CN116333052A - Immunomodulatory peptide Ha-CATH and application thereof - Google Patents
Immunomodulatory peptide Ha-CATH and application thereof Download PDFInfo
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- CN116333052A CN116333052A CN202310593140.3A CN202310593140A CN116333052A CN 116333052 A CN116333052 A CN 116333052A CN 202310593140 A CN202310593140 A CN 202310593140A CN 116333052 A CN116333052 A CN 116333052A
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Chemistry (AREA)
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- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an immunomodulatory peptide Ha-CATH and application thereof. Belongs to the biomedical field. The immunomodulatory peptide Ha-CATH is a cyclic polypeptide of which the eighth cysteine residue and the thirteenth cysteine residue form a pair of intramolecular disulfide bonds, the molecular weight is 2361.8 daltons, the isoelectric point is 6.04, and the amino acid sequence is shown as SEQ ID NO. 1, wherein the eighth cysteine residue and the thirteenth cysteine residue are coded by the skin repair peptide gene of the Chinese amphibian rana. The gene (GenBank accession: OQ 992774) encoding the immunomodulatory peptide Ha-CATH precursor consists of 714 nucleotides, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2; wherein the 445 th to 504 th nucleotides are immunomodulatory peptide Ha‑The coding gene of CATH. Immunomodulatory peptide Ha‑CATH in preparation of skin wound repair promoting treatmentThe application in medicines.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an immunomodulatory peptide Ha-CATH and application thereof.
Background
Amphibious animals are used as a transition type from aquatic vertebrates to terrestrial vertebrates, and living environments are complex and various. The skin of the amphibian is bare, smooth, without hair or scale coverage, and faces more threats and challenges. To accommodate the complex living environment in land and aquatic, amphibians have evolved a unique and powerful set of skin defense systems. The skin serves as an important physical barrier, and has the functions of preventing invasion of microorganisms, and maintaining body temperature and fluid homeostasis. Skin wounds can instantaneously destroy this barrier, posing a great threat to health. Skin lesion healing is therefore critical to the body's maintenance of the external barrier. Skin wound healing is a dynamic process of self-repair and reconstruction of intact skin after tissue injury, which is tightly regulated by a variety of cell types and numerous factors.
The skin of an amphibian plays a very important role in maintaining its own survival and adapting to the ecological environment of diverse habitats, and must provide necessary guarantee for its survival. The skin of amphibians is capable of secreting large amounts of bioactive molecules to combat biological and non-biological attacks in the environment. These active molecules are widely involved in various physiological activities of the body, and have various pharmacological activities such as antimicrobial, antitumor, antioxidant, wound repair and the like. Hua Xiyu frogHyla annectans) Belonging to genus Rana of family Ranidae of order Rana of class Amphiza, at presentThe report of Hua Xiyu frog is mainly focused on the effects of relieving pain and resisting oxidation of nerves except for being integrally used for treating traumatic injury, but the identification and mechanism research of the active ingredients for promoting skin repair are not reported.
Disclosure of Invention
A first object of the present invention is to provide an immunomodulatory peptide Ha-CATH; a second object is to provide the use of said immunomodulatory peptide Ha-CATH.
The first object of the invention is realized in that the nucleotide sequence of the immunomodulatory peptide Ha-CATH is shown in a sequence table SEQ ID NO: 1.
The immunomodulatory peptide Ha-CATH is a cyclic polypeptide of which a pair of intramolecular disulfide bonds is formed by an eighth cysteine residue and an eleventh cysteine residue which are encoded by a Chinese amphibian Chinese toad defensive peptide gene, and the cyclic polypeptide consists of 20 amino acid residues, has a molecular weight of 2361.8 daltons, an isoelectric point of 6.04 and an amino acid sequence of KKTHKEECLECIITLLPGHP, and is shown as SEQ ID NO 2: lys Lys Thr His Lys Glu GluCys Leu Glu Cys Ile Ile Thr Leu Leu Pro Glu His Pro.
