CN112125968A - Shibata skin antioxidant peptide antioxidant-NV, and gene and application thereof - Google Patents

Abstract

The invention relates to a skin antioxidant peptide antioxidant-NV of rana obovata (Nanora ventrriputa), a gene and application thereof, belonging to the field of biomedicine. The skin antioxidant peptide antioxidant-NV is a polypeptide encoded by a defense peptide gene of a specific species of the China amphibian rana obovata, the molecular weight is 1963.7 daltons, the isoelectric point is 9.31, and the amino acid sequence is shown in SEQ ID NO. 1. The gene for coding the antioxidant peptide antioxidant-NV precursor of the skin of the rana obovata consists of 299 nucleotide sequences, and the nucleotide sequence is shown in SEQ ID NO. 2; wherein the 156-th 216 th nucleotide is a coding gene of mature bonobo peelii frog skin antioxidant peptide antioxidant-NV. Application of the Japanese frog skin antioxidant peptide antioxidant-NV in preparing medicine for resisting skin photoaging is provided. Has the advantages that: has strong skin photoaging resistance effect on skin, and can remarkably inhibit the occurrence of skin photoaging.

Description

Shibata skin antioxidant peptide antioxidant-NV, and gene and application thereof

Technical Field

The invention relates to a skin antioxidant peptide antioxidant-NV of rana japonica (rana ventricuta), a gene and application thereof, belonging to the field of biomedicine. .

Background

Skin photoaging (Skin photoaging) refers to the characteristic changes in Skin structure and function resulting from repeated exposure of the Skin to ultraviolet light, characterized by premature aging of the Skin, multiple wrinkles, rough leathery appearance, pigmentation, telangiectasia, associated precancerous lesions and malignancies. Because photoaging not only seriously affects the appearance of the skin of a patient, greatly reduces the quality of life of the patient, but also induces skin malignant tumors (such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and the like) [1], in recent years, because of the damage of the atmospheric ozone layer, the ultraviolet rays radiated to the earth surface are gradually increased, the incidence rate of the skin photoaging caused by the ultraviolet rays is sharply increased, in 2015, the huge medical expenses for treating the serious skin photoaging and the skin cancer caused by the serious skin photoaging all year round are over 291 billion dollars, wherein the U.S. is as high as 114 billion dollar [1], so how to effectively prevent and treat the skin photoaging caused by the ultraviolet rays, and a novel high-efficiency antioxidant drug which is safe, low in toxicity and low in cost is found to be urgently solved in the prevention and treatment process of.

Amphibians are the most important resource in the discovery of bioactive polypeptide resources, have exposed skins, and can secrete various bioactive polypeptides with novel molecular structures and complex and diverse functions in order to resist various severe environments in the environment. The active polypeptides are widely involved in various physiological activities of organisms, and have various pharmacological activities, such as antimicrobial, anti-tumor, antioxidant, immunoregulation, wound repair, analgesia and the like. At present, the screening of some pharmacological active monomer compounds from the skin of amphibians is the hotspot of the new drug invention. According to the literature reports at home and abroad, different active polypeptides have been separated from various biological sources, and some active polypeptides enter the clinical stage. The living environment of the rana obovata is extremely harsh compared with other frogs, and the rana obovata has long-lasting sunlight irradiation, strong ultraviolet radiation and climatic characteristics of cold plateau, low pressure and oxygen deficiency, dryness and strong wind. The specific harsh ecological environment makes the internal and external stressors and external microbial attacks on the amphibians different. The special high-altitude strong ultraviolet environment necessarily endows the species with special substances for adapting to the severe environment. The rana ventricosa (Nanora ventrriparia), which is an amphibian of the genus Shibata of the order Anophiala, is a species specific to China, is a species specific to Qinghai-Tibetan plateau, and has no report on the effect of antioxidant peptides in skin secretions to resist skin photoaging.

Disclosure of Invention

The invention aims to provide a novel rana japonica (rana ventriculriputa) skin antioxidant peptide antioxidant-NV with a strong skin photoaging resistance effect, and a gene and application thereof.

The skin antioxidant peptide antioxidant-NV of the invention is a polypeptide encoded by a defense peptide gene of a specific species of the amphibian China squab frog, the molecular weight is 1963.7 daltons, the isoelectric point is 9.31, and the full sequence primary structure (amino acid sequence SEQ ID NO:1) of the polypeptide is as follows:

the coding gene of the skin antioxidant peptide precursor consists of 299 nucleotides (SEQ ID NO:2), and the sequence from the 5 'end to the 3' end is as follows:

wherein, the 156-th 216-th nucleotide is a coding gene of mature skin antioxidant peptide antioxidant-NV.

