CN106478811B - Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy - Google Patents

Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy Download PDF

Info

Publication number
CN106478811B
CN106478811B CN201610915371.1A CN201610915371A CN106478811B CN 106478811 B CN106478811 B CN 106478811B CN 201610915371 A CN201610915371 A CN 201610915371A CN 106478811 B CN106478811 B CN 106478811B
Authority
CN
China
Prior art keywords
rana
inhibitory peptide
protease
protease inhibitory
rugulosa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610915371.1A
Other languages
Chinese (zh)
Other versions
CN106478811A (en
Inventor
徐学清
陈新
曾白霜
张贝
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southern Medical University
Original Assignee
Southern Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southern Medical University filed Critical Southern Medical University
Priority to CN201610915371.1A priority Critical patent/CN106478811B/en
Publication of CN106478811A publication Critical patent/CN106478811A/en
Application granted granted Critical
Publication of CN106478811B publication Critical patent/CN106478811B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Birds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to an active polypeptide, a gene thereof and application thereof in pharmacy, wherein the rana rugulosa protease inhibitory peptide is cyclic peptide consisting of 33 amino acids, the molecular weight is 3768.33 daltons, the isoelectric point is 6.303, the amino acid sequence is SEQ ID NO.1, and the third cysteine and the eighth cysteine of the polypeptide form an intramolecular disulfide bond. The gene sequence of the rana nigromaculata protease inhibitory peptide consists of SEQ ID NO.4, wherein the mature rana nigromaculata protease inhibitory peptide with the coding function is 379-477 nucleotide. The rana rugulosa protease inhibitory peptide has the characteristics of simple structure, convenient artificial synthesis, wide antibacterial spectrum and strong activity, and can be used for preparing medicaments for treating pathogenic microorganism infectious diseases and beautifying and caring skin medicaments.