The encoding gene of the immunoregulatory peptide precursor consists of 714 nucleotides, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the sequence from the 5 'end to the 3' end is as follows:
atggacggctccttgaagtatctcgtcctcttcggacacttactcgccgtcgccgctgcccccattcactcttccctagaagacattgcagaagttattcattttaacaacaaatatttgggcacaaatttcctgtttaagctcttaagggctgaagatgaaggcgactactacttgtcggaaaactccacaataatgaagagtctgaaattcttcataaaggagacgacttgtacgatctctgacaccgaggatccggaaaactgtgactttaaacccgatggggtggtgatggcgtgtgtggccgatattattactgataaaaatgagaaaaaaatcacaggacaatgtgtgaaagaaaacaacacatcgaaaggagcgcacgctatgaagagggagcttgcggacaaaacacgagtgacatttgagatatctgtggacctcaagaagacccataaggaagaatgtctggaatgtatcatcaccctccttccggagcacccctgacttacagtcatgacctctcggagcacccctgacttacagtcatcacctctcggagcacccctgactcacagtcatcacctcttggagcacccctgacttacagtcatcacctcccggagcactcatgactcacagtcatcacctcctggagcacccctgacaataaatcacagtcatcacgtcagaaaaaaaaaaaaaaaaaaaaaa。
wherein, the 445 th to 504 th nucleotides are the coding genes of the mature immunomodulatory peptide Ha-CATH.
The invention relates to an application of an immunomodulatory peptide Ha-CATH in preparing a medicament for promoting skin wound repair and treatment.
The invention has the beneficial effects that: provides a novel immunomodulatory peptide Ha-CATH, which has remarkable function of promoting skin wound repair and can be applied to preparing skin wound repair promoting therapeutic drugs.
Drawings
FIG. 1 is a comparative schematic of the immunomodulatory peptide Ha-CATH of the invention to promote wound healing in mice;
wherein A is immunomodulatory peptide Ha-CATH for promoting wound healing of mice, and is topically administered dailyHa-CATHComparison of pictures after EGF, sterile water (vehicle negative control group) treatment;
b is 2 mg/mLHa-CATH20 [ mu ] L of each of the polypeptide (sample treatment group), 100 mg/mL Epidermal Growth Factor (EGF (positive control group) and sterile water (Vehicle (negative control group)) was applied to a mouse wound comparison chart;
in comparison with the vehicle negative control group,Ha-CATHthe skin wound area of the mice treated by EGF is obviously reduced, and the numerical value is mean value + -standard deviation, ns and p>0.05，*p<0.05，**p<0.01。
Detailed Description
The invention is further described below with reference to examples and figures, but is not limited in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The nucleotide sequence of the immunomodulatory peptide Ha-CATH is shown in a sequence table SEQ ID NO: 1.
The amino acid sequence of the immunomodulatory peptide Ha-CATH is shown as SEQ ID NO: 2.
The application of the immunomodulatory peptide Ha-CATH provided by the invention is the application of the immunomodulatory peptide Ha-CATH in preparing medicaments for promoting skin wound repair.
The invention is further illustrated by the following examples:
example 1
Immunomodulatory peptidesHa-CATHChemical synthesis method of (2)
(1) Synthesis of Ha Using an automated polypeptide synthesizer (433A,Applied Biosystems, USA)-Amino acid full sequence of CATH, C by high performance liquid chromatography HPLC (Waters, USA) 18 The molecular weight of the synthesized sample (2) is measured by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) method, and the purity of the synthesized sample is identified by High Performance Liquid Chromatography (HPLC). MALDI-TOF determination the molecular weight of the chemically synthesized polypeptide was 2361.80 Da, isoelectric point of the synthesized polypeptide was 6.04 as determined by isoelectric focusing electrophoresis, and purity of the synthesized sample as determined by HPLC>95%. The amino acid sequence structure of the synthesized Ha-CATH is determined to be consistent with that of the natural Ha-CATH by using an automatic amino acid sequencer.
EXAMPLE 2 Gene cloning of the immunomodulatory peptide Ha-CATH
(1) Hua Xiyu frog skin total RNA extraction:
washing Hua Xiyu frog back skin with deionized water, quick-freezing in liquid nitrogen for 10 hr, collecting 100 mg skin tissue, adding 1ml Trizol solution, and homogenizing in 20 ml glass homogenizer for 30 min. An equal volume of phenol/chloroform solution was added, vigorously mixed, left at room temperature for 10 minutes, centrifuged at 12000 rpm for 10 minutes at 4℃and the precipitate discarded. Adding isopropyl alcohol into the supernatant, standing at room temperature for 10 minutes, centrifuging at 12000 rpm for 10 minutes at 4 ℃, washing the precipitate once with 75% ethanol, and airing to obtain the total RNA of the skin as the precipitate at the bottom of the tube.