The invention relates to application of skin antioxidant peptide antioxidant-NV in preparing a medicament for treating skin photoaging.

The invention has the beneficial effects that: the Japanese frog skin antioxidant peptide antioxidant-NV has obvious skin photoaging resisting effect, can inhibit skin photoaging obviously and may be used in preparing medicine for treating skin photoaging.

Drawings

FIG. 1 shows the use of a reversed phase high pressure liquid phase for the separation of purified Japanese frog skin exudate, arrows indicate components with anti-skin photoaging properties.

FIG. 2 shows the further purification of the fraction using a reverse phase high pressure liquid phase, and the arrow indicates the purified skin antioxidant peptide, antioxidant-NV.

FIG. 3 shows that the oxidation resisting peptide antioxidant-NV of the skin of the Japanese butterflybush has extremely strong skin photoaging resistance.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Preparation and amino acid sequence determination of anti-oxidation peptide (antioxidant-NV) of Japanese butterflies

1. Collecting skin exudate of squash

The live frogs are cleaned by water and placed in a cylindrical sample bottle with the diameter of 10 cm and the height of 10 cm. After 2 minutes of ether stimulation, the frothy liquid was rinsed from the back of the frog with 10ml of 0.1M Phosphate Buffered Saline (PBS), pH 6.0. The secretion was centrifuged at 12000rpm at 4 ℃ for 15 minutes, and the supernatant was stored at-80 ℃.

2. Separation and purification of antioxidant peptide antioxidant-NV of Japanese butterflybush skin

The collected supernatant is used as a raw material, the antioxidant peptide antioxidant-NV of the skin of the squash is purified according to the following procedures, and each separated and purified component is subjected to antioxidant and anti-skin photoaging activity detection, and the specific method is as follows:

(a) using Millipore corporation of America

Separating the supernatant with ultrafilter tubeSubstances with a molecular weight greater than 10kDa are removed. Adding the supernatant

The first centrifugation (3000 Xg, 40 min) was performed in an ultrafiltration tube (10kDa, 15ml), the filtrate was collected, the second centrifugation (3000 Xg, 10 min) was performed, the filtrate was collected, the third centrifugation (3000 Xg, 5 min) was performed, and the remaining filtrate was collected and stored at-20 ℃.

(b) The filtrate is subjected to reversed phase high pressure liquid phase (RP-HPLC) C₁₈Purifying with chromatographic column, which is performed in 2795 quaternary gradient high performance liquid chromatograph of Waters corporation, U.S. at a flow rate of 0.7ml/min with linear gradient of 0-80% acetonitrile, collecting each eluted fraction as shown in FIG. 1, and determining antioxidant and anti-skin photoaging activities. Lyophilizing the fraction with antioxidant and skin photoaging resisting activity shown by arrow in figure 1, and further using RP-HPLC C₁₈Further purifying the chromatographic column to obtain the purified oxidation resisting peptide antioxidant-NV of the Japanese butterflybush skin as shown by an arrow in figure 2.

3. Amino acid determination of antioxidant peptide antioxidant-NV of skin of populus ventricosum

Purified Japanese butterflybush skin antioxidant peptide antioxidant-NV was sequenced by Edman degradation method using Proscise of ABI, USA^TM491 in a protein sequencer. Sequencing results show that the full-sequence primary structure of the antioxidant peptide of the skin of the squaliobarum kurosou is as follows: GWANTLKNVAGGLCKMTGAA are provided.

Cloning of anti-oxidant peptide antioxidant-NV gene of Japanese squash skin

1. Total RNA extraction from skin of Shibataea kumasiana

The living rana obovata was washed with water, put into liquid nitrogen to be quickly frozen for 10 hours, and 300mg of skin tissue was taken, and 3ml of Trizol solution was added to the skin tissue, and homogenized in a 20ml glass homogenizer for 30 minutes. Adding equal volume of phenol/chloroform solution, mixing vigorously, standing at room temperature for 10 min, centrifuging at 12000rpm at 4 deg.C for 10 min, and removing precipitate. Adding isopropanol with the same volume into the supernatant, standing at room temperature for 10 minutes, centrifuging at 12000rpm for 10 minutes at 4 ℃, washing the precipitate with 75% ethanol once, and drying in the air to obtain the tube bottom precipitate, namely the total RNA of the skin of the Shibataea kumasasa.

