CO at 37 ℃₂And culturing in an incubator overnight.

(3) After 24h of culture, the yellow tip was placed closer to the ruler than to the rear transverse line, and the tip was perpendicular and could not be tilted.

(4) Cells were washed 3 times with DPBS, and the scraped cells were removed, and 100. mu.g/ml of EP-1-G4S-KGF2 fusion protein, an equal volume of physiological saline, and 3% FBS-containing cell culture medium were added to each of the experimental and control groups.

(5) 5% CO at 37 ℃₂And (5) an incubator for culture. Samples were taken at 0, 24 hours, observed under a microscope and photographed, and the results are shown in FIG. 5.

As can be seen from FIG. 5, the fusion protein promoted migration of fibroblasts.

Example 4

Fusion protein transmembrane

Cell culture:

the purchased human skin fibroblast cell line was cultured in DMEM (containing 10% fetal bovine serum and 1% double antibody mixture) and placed at 37 ℃ in 5% CO₂Culturing in an incubator, wherein cells grow to be more than 80%, washing for 2-3 times by PBS, adding 1mL of 0.25% trypsin, digesting at normal temperature for 30-60 s, slightly tapping the bottle wall to enable the cells to fall off, adding 2 times of volume of culture medium containing fetal calf serum to stop digestion, uniformly blowing by a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding a proper amount of complete culture medium to prepare single cell suspension, adjusting concentration, and using the obtained cells in logarithmic phase with good conditions for experiments. Taking human skin fibroblast of logarithmic growth phase at 1 × 10⁵One cell/mL of the cells was inoculated into a 24-well plate culture in an amount of 900. mu.l/well, and placed in a cell culture chamber at 37 ℃ in 5% CO₂Culturing for 24h, and taking out to establish a fusion protein PEP-1-G4S-KGF2 experimental group and a KGF2 control group. The experimental groups were added with fusion proteins PEP-1-G4S-KGF 2100. mu.l/well, respectively, to end upThe concentration was 100. mu.g/mL, and 100. mu.g/mL of KGF2 protein (encoded by SEQ ID No.5) was added to each well of the control group. Each group is provided with 3 multiple holes, and after being uniformly shaken, the groups are placed in a standard incubator to be continuously cultured for 24 hours.

Immunofluorescence staining

(1) The medium was discarded and washed 3 times with 1 × PBS for 3-5 min each time.

(2) The PBS is discarded, and the solution is fixed with 4% paraformaldehyde for 15-20 min.

(3) Washing with 1 × PBS for 2 times, each time for 3-5 min.

(4) Cells were permeabilized with 0.1% PBST for 10 min.

(5) Washing with 1 × PBS for 2 times, each time for 3-5 min.

(6) 5% BSA (dissolved in 1 XPBS and treated at 60 ℃ for 10min) was added and blocked at room temperature for 30 min.

(7) The cells were blotted dry and prepared for 1:50 dilution of KGF2 antibody (anti-FGF 10 from Abcam) in blocking medium at 100. mu.l/well and incubation at room temperature for 1 h.

(8) Primary antibody was aspirated and washed 3 times with 1 XPBS for 5min each.

(9) Blocking with 5% BSA blocking solution for 30min at room temperature.

(10) The blocking solution was aspirated off and washed 3 times with 1 × PBS for 5min each time.

(11) Adding 100 μ l/well of PE goat anti-rabbit secondary antibody diluted at 1:500 in 1 XPBS, and incubating for 1h at room temperature in the dark.

(12) The secondary antibody was aspirated and washed 3 times with 1 × PBS for 10min each.

(13) DAPI (5ng/ml) stained nuclei at 100. mu.l/well and incubated for 2min at room temperature.

(14) Wash 3 times with 1 × PBS for 5min each.

(15) The distilled water is rinsed once.

(16) The film is read and photographed under a fluorescence microscope, and the result is shown in figure 6.

As can be seen from FIG. 6, the fusion protein PEP-1-G4S-KGF2 has a significant membrane penetration effect compared with KGF 2.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.