Description

Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy
Technical Field
The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissues.
Background
The most essential function of serine protease inhibitors (serpins) is to prevent proteolysis and to regulate the hydrolytic equilibrium of serine proteases. By regulating serine proteases, serine protease inhibitors have important effects on physiological and biochemical functions in organisms. For example, they play an important role in blood coagulation, complement formation, fibrinolysis, protein folding, cell migration, cell differentiation, cell matrix reconstitution, hormone formation and transport, intracellular proteolysis, blood pressure regulation, tumor suppression, and the development of viral or parasitic pathogenicity. Serine protease inhibitors regulate such numerous physiological processes that they have wide clinical application values, e.g., aprotinin is widely used clinically in the treatment of gastritis, pancreatitis, etc., and also in chest surgery for anti-fibrinolysis, inhibition of contact activation, anti-inflammation, etc. (Br J anasth.2013, 110(5): 675-8). Serine protease analogs such as Kallikrein and tryptase play an important role in the development of inflammatory diseases such as rheumatoid arthritis and rhinitis, conjunctivitis, asthma, gastroenteritis, and cardiovascular inflammation (Biol chem.2004,385(11): 989-96). Therefore, serine protease inhibitors have become hot spots in international research, and the research and development thereof imply great preparation value of clinical therapeutic drugs.
Amphibians have been the source of traditional pharmaceuticals. Amphibian animals such as Bufo gargarizans, Bombina maxima, Rana nigromaculata, and Rana nigromaculata (Euphlyctis limnocharis) are widely used as traditional Chinese medicinal materials in China. Modern research shows that: the skin and internal organs of these amphibians have a wide range of pharmacological activities, such as broad-spectrum antibacterial action, antitumor, local anesthesia, analgesia, immunomodulation, action on the cardiovascular system, etc. (Dongwuxue Yanjiu,2015,36(4): 183-222). The complexity of the traditional Chinese medicine components and the limitation of the processing method thereof cause that the active ingredients of the medicine can not play a better role, and the search of specific active monomer compounds from the traditional medicines is one of the important contents of the modernization of the traditional Chinese medicine. In foreign countries, the search for specific pharmacologically active monomeric compounds of amphibious skin has become a hot spot of new drug invention. A number of protease inhibitor polypeptides with a molecular weight of less than 10kDa have been identified from amphibian skin. These protease inhibitors of amphibian skin are functional in combating microbial infections in addition to their role in the processing and degradation of precursor proteins, and thus many of the protease inhibitors from amphibians have both antibacterial and protease inhibitor activity, including ranacyclin-B, KPHTI, HV-BBI, HJTI, hyllaserpin S2, OGTI, PYR, PSKP-1, PSKP-2, BOTI, BVTI, BMTI, BPTI, pLR and BSTI, etc. (Chem Rev. 2015176115 (4): 0-1846).
In addition, oxidative stress is considered to be associated with various diseases such as senile dementia, Parkinson's disease, which are caused by diabetes, complications caused by diabetes, rheumatoid arthritis and amyotrophic lateral sclerosis (Food Chemistry,2008,107, 1485-.
China has a long history of application of amphibian drugs, but researches on active ingredients and pharmacological properties of the amphibian drugs mainly focus on organic small molecules such as alkaloid and the like, and few researches on skin active peptide substances are carried out. Frog (Huwen)Hoplobatrachus_ rugulosus) Is one of the important economic frogs which are artificially fed, and the national level II focuses on protecting animals. At present, few studies on pharmacologically active substances for the skin are made.
Since the 21 st century, the rapid development of genomics and the rise of synthetic biology have greatly promoted the research on the structure and function of natural products; the invention is obtained by using a transcriptomics method and pharmacological researchGiant knotweed frog protease inhibiting peptideThe complete sequence amino acid structure is searched and compared by a protein database, and no identical polypeptide is found. The inventor will inventGiant knotweed frog protease inhibiting peptideThe coding genes are searched and compared by a gene database, and any identical gene is not found.
Disclosure of Invention
The invention aims to provide a rana rugulosa protease inhibiting peptide with broad-spectrum antimicrobial (including gram negative and gram positive bacteria, fungi and viruses), antioxidant and pancreatin inhibitor activities, a gene thereof and application thereof in preparing medicaments for treating pathogenic microorganism infectious diseases and beautifying and skin-care medicaments.
In order to solve the technical problems, the invention adopts the technical scheme that:
one aspect of the invention provides a rana rugulosa protease inhibitory peptide, wherein the sequence of the polypeptide is shown as SEQ ID No. 1.
KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ SEQ ID NO.1
The invention also provides a rana rugulosa protease inhibitory peptide, which is characterized by consisting of 33 amino acids, having a molecular weight of 3768.33 daltons, an isoelectric point of 6.303 and an amino acid sequence as follows: lys Glu Cys Lys Asp Tyr Tyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr SerHis Ile Asp Ser Leu Asp Leu Gly Leu Ser His Glu Ala Gln Gln (KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ) (SEQ ID NO.1), the third cysteine and the eighth cysteine of which form an intramolecular disulfide bond.
In another aspect, the present invention provides a nucleotide sequence of a rana gherai protease inhibitory peptide gene, which is characterized in that: the cDNA consists of 480 nucleotides, and the sequence from the 5 'end to the 3' end of the cDNA is shown in SEQ ID NO. 4.
Figure BDA0001135186770000031
The 379-477 th nucleotide in the sequence codes the mature rana rugulosa protease inhibiting peptide with functions.
In yet another aspect, the present invention provides a nucleotide encoding the rana rugulosa protease inhibitory peptide of claim 1.
In another aspect, the invention provides application of the rana rugulosa protease inhibitory peptide in preparation of medicines for treating pathogenic microorganism infection diseases and beautifying and skin-care medicines.
In another aspect, the invention provides the application of the rana rugulosa protease inhibitory peptide in preparing free radical scavenging medicaments or cosmetic products.
In another aspect, the invention provides application of the rana rugulosa protease inhibitory peptide in preparation of an antioxidant drug or a cosmetic product.
In a further aspect of the invention, the rana nigromaculata protease inhibitory peptide is used for preparing a medicine for preventing or treating gastritis or pancreatitis, preferably, the gastritis or the pancreatitis is inflammation-related gastritis or inflammation-related pancreatitis.
In a further aspect the invention provides the use of the aforementioned rana gherai protease inhibitory peptides in inhibiting bacteria for non-diagnostic or therapeutic purposes, preferably the bacteria are escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and candida albicans.
In a further aspect, the invention provides the use of the rana nigromaculata protease inhibitory peptide in the preparation of a medicament or a cosmetic product for inhibiting bacteria, preferably escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and candida albicans.
The invention has the beneficial effects that:
the amino acid structure of the rana rugulosa protease inhibitory peptide is deduced from the rana rugulosa protease inhibitory peptide coding gene, and the synthesized rana rugulosa protease inhibitory peptide has obvious effects of inhibiting the growth of bacteria and fungi, inhibiting the activity of pancreatin and resisting oxidation. The rana rugulosa protease inhibitory peptide has the beneficial characteristics of simple structure, convenience in artificial synthesis, wide antibacterial spectrum and strong activity.
Drawings
FIG. 1 shows HPLC purification identification results of rana rugulosa protease inhibitory peptide of the present invention;
FIG. 2 shows the results of mass spectrometric identification of rana rugulosa protease inhibitory peptides of the present invention;
FIG. 3 shows the results of the activity of the peptide serine protease inhibitor of rana rugulosa protease inhibitor of the present invention;
FIG. 4 is a relation curve of the amount-effect of the rana rugulosa protease inhibitory peptide for eliminating DPPH and ABTS free radicals.
Detailed Description
The invention is described in further detail below with reference to the following figures and detailed description:
the rana rugulosa protease inhibitory peptide is a polypeptide consisting of 33 amino acids, has the molecular weight of 3768.33 daltons, the isoelectric point of 6.303 and the amino acid sequence of: lys Glu Cys Lys Asp TyrTyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp Ser Leu Asp LeuGly Leu Ser His Glu Ala Gln Gln (KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ) (SEQ ID NO.1), the third cysteine and the eighth cysteine of which form an intramolecular disulfide bond to form a loop.
The 379-477 nucleotide code of the rana nigromaculata protease inhibitory peptide gene sequence SEQ ID NO. 4. The preparation process of the rana rugulosa protease inhibitory peptide and the gene thereof comprises the following steps:
example 1 cloning of a tiger frog protease inhibitory peptide gene:
I. extracting total RNA of rana rugulosa skin: cleaning living Rana rugulosa with water, placing into liquid nitrogen, quickly freezing for 4h, collecting skin tissue, weighing, collecting 300mg skin tissue, adding 10m1 total RNA extraction buffer (Trizol solution, product of GIBCOBRL company, USA), and homogenizing for 30min in 20m1 glass homogenizer. Adding equal volume of phenol/chloroform solution, mixing vigorously, standing at room temperature for 10min, centrifuging at 4 deg.C and 12000rpm for 10min, and removing precipitate. Adding isopropanol with the same volume into the supernatant, standing at room temperature for 10min, centrifuging at 12000rpm for 10min at 4 ℃, washing the precipitate with 75% ethanol once, and air drying to obtain the precipitate at the bottom of the tube, namely the total RNA of the rana rugulosa skin.