2. Purification of Japanese butterhood skin mRNA

mRNA isolation and purification Using Promega, USA

mRNA Isolation Systems kit.

The extracted total RNA from the skin of the Shibataea kumasasa is dissolved in 500. mu.l of DEPC water, placed in a water bath at 65 ℃ for 10 minutes, added with 3. mu.l of oligo (dT) probe and 13. mu.l of 20 XSSC solution, mixed well, and left to cool at room temperature, called solution A. The magnetic beads (SA-PMP) were flicked and mixed until 30 seconds of magnetic shelf adsorption, the supernatant was discarded, 0.5 XSSC 0.3m1 was added until 30 seconds of magnetic shelf adsorption, and 0.1ml of 0.5 XSSC was added for suspension, called solution B. Adding the solution A into the solution B, standing at room temperature for 10 minutes until the solution is adsorbed by a magnetic rack for 30 seconds, abandoning the supernatant, washing with 0.1 XSSC for 4 times, abandoning the supernatant finally, adding 0.1ml of DEPC water for suspension, adsorbing by the magnetic rack for 30 seconds, transferring the supernatant to a new test tube, adding 0.15m of 1 DEPC water for resuspension until the solution is adsorbed by the magnetic rack for 30 seconds, and transferring the supernatant to the test tube, thus obtaining the purified mRNA of the skin of the Japanese frog. 1/10 volumes of 3M sodium acetate, pH5.2, equal volume of isopropanol were added, left at-70 ℃ for 30 minutes, centrifuged at 12000rpm for 10 minutes at 4 ℃, the supernatant discarded and the pellet dissolved in 10. mu.l of DEPC water.

3. Construction of a Japanese butterhood skin cDNA library

The Creator of CLONTECH is adopted^TM SMART ^TMcDNA Library Construction Kit of cDNA Library restriction Kit.

(a) First Strand cDNA Synthesis (reverse transcription of mRNA)

Mu.l of Japanese butterflybush skin mRNA, 1. mu.l of SMART IV oligonucleotide, 1. mu.l of CDS III/3' PCR primer, and 2. mu.l of deionized water were added to a 0.5ml sterile centrifuge tube to make the total volume 5. mu.l. Mix the reagents in the centrifuge tubes and centrifuge at 12000rpm for 15 seconds and incubate at 72 ℃ for 2 minutes. The centrifuge tubes were incubated on ice for 2 minutes. The following reagents 2.0. mu.l of 5 Xprimary strand buffer, 1.0. mu.l of 20mM dithiothreitol, 1.0. mu.l of 10mM dNTP mix, and 1.0. mu.l of PowerScript reverse transcriptase were added to the centrifuge tube. The reagents were mixed in the centrifuge tubes and centrifuged at 12000rpm for 15 seconds and incubated at 42 ℃ for 1 hour. The first strand synthesis was stopped by placing the centrifuge tube on ice. Mu.l of the first strand of the synthesized cDNA was taken from the centrifuge tube and used.

(b) Amplification of the second Strand Using Long-terminal polymerase chain reaction (LD-PCR)

The PCR instrument was preheated to 95 ℃ and reacted with 2. mu.l of the first strand of cDNA (reverse transcription of mRNA), 80. mu.l of deionized water, 10. mu.l of 10 × Advantage 2PCR buffer, 2. mu.l of 50 × dNTP mix, 2. mu.l of 5 'PCR primers, 2. mu.l of CDS III/3' PCR primers, and 2. mu.l of E.coli polymerase centrifuge tubes. Amplification was performed in a PCR instrument according to the following procedure: at 95 ℃ for 20 seconds; 22 cycles (95 ℃ C., 5 seconds; 68 ℃ C., 6 minutes). After completion of the circulation, the double strand cDNA synthesized in the centrifuge tube was recovered.

(c) PCR product recovery

By PROMEGA

The SV Gel and PCR Clean-Up System kit is extracted and recovered, and the steps are as follows:

adding cDNA double chains obtained by PCR into equal-volume membrane binding buffer, reversing and uniformly mixing, transferring the mixed solution into a centrifugal purification column, and standing at room temperature for 5 minutes to ensure that the DNA is fully bound with a silica gel membrane. Centrifuge at 12000rpm for 30 seconds and discard the waste liquid from the collection tube. Add 700. mu.l of eluent (containing ethanol) to the centrifugation and purification column, centrifuge at 12000rpm for 30 seconds, and discard the waste liquid from the collection tube. And (5) repeating the step (2). Centrifuge at 12000rpm for 5 minutes, and place the centrifugal purification column in a new centrifuge tube. 30. mu.l of ultrapure water was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes. Centrifuging at 12000rpm for 30 seconds, and obtaining the purified cDNA double strand at the bottom of the tube.