(2) Hua Xiyu construction of frog skin cDNA library:
by using a Creator from CLONTECH company TM SMART TM cDNA Library Construction Kit plasmid cDNA library construction kit, the specific procedures were as per the instructions.
By using a Creator from CLONTECH company TM SMART TM cDNA Library Construction Kit plasmid cDNA library construction kit, the specific procedures were as per the instructions. Using SMARTScribe TM Reverse transcriptase and SMART V oligonucleotides and 3' In-Fusion SMARTer CDS primerFirst strand cDNA was synthesized. Second Strand cDNA Synthesis was performed by long-distance PCR using Advantage 2 Polymerase Mix, 5'PCR primer II A, and 3' In-FusionSMARTer PCR primer. The synthesized second strand cDNA was used as a template for the following PCR to screen cDNA encoding the polypeptide (Ha-CATH).
(3) Cloning and screening of immune regulating peptide Ha-CATH gene:
according to the reported highly conserved cathelin domain sequences of the cathelicidins, the 3' -end reverse primer Nv-CATH-R1 (5 ' -WSCRCAGRYCTTCACCTCC-3' (W=A/T; S=G/C; R=A/G; Y=C/T) was designed and combined with the kit-provided 5' PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3'), the 5' -end fragment of the cathelicidin-encoded cDNA was amplified, the PCR conditions were 95℃for 2 min pre-denaturation, 94℃for 30S, 52℃for 40S, 72℃for 45S for 30 cycles, and then 72℃for 10 min more extension the PCR products were purified by gel electrophoresis and cloned into pMD19-T vector (Takara, japan) for DNA sequencing.
According to the sequence of the 5 'end, a primer 5'-CCATGAGGAGCTGGTGGCTGT-3'is designed and combined with a 3' PCR primer in a library-building kit: 5'-ATTCTAGAGGCCGAGGCGGCCG-3' PCR amplification was performed under the same conditions as above. And (3) recovering the target fragment through a DNA gel recovery kit after amplification is finished, and verifying the size of the strip through gel electrophoresis. The gel recovery product was connected to pMD18-T vector overnight and transformed into E.coli DH 5. Alpha. Competent cells prepared in advance by the calcium chloride method. The next day, single clone is selected for bacterial liquid PCR, positive clone is selected for bacterial inoculation, and plasmid is extracted. The plasmid fragment size was then verified by agarose gel electrophoresis and the plasmid corresponding to the band size was sequenced.
(4) Determination of the sequence of the immunomodulatory peptide Ha-CATH gene:
extracting plasmid DNA, determining nucleotide sequence by dideoxy method, using full-automatic nucleotide sequence determination instrument of U.S. Applied Biosystems373A, sequencing primer BcaBEST TM Sequencing Primer RV-M and BcaBEST TM Sequencing Primer M13-47，BcaBEST TM Sequencing Primer RV-M sequence: 5' GAGCGGATAACAATTTCAC ACAGG 3’，BcaBEST TM Sequencing Primer M13-47：5’CGCCAGGGTTTTC CCAGTCACGAC 3’。
The gene sequencing result shows that the gene for encoding the Hua Xiyu frog immunoregulatory peptide precursor consists of 714 nucleotides (SEQ ID NO: 1) (GenBankAccession Number: OQ 992774), and the sequence from the 5 'end to the 3' end is as follows:
atggacggctccttgaagtatctcgtcctcttcggacacttactcgccgtcgccgctgcccccattcactcttccctagaagacattgcagaagttattcattttaacaacaaatatttgggcacaaatttcctgtttaagctcttaagggctgaagatgaaggcgactactacttgtcggaaaactccacaataatgaagagtctgaaattcttcataaaggagacgacttgtacgatctctgacaccgaggatccggaaaactgtgactttaaacccgatggggtggtgatggcgtgtgtggccgatattattactgataaaaatgagaaaaaaatcacaggacaatgtgtgaaagaaaacaacacatcgaaaggagcgcacgctatgaagagggagcttgcggacaaaacacgagtgacatttgagatatctgtggacctcaagaagacccataaggaagaatgtctggaatgtatcatcaccctccttccggagcacccctgacttacagtcatgacctctcggagcacccctgacttacagtcatcacctctcggagcacccctgactcacagtcatcacctcttggagcacccctgacttacagtcatcacctcccggagcactcatgactcacagtcatcacctcctggagcacccctgacaataaatcacagtcatcacgtcagaaaaaaaaaaaaaaaaaaaaaa。
wherein, the 445 th to 504 th nucleotides are the coding genes of the mature immunomodulatory peptide Ha-CATH.