II. And (3) purifying the giant frog skin mRNA: the mRNA of the skin of tiger frog is separated and purified by PROMEGA of America
Figure BDA0001135186770000041
mRNA Isolation Systems kit. The method comprises the following specific steps: dissolving 500 μ g of total RNA of Rana rugulosa skin in 500 μ l of DEPC water, placing in 65 deg.C water bath for 10min, adding 3 μ l of Oligo (dT) probe and 13 μ l of 20 XSSC solution, mixing, standing at room temperature, and cooling to obtain solution A. Mixing the magnetic beads, adsorbing with magnetic frame for 30S, discarding supernatant, adding 0.5 XSSC 0.3m1, adsorbing with magnetic frame for 30S, and adding 0.1ml 0.5 XSSC to suspend, and named as solution B. Adding solution A into solution B, standing at room temperature for 10min, adsorbing with magnetic frame for 30sec, discarding supernatant, washing with 0.1 XSSC for 4 times, discarding supernatant, adding 0.L DEPC water, suspending, adsorbing with magnetic frame for 30sec, transferring supernatant to new test tube, adding 0.15m1DEPC waterResuspending, adsorbing with magnetic frame for 30S, transferring supernatant to the test tube, and collecting the supernatant as purified skin mRNA of Rana rugulosa. 1/10 volumes of 3M sodium acetate, pH5.2 and an equal volume of isopropanol were added, the mixture was left at-70 ℃ for 30 minutes, centrifuged at 12000rpm for 10 minutes at 4 ℃, the supernatant was discarded, and the precipitate was dissolved in 10. mu.l of DEPC water to obtain the skin mRNA of Rana nigromaculata.
III, constructing a skin cDNA library of the tiger frog: a Kit was constructed using the plasmid cDNA library of the CreatorTM SMART TM cDNAlibrary Construction Kit of CLONTECH.
First strand cDNA Synthesis (reverse transcription of mRNA): mu.l of Rana rugulosa 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 15sec, and incubate at 72 ℃ for 2 min. Centrifuge tubes were incubated on ice for 2 min. 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. Mix the reagents in the centrifuge tubes and centrifuge at 12000rpm for 15sec, incubate at 42 ℃ for 1 h. 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. Amplifying the second strand by using a long-terminal polymerase chain reaction (LD-PCR) method: the PCR instrument was preheated to 95 ℃. Mu.l of the first strand of the cDNA (reverse transcription of mRNA), 80. mu.l of deionized water, 10. mu.l of 10 × Advantage 2PCR buffer, 2. mu.l of a 50 × dNTP mix, 2. mu.l of a 5 'PCR primer, 2. mu.l of a CDS III/3' PCR primer, and 2. mu.l of an E.coli polymerase centrifuge tube were reacted. Amplification was performed in a PCR instrument according to the following procedure: 20sec at 95 ℃, 5sec at 95 ℃, 6min at 68 ℃ and 22 cycles. After the circulation was completed, the double strand cDNA synthesized in the centrifuge tube was extracted.
PCR products from PROMEGA
Figure BDA0001135186770000051
The SV Gel and PCR Clean-Up System kit is extracted and recovered, and the steps are as follows: adding cDNA double chain obtained by PCR into equal volume of membrane combined buffer, reversing and mixing, transferring the mixture into a centrifugal purification column, standing at room temperature for 5minAnd (5) fully combining the DNA with the silica gel membrane. The collection tube was centrifuged at 12000rpm for 30sec to discard the waste liquid. Add 700. mu.l of eluent (containing ethanol) to the centrifugation and purification column, centrifuge at 12000rpm for 30sec, and discard the waste liquid from the collection tube. And repeating the steps. Centrifuge at 12000rpm for 5 min. The centrifugal purification column was placed 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 min. Centrifuging at 12000rpm for 30sec, and obtaining the purified cDNA double strand in the bottom of the tube.
D. Adding 1 mul Takara pMD18-T vector and 4 mul tiger frog cDNA double-chain solution into a microcentrifuge tube, adding 5 mul ligase buffer mixture, reacting at 16 ℃ for 2h, adding 10 mul into 100 mul DH5 α competent cells, placing in ice for 30min, heating at 42 ℃ for 90Sec, placing in ice for 1 min, adding LB culture medium 890 mul incubated at 37 ℃, slowly shaking for culturing at 37 ℃ for 60min, taking 200 mul LB culture medium coated on X-Gal, IPTG and Amp, culturing at 37 ℃ for 16h to form single colonies, washing colonies with 5m1LB liquid culture medium on each dish, adding 30% glycerol, freezing, and storing the constructed cDNA about 1 × 106Individual clones.
IV, cloning and screening the rana rugulosa protease inhibitory peptide gene: the amplification primer is 25 nucleotides in length and has a sequence of 5 'ATGAAGGTCTGGCAGTGCGCGCTC 3' (SEQ ID NO.2), and another amplification primer in PCR is SMART (CLONTECH Co., Ltd.)TM3 ' PCR Primer in cDNA Library Construction Kit, the sequence is 5 ' ATTCTAGAGGCCGAGGCGGCCGACATG 3 ' (SEQ ID NO. 3). The PCR reaction was performed under the following conditions: 30sec at 94 ℃, 45sec at 50 ℃ and 2.5min at 72 ℃ 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), plated in an 8X 8 matrix (64 wells in total, 100. mu.1 per well) on 96-well plates, and incubated overnight at 37 ℃. And respectively combining the bacterial culture solutions according to rows and columns, carrying out PCR identification on 16 samples, and carrying out second round screening on the bacteria samples with the crossed positive holes.