(d) Preparation of E.coli DH5 alpha competent cells

A single DH5 alpha colony was picked and inoculated into 3ml Luria-Bertani (LB) medium without ampicillin, cultured overnight at 37 ℃, and the bacterial solution was re-inoculated into 50ml LB medium the next day at a ratio of 1:100 and shaken at 37 ℃ for 2 hours. When OD is reached₆₀₀When the value reached 0.35, the bacterial culture was harvested. Transfer of bacteria to a sterile, single-use, ice-pre-cooled 50m1 polypropyleneThe tubes were placed on ice for 10 minutes and the cultures were cooled to 0 ℃. The cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The broth was decanted and the tube inverted lmin to allow the last traces of broth to drain. 30ml of precooled 0.1mol/LCaCl per 50ml of initial broth₂-MgCl₂Solution (80mmol/L MgCl)₂,20mmol/L CaCl₂) Resuspend each cell pellet. The cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The medium was decanted and the tube inverted for l minutes to allow the last traces of medium to drain. 0.1mol/L CaCl precooled with ice at 2m1 per 50m1 primary culture₂Resuspend each cell pellet and dispense for use.

(e) Enzyme cleavage, ligation and transformation of ligation products

Mu.l of Takara pMD18-T vector and 4. mu.l of Shibataea kumasasa cDNA double-stranded solution were added to a microcentrifuge tube, and the total amount was 5. mu.l. Mu.l (equal amount) of ligase buffer mixture was added. The reaction was carried out at 16 ℃ for 2 hours. A total of 10. mu.l was added to 100. mu.l of DH 5. alpha. competent cells and left on ice for 30 minutes. After heating at 42 ℃ for 90 seconds, the mixture was left on ice for 1 minute. Then, 890. mu.l of LB medium incubated at 37 ℃ was added thereto, and the mixture was incubated at 37 ℃ for 60 minutes with slow shaking. 200. mu.l of the suspension was spread on LB medium containing X-Gal, IPTG and Amp and cultured at 37 ℃ for 16 hours to form a single colony. Colonies were washed with 5ml LB liquid medium per LB plate and frozen with 30% glycerol. The constructed cDNA contained approximately 1X 10⁶Individual clones.

4. Cloning and screening of antioxidant peptide gene of skin of squash

The sequence of the amplification primer is 5 'AAGCAGTGGTATCAACGCAGAGT 3', and the other amplification primer of the PCR is SMART (CLONTECH Co., Ltd.)^TMThe 3 ' PCR Primer in the cDNA Library Construction Kit has the sequence of 5 ' ATTCTAGAGGCCGAGGCGGCCGACATG 3 '. The PCR reaction was performed under the following conditions: 30 seconds at 94 ℃; 45 seconds at 52 ℃; at 72 ℃ for 2 min 30 s for 35 cycles. The constructed bacterial cDNA library was first titrated, then diluted to the appropriate bacterial concentration with LB medium containing 100. mu.g/ml ampicillin (approximately 5000 bacteria/ml, and 30 bacteria/ml for the first round of selection and the second round of selection, respectively), and plated in an 8X 8 matrix (64 wells total, 10 per well) on a 96-well plate0. mu.l), incubated overnight at 37 ℃. And respectively combining the bacterial culture solutions according to rows and columns, carrying out PCR identification on 10 samples, and carrying out second round screening on the bacteria samples with the crossed positive holes.

5. Determination of antioxidant peptide gene sequence of skin of squash

Extracting plasmid DNA and determining nucleotide sequence by dideoxy method, using an apparatus of U.S. Applied Biosystems373A full-automatic nucleotide sequence determination apparatus, sequencing primer BcaBEST^TMSequencing Primer RV-M and BcaBEST^TM Sequencing Primer M13-47，BcaBEST^TMSequence 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 coding the precursor of the antioxidant peptide of the skin of the Japanese butterflybush is composed of 299 nucleotides (SEQ ID NO:2), and the sequence from the 5 'end to the 3' end is as follows:

wherein the 156 th-216 th nucleotide is a coding gene of mature bonobo shibata skin antioxidant peptide antioxidant-NV.