EXAMPLE 3 chemical Synthesis of immunomodulatory peptide Ha-CATH
(1) Synthesis of Ha Using an automated polypeptide synthesizer (433A,Applied Biosystems, USA)-Amino acid full sequence of CATH, C by high performance liquid chromatography HPLC (Waters, USA) 18 Desalting and purifying the synthesized sample by reversed phase column chromatography;
(2) Determining the molecular weight of the synthesized sample by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) method;
(3) The purity of the synthesized sample was identified by high performance liquid chromatography HPLC. MALDI-TOF determination the molecular weight of the chemically synthesized polypeptide was 2361.80 Da, isoelectric point of the synthesized polypeptide was 6.04 as determined by isoelectric focusing electrophoresis, and purity of the synthesized sample was >95% as determined by HPLC. The amino acid sequence structure of the synthesized Ha-CATH is determined to be consistent with that of the natural Ha-CATH by using an automatic amino acid sequencer.
Example 4 use of immunomodulatory peptide Ha-CATH in the preparation of a medicament for promoting repair of skin wounds
A6 week old BALB/c mouse (20-30 g) was anesthetized with 1% sodium pentobarbital (0.1 mL/20 g), the mid-dorsal hair was removed and sterilized with alcohol cotton pads), and two symmetrical circular full-cortical excision wounds were made on the back with a skin biopsy device, the wound diameter being 8 mm. The mice were back wound instilled with 20 μl of antimicrobial peptide Ha-CATH (20 μl,2 mg/mL) per wound instillation volume, and sterile water (20 μl) and EpidermalGrowth Factor (EGF, 20 μl,100 mg/mL) were negative and positive controls, respectively. Each experimental mouse was placed with a separate cage to prevent wound contact infection. During the feeding process, sufficient food and water is provided, and a proper temperature and humidity are maintained, and 12 h/12 h illumination is maintained. Dosing was fixed once a day for a period of time and wound healing was recorded. Mice were observed for wound healing at postoperative points 0 d, 2 d, 4 d, 8 d and recorded with digital cameras.
The results are shown in FIG. 1: compared with the veccle group, the Ha-CATH treatment group and the positive control EGF group can effectively promote wound healing of mice (fig. 1A). Wound healing rates in the 2 nd d th, 4 d th, 6 d th, 8 d Ha-CATH group were 57.68%, 66.67%, 82.67%, 93%, respectively; the wound healing rates of the vehicle groups are respectively as follows: 25.45%, 35.23%, 37.31%, 53.9%; the wound healing rates of the EGF groups are respectively as follows: 50.67%, 51.95%, 67%, 75.31% (fig. 1B). On day 8 or so, the wounds in the treatment group were substantially healed, with significant differences compared to the control group. The experimental results show that: the immunomodulatory peptide Ha-CATH has a remarkable effect of promoting skin wound healing, and the effect of promoting skin wound healing is better than that of Epidermal Growth Factor (EGF). The immunomodulatory peptide Ha-CATH can be used for preparing medicaments for promoting skin wound repair.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. The immune regulation peptide Ha-CATH is characterized in that the nucleotide sequence of the immune regulation peptide Ha-CATH is shown in a sequence table SEQ ID NO: 1.
2. The immunomodulatory peptide Ha-CATH of claim 1, wherein the amino acid sequence encoded by the immunomodulatory peptide Ha-CATH is as set forth in SEQ ID NO: 2.
3. Use of the immunomodulatory peptide Ha-CATH of claim 1 or 2, for the preparation of a medicament for promoting skin wound repair.
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