V, determining the gene sequence of the tiger frog protease inhibitory peptide and obtaining the results: plasmid DNA extraction nucleotide sequences were determined by dideoxy using an apparatus, US Applied Biosystems 373A full-automatic nucleotide sequencer, Sequencing primers were BcaBESTTM Sequencing Primer RV-M and BcaBESTTM Sequencing Primer M13-47, BcaBESTTM Sequencing Primer RV-M sequences: 5 'GAGCGGATAACAATTTCACACAGG 3' (SEQ ID NO.5), BcaBESTTM Sequencing Primer M13-47: 5 'CGCCAGGGTTTTCCCAGTCACGAC 3' (SEQ ID NO. 6). The sequence from 5 'end to 3' end of the gene sequencing result is (SEQ ID NO.4):
Figure BDA0001135186770000061
the sequence table of the tiger frog protease inhibitory peptide gene nucleotide is as follows: the sequence length is 480 bases; sequence types: a nucleic acid; number of chains: single-stranded; topology: straight-chain; the sequence types are as follows: cDNA; the source is as follows: skin of tiger frog.
The gene of the rana rugulosa protease inhibitory peptide is used for deducing that the mature active multifunctional protease inhibitory peptide with the function is 379-477 th nucleotide and has the amino acid sequence as follows: KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ (see sequence SEQID NO.1)
Example 2 preparation of rana rugulosa protease inhibitory peptides:
i, a preparation method of rana rugulosa protease inhibitory peptide: the amino acid sequence of the mature multifunctional protease inhibitory peptide with the coding function is deduced according to the gene of the rana gherai protease inhibitory peptide, and then the polypeptide is synthesized by an automatic polypeptide synthesizer. Desalting by HPLC reverse phase C18 column chromatography, and purifying. The formation of disulfide bonds was carried out by air oxidation, specifically by dissolving the polypeptide in 0.1mg/ml in 0.1% acetic acid solution in a flask, titrating to pH 7.8 with ammonium hydroxide, and stirring overnight at room temperature. Desalting by HPLC reverse phase C18 column chromatography, and purifying. The purification was carried out with a solution A of 0.05% TFA + 2% CH3CN, a solution B of 0.05% TFA + 90% CH3CN, a concentration gradient of 25-40% in 15min, a detection wavelength of 220nm, and the presence of the polypeptide at 9.248 min.
II, measuring the molecular weight by adopting a Fast atom bombardment mass spectrometry (FAB-MS), taking glycerol, m-nitrobenzyl alcohol, dimethyl sulfoxide (1:1: l, V: V: V, volume ratio) as a substrate, taking Cs + as a bombardment particle, and taking the current as 1 muA and the emission voltage as 25 Kv.
III, identifying the purity of the purified rana rugulosa protease inhibitory peptide by using a High Performance Liquid Chromatography (HPLC) method, determining an isoelectric point by using isoelectric focusing electrophoresis, and determining an amino acid sequence structure by using an automatic amino acid sequencer.
The tiger frog protease inhibitory peptide is a polypeptide encoded by China amphibian tiger frog protease inhibitory peptide gene, has the molecular weight of 3768.33 daltons, the isoelectric point of 6.303 and the amino acid sequence of: lys Glu Cys Lys Asp Tyr TyrCys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp Ser Leu Asp Leu GlyLeu Ser His Glu Ala Gln Gln (KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ) (SEQ ID NO.1), the third cysteine and the eighth cysteine of which form an intramolecular disulfide bond.
Example 3 Activity assay of Rana rugulosa protease inhibitory peptides
I, determination of antibacterial ability
The antibacterial activity is detected by adopting a cup-dish method, the bacterial culture adopts a common agar culture medium, and the fungal culture adopts a modified Sabouraud (Sabousand) culture medium. Respectively injecting 20ml of the heated and melted culture medium into the flat dishes to serve as a bottom layer, uniformly spreading the culture medium in the dish bottoms, solidifying, heating and melting another proper amount of the culture medium, respectively adding 5ml of bacterial suspension into each dish, and uniformly shaking to uniformly spread the culture medium on the bottom layer to serve as a bacterial layer. After cooling, 6 sterilized stainless steel cups were placed in a dish at equal distances. Adding 0.3mg/ml sample solution to be measured into the first steel cup, adding 0.l ml sample solution into the other steel cups by a two-fold dilution method, culturing at 37 ℃, and observing the size of the inhibition zone after 24-48 h. The inhibition zone l0mm or more is defined as the Minimum Inhibition Concentration (MIC). All the tested strains are from Guangdong microbiological research institute, the test is repeated for four times, the average value is taken, the result is shown in table 1, the rana nigromaculata protease inhibitory peptide has obvious inhibitory action on escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and candida albicans, and the rana nigromaculata protease inhibition has good broad-spectrum bacterial growth inhibition effect.