Application of anti-oxidation peptide (antioxidant-NV) of Japanese butterflybush skin in preparation of anti-skin photoaging treatment medicine

Hairless mice were randomly divided into a normal control group (no ultraviolet UVB irradiation), a UVB model group (ultraviolet UVB irradiation with PBS given simultaneously), an antioxidant-NV group (UVB irradiation + antioxidant-NV group, ultraviolet UVB irradiation with antioxidant-NV given simultaneously), and a positive drug control group (UVB irradiation + vitamin C group, ultraviolet UVB irradiation with vitamin C given simultaneously). Coating PBS solution, antioxidant-NV group and positive drug control group on the back of hairless mice in a normal control group and a UVB model group, respectively coating 40 mul of skin antioxidant peptide antioxidant-NV and vitamin C (the concentration is 200 mug/ml) dissolved in the PBS solution on the back of the hairless mice, then placing the hairless mice at a position 50cm under a UVB light source, irradiating the other groups once a day for the first week except the normal control group for 5 times, wherein the irradiation dose of UVB is 100 every timemJ/cm²After 2 weeks, the other groups except the normal control group were irradiated with UVB once every other day at a dose of 200mJ/cm²For a total of 12 weeks.

The results are shown in FIG. 3: after 12 weeks of UVB irradiation, the model group hairless mice were visually observed for the irradiated area of their backs, and a typical skin photoaging phenomenon was observed: the appearance of the leather is changed by progressive elasticity loss, the wrinkles become thick and deep, and the appearance of the leather is accompanied with photoaging such as desquamation; the skin color of the normal group is normal and has good elasticity, the change of the skin photoaging after the skin antioxidant peptide antioxidant-NV is given is obviously lighter than that of the model group, and the photoaging resistant effect of the skin photoaging is even stronger than that of the vitamin C group. Further histologically observing the pathological changes of the skin of the hairless mouse photoaging model, obviously thickening the epidermis of the model group, and eliminating the epicondyle and dermal papilla; dermal fibrosis, disorganization, and uneven or sparse distribution. The normal group of epidermal cells are regularly arranged, the dermis layer can see wavy fibrous tissues, the arrangement is orderly, the distribution is uniform, the density is dense, and the cell component quantity is moderate. After the skin antioxidant peptide antioxidant-NV is given, the epidermis thickness and collagen fiber are denatured, and the damage degree is lighter than that of a model group and a vitamin C group.

The experimental results show that: the skin antioxidant peptide antioxidant-NV has stronger skin photoaging resistance effect, and the skin photoaging resistance effect is stronger than that of vitamin C (detailed shown in figure 3).

SEQUENCE LISTING

<110> university of Kunming medical science

<120> Japanese butterfrog skin antioxidant peptide antioxidant-NV, gene and application thereof

<130> 2020-6-29

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> PRT

<213> Nanorana N. ventripunctata

<400> 1

Gly Trp Ala Asn Thr Leu Lys Asn Val Ala Gly Gly Leu Cys Lys Met

1 5 10 15

Thr Gly Ala Ala

20

<210> 2

<211> 299

<212> DNA

<213> Nanorana N. ventripunctata

<400> 2

atgttcacct tgaagaagtc cctgttcctg gttttctttc tggggatggt ctccttatct 60

ctctgcaggt ctgagagcca cgcccatgaa gagtccagca ctgatcccac agaggaggaa 120

aatgcagccg aaaatgagga aagcgtagag aaaagaggct gggccaatac actaaagaac 180

gttgctggtg gattgtgtaa aatgactggg gctgcttgat tgcgaattgg aatcctaaac 240

agatgtctaa taaaacagca aaattaattc aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 299

Claims (3)

1. The bonobo frog skin antioxidant peptide antioxidant-NV is characterized in that the skin antioxidant peptide is a polypeptide encoded by a bonobo frog defense peptide gene of a species specific to the amphibian China, the molecular weight is 1963.7 daltons, the isoelectric point is 9.31, and the amino acid sequence is shown in SEQ ID NO. 1.

2. The gene for coding the precursor of the antioxidant peptide antioxidant-NV of the skin of the Japanese frog is characterized in that the precursor gene consists of 299 nucleotides, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2; wherein the 156-th 216 th nucleotide is a coding gene of mature rana japonica skin antioxidant peptide antioxidant-NV.

3. Use of the rana obo skin antioxidant peptide antioxidant-NV as claimed in claim 1 in the preparation of a medicament for treating skin photoaging.

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