Sequence listing

<110> Beijing Congregation science and technology Co., Ltd

<120> PEP-1-G4S-KGF2 fusion protein and encoding gene and application thereof

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aaggagacct ggtgggagac ctggtggacc gagtggagcc agcccaagaa gaagaagaag 60

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tcctcctcct tctccagccc ttccagcgcg ggcaggcacg tgcggagcta caaccacctg 180

cagggcgatg tccgctggcg caagctgttc agcttcacca agtactttct caagatcgag 240

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atgaacaaga aggggaagct ctacggcagc aaggagttta acaacgactg taagctgaag 420

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cgcaagctgt tcagcttcac caagtacttt ctcaagatcg agaagaacgg gaaggtcagc 180

gggaccaaga aggagaactg cccgtacagc atcctggaga tcacaagcgt ggagatcggc 240

gtggtggccg tcaaggccat caacagcaac tactacctgg ccatgaacaa gaaggggaag 300

ctctacggca gcaaggagtt taacaacgac tgtaagctga aggagaggat cgaggagaac 360

ggctacaaca cctacgccag ctttaactgg cagcacaacg ggaggcagat gtacgtggcc 420

ctgaacggca agggcgctcc aaggcgcggc cagaagacac gcaggaagaa caccagcgct 480

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<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctgggccagg acatggtgag 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaagtcgact cagctgtgga c 21

<210> 11

<211> 624

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtggaaat ggatactgac acattgtgcc tcagcctttc cccacctgcc cggctgctgc 60

tgctgctgct ttttgttgct gttcttggtg tcttccgtcc ctgtcacctg ccaagccctt 120

ggtcaggtca tggtgtcacc agaggccacc aactcttctt cctcctcctt ctcctctcct 180

tccagcgcgg gaaggcatgt gcggagctac aatcaccttc aaggagatgt ccgctggaga 240

aagctattct ctttcaccaa gtactttctc aagattgaga agaacgggaa ggtcagcggg 300

accaagaagg agaactgccc gtacagcatc ctggagataa catcagtaga aatcggagtt 360

gttgccgtca aagccattaa cagcaactat tacttagcca tgaacaagaa ggggaaactc 420

tatggctcaa aagaatttaa caatgactgt aagctgaagg agaggataga ggaaaatgga 480

tacaatacct atgcatcatt taactggcag cataatggga ggcaaatgta tgtggcattg 540

aatggaaaag gagctccaag gagaggacag aaaacacgaa ggaaaaacac ctctgctcac 600

tttcttccaa tggtggtaca ctca 624

<210> 12

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Lys Lys Val

20

<210> 13

<211> 169

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser

1 5 10 15

Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn

20 25 30

His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys

35 40 45

Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys

50 55 60

Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly

65 70 75 80

Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn

85 90 95

Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys

100 105 110

Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe

115 120 125

Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys

130 135 140

Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala

145 150 155 160

His Phe Leu Pro Met Val Val His Ser

165

Claims

1. The PEP-1-G4S-KGF2 fusion protein is characterized in that the amino acid sequence of the PEP-1-G4S-KGF2 fusion protein is shown as SEQ ID No. 1.

2. A gene encoding the PEP-1-G4S-KGF2 fusion protein of claim 1, wherein the nucleotide sequence of the gene is represented by SEQ ID No. 2.

3. The use of the PEP-1-G4S-KGF2 fusion protein of claim 1 in the preparation of a medicament for increasing the ability to penetrate cell membranes.

4. The use of the PEP-1-G4S-KGF2 fusion protein of claim 1 in the preparation of a medicament for repairing epithelial tissue damage.

5. The use of the PEP-1-G4S-KGF2 fusion protein of claim 1 in the preparation of a medicament for promoting cell proliferation.

6. The use of claim 5, wherein the cells comprise epithelial cells.

7. The use of the PEP-1-G4S-KGF2 fusion protein of claim 1 in the preparation of a medicament for promoting cell migration.

8. The use of claim 7, wherein the cells comprise epithelial cells.

9. The use of the PEP-1-G4S-KGF2 fusion protein of claim 1 for the preparation of a medicament for the repair of acute and chronic inflammation, ulcers and physical and chemical burns.