TABLE 1 antibacterial Activity of rana rugulosa protease inhibitory peptides
Figure BDA0001135186770000071
Figure BDA0001135186770000081
II, determination of serine protease inhibitor Activity
Varying amounts of rana gheraria protease inhibiting peptide were dissolved in 0.05M Tris-HCl buffer and a certain amount of trypsin (final concentration 40. mu.g/ml) in 0.05M Tris-HCl buffer at room temperature for 2min, and the reaction was initiated by addition of chromogenic substrate S-2238 (final concentration 40. mu.g/ml), followed by monitoring the change in absorbance for 2min using a spectrophotometer manufactured by PERKIN ELMER (USA), and addition of the same volume of 0.05M Tris-HCl buffer with a blank, and the inhibition constant Ki ═ I/(V0/VI + 1). [I] The molar concentration of the tiger frog protease inhibitory peptide is shown, V0 is blank control and is the reaction speed of trypsin and chromogenic substrate, and V1 is the reaction speed of trypsin and chromogenic substrate after the tiger frog protease inhibitory peptide is added. This experiment was repeated six times and the average was taken.
As shown in FIG. 3, the concentration of the peptide inhibiting sansevieria trifasciata protease required for inhibiting the activity of half of trypsin was 0.368. mu.M, and the inhibition constant Ki against trypsin was 3.2X 10-7M。
Serine protease inhibitors have important effects on physiological and biochemical functions in organisms. For example, they play an important role in blood coagulation, complement formation, fibrinolysis, protein folding, cell migration, cell differentiation, cell matrix reconstitution, hormone formation and transport, intracellular proteolysis, blood pressure regulation, tumor suppression, and the development of viral or parasitic pathogenicity. The rana rugulosa protease inhibiting peptide can effectively inhibit trypsin, and is proved to be capable of regulating a plurality of physiological processes in vivo and to have wide clinical application value. The pathogenesis of gastritis and pancreatitis involves the hydrolysis of many serine proteases, so inhibiting the hydrolysis of these enzymes can prevent the disease and reduce the subsequent damage of inflammatory factors to the body. The rana rugulosa protease inhibitory peptide with serine protease inhibitor activity can be applied to gastritis and pancreatitis related to inflammation.
III, determination of antioxidant Capacity
1) Determination of DPPH radical scavenging Capacity
Antioxidant polypeptides were studied using DPPH (1, 1-diphenylyl-2-picryl-hydrzyl) free radical scavenging assay. Preparing a DPPH ethanol solution with the concentration of 1 multiplied by 10 < -5 > mol/L, and storing the solution in a dark place. 2ml of 0.1mM solution of DPPH in absolute ethanol are added to a clean tube containing 2ml of the different enzymatic samples and mixed well. After standing at room temperature for 30min, absorbance at 517nm was measured, and the smaller the absorbance, the stronger the radical scavenging ability was.
Clearance (%) ═ 1- (a)i-Aj)/A0】*100％
In the formula, A02ml,0.1mM DPPH in absolute ethanol +2ml of sample reagent, blank, Ai2ml,0.1mM DPPH in absolute ethanol +2ml of sample, Aj2ml of absolute ethanol +2ml of sample.
2) Determination of ABTS free radical scavenging Activity
Dissolving ABTS with deionized water to make ABTS concentration reach 7mmol/L, adding potassium persulfate to make potassium persulfate concentration be 2.45 nmol/L. The solution was then left overnight at room temperature for 12-16 h in the dark. The resultant ABTS radical solution was diluted with phosphate buffer (PBS, 0.2mol/L, pH 7.4) to give an absorbance of 0.70 at 734 nm. 0.1ml of the enzymatic hydrolysate was mixed with 2.9ml of ABTS free radical solution, shaken for 30 seconds, reacted in the dark for 10 minutes, and then the absorbance of the reaction solution was measured at 734 nm. Distilled water is used as a blank to replace hydrolysate.
Clearance (%) ═ ai-Aj)/A0*100％
In the formula, A02.9ml of ABTS reagent was mixed with 0.1ml of distilled waterAbsorbance of the liquid, AjThe absorbance was 2.9ml ABTS +0.1ml enzymolysis solution mixture.
As shown in fig. 4, the rana gherai protease inhibitory peptide can significantly scavenge DPPH and ABTS free radicals. Free radical oxidation plays an important role in neurodegenerative diseases caused by senile dementia, Parkinson's disease, diabetes, rheumatoid arthritis and amyotrophic lateral sclerosis. The rana rugulosa protease inhibitory peptide can well remove free radicals, so that the rana rugulosa protease inhibitory peptide can be applied to treatment of related diseases caused by free radical oxidation. In addition, in order to prevent damage to the skin caused by free radicals, it is essential to add a free radical scavenger to a cosmetic skin care product. Therefore, the rana rugulosa protease inhibiting peptide can also be applied to beauty skin care products.
Figure IDA0001135186840000011
Figure IDA0001135186840000021

Claims (7)

1. The rana rugulosa protease inhibiting peptide is characterized by consisting of 33 amino acids, wherein the molecular weight of the rana rugulosa protease inhibiting peptide is 3768.33 daltons, the isoelectric point of the rana rugulosa protease inhibiting peptide is 6.303, and the amino acid sequence SEQ ID number 1 of the rana rugulosa protease inhibiting peptide is: lysglucyslys Asp Tyr Tyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp SerLeu Asp Leu Gly Leu Ser His Glu Ala Gln Gln; the third and eighth cysteines of the polypeptide form an intramolecular disulfide bond.
2. A nucleotide encoding the rana rugulosa protease inhibitory peptide of claim 1.
3. Use of rana nigromaculata protease inhibitory peptide according to claim 1 for the preparation of a therapeutic drug and a cosmetic skin care drug for pathogenic microorganism infection diseases selected from escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and candida albicans.
4. Use of the rana rugulosa protease inhibitory peptide of claim 1 in the preparation of a free radical scavenging medicament or a free radical scavenging cosmetic product.
5. Use of the rana rugulosa protease inhibitory peptide of claim 1 in the preparation of an antioxidant drug or an antioxidant cosmetic product.
6. Use of a rana nigromaculata protease inhibiting peptide according to claim 1 for inhibiting bacteria, which are escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and candida albicans, for non-diagnostic or therapeutic purposes.
7. Use of the rana nigromaculata protease inhibitory peptide of claim 1 for the preparation of a medicament or cosmetic product for inhibiting bacteria, such as escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa, and candida albicans.
CN201610915371.1A 2016-10-20 2016-10-20 Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy Active CN106478811B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610915371.1A CN106478811B (en) 2016-10-20 2016-10-20 Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610915371.1A CN106478811B (en) 2016-10-20 2016-10-20 Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy

Publications (2)

Publication Number Publication Date
CN106478811A CN106478811A (en) 2017-03-08
CN106478811B true CN106478811B (en) 2020-04-17

Family

ID=58271054

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610915371.1A Active CN106478811B (en) 2016-10-20 2016-10-20 Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy

Country Status (1)

Country Link
CN (1) CN106478811B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107513100A (en) * 2017-10-19 2017-12-26 南方医科大学 Mercuric chloride antibacterial peptide and its gene and application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1883703A (en) * 2006-05-30 2006-12-27 中国科学院昆明动物研究所 Serine protease inhibitor of Rana grahami, its gene and application
CN104558121A (en) * 2014-12-31 2015-04-29 常熟理工学院 Tiger frog antibacterial peptide, and coding gene and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1883703A (en) * 2006-05-30 2006-12-27 中国科学院昆明动物研究所 Serine protease inhibitor of Rana grahami, its gene and application
CN104558121A (en) * 2014-12-31 2015-04-29 常熟理工学院 Tiger frog antibacterial peptide, and coding gene and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides;Li 等;《The FASEB Journal》;20070930;第21卷(第10期);第2466-2473页 *
虎纹蛙皮肤组织cDNA文库的构建及抗菌肽基因tigerinin-HRs的克隆;王辉 等;《华北农学报》;20100228;第25卷(第1期);第61-67页 *

Also Published As

Publication number Publication date
CN106478811A (en) 2017-03-08

Similar Documents

Publication Publication Date Title
Noga et al. Piscidin 4, a novel member of the piscidin family of antimicrobial peptides
Rolland et al. Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris
Sun et al. A novel cathelicidin from Bufo bufo gargarizans Cantor showed specific activity to its habitat bacteria
WO2015161820A1 (en) Amphiphilic synthetic antimicrobial peptide, and pharmaceutical composition and use thereof
CA2325658A1 (en) Gene coding for heliomicine and use thereof
CA2776883A1 (en) Antimicrobial and antibiofilm activity of cathelicidins
Luo et al. A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var
Kim et al. Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii
CN106478811B (en) Giant knotweed frog protease inhibitory peptide, gene thereof and application thereof in pharmacy
CN106432483B (en) Rana chensinensis skin serine protease inhibitory peptide, gene and pharmaceutical application thereof
CN100581584C (en) Serine protease inhibitor of Rana grahami, and its application
CN113527461B (en) Horseshoe head bats source antibacterial peptide RF-CATH1 and application thereof
CN106496317B (en) Rana chensinensis secretory peptide, gene thereof and application thereof in pharmacy
KR102146935B1 (en) Poecilocorisin-1 peptide isolated from Poecilocoris lewisi or antimicrobial, antimycotic and antiinflammatory composition comprising it
CN106397564B (en) Black-frame toad antioxidant peptide, gene thereof and application thereof in pharmacy
CN106432459B (en) Antibacterial peptide of hypsizigus marmoreus, gene thereof and application thereof in pharmacy
CN101570760A (en) Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof
CN101186646A (en) Application of king cobra toxin protease inhibitor and its derivatives
CN107513100A (en) Mercuric chloride antibacterial peptide and its gene and application
CN113480628B (en) Guizhou wart mud eel polypeptide TK-CATH and coding gene and application thereof
TW200825100A (en) Protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (GFD), alcohol dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata, manufacturing method and uses therefor
KR20130105545A (en) A USE OF RORα FOR CONTROLLING ACTIVITY OF TH17 CELL
CN100389125C (en) Rana grahami propyl sperm peptide, gene and variant and its pharmaceutical use
KR102146937B1 (en) Tenoderasin-1 peptide isolated from Tenodera sinensis or antimicrobial, antimycotic and antiinflammatory composition comprising it
JP2005508151A (en) Mekuna eel caterin-related antimicrobial peptides and genes

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant