CN102875659A - Antibacterial peptide secreted by skins of northeast forest frog, and coding sequence and application thereof - Google Patents
Antibacterial peptide secreted by skins of northeast forest frog, and coding sequence and application thereof Download PDFInfo
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- CN102875659A CN102875659A CN2012103816046A CN201210381604A CN102875659A CN 102875659 A CN102875659 A CN 102875659A CN 2012103816046 A CN2012103816046 A CN 2012103816046A CN 201210381604 A CN201210381604 A CN 201210381604A CN 102875659 A CN102875659 A CN 102875659A
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Abstract
The invention relates to an antibacterial peptide secreted by skins of a northeast forest frog, and a coding sequence and an application thereof. The amino acid sequence of the antibacterial peptide is shown in the sequence table of SEQID No. 1; the molecular weight is 3000.6 Daltons; the isoelectric point is 9.7; and the full DNA sequence of the antibacterial peptide is shown in the sequence table of SEQID No. 2. The antibacterial peptide can be prepared through an amino acid synthesizer, has significant antibacterial activity, can not generate hemolytic effect to red blood cells of mammals, and can be applied to explore and prepare antibacterial drugs.
Description
Technical field
The invention belongs to antibacterial peptide and preparation field thereof, particularly a kind of antibacterial peptide (ranatuerin-2YJ) and encoding sequence and application of skin of Northeast China brown frog secretion.
Background technology
Antibacterial peptide claims again peptide antibiotic, it is the immunological molecule the earliest that living organism produces for conforming in resisting the defensive raction process of pathogenic micro-organism, comprise pathogenic micro-organism played inhibiting all peptide matters to have except the enzyme of hydrolytic activity such as N,O-Diacetylmuramidase, chitinase, dextranase etc.Antibacterial peptide is distributed widely in the occurring in nature different kind organism body, and is in the majority with insect, batrachians, mammals in the animal, is mainly derived from the histoorgans such as mucocutaneous, blood, gi tract, liver, spleen, has antipathogen invasion effect.
Ranid is the occurring in nature the most widely amphibian animal that distributes, various places all over the world, and its moist skin, exposed provides the good environment that grows and multiply for the pathogenic micro-organisms such as bacterium, virus.Ranine skin plays an important role in keeping its existence and adaptation habitat, in order to adapt to living environment, distributed in the ranine skin of a great variety, molecular structure is special, the biologically active substance of diverse in function, mainly contain biogenic amine, alkaloid, Bufodienolides and polypeptide 4 large class biochemical compositions, formed a huge bioactive molecules resources bank and drug resource storehouse.Wherein, ranid antibacterial skin peptide is that the opposing pathogenic micro-organism is invaded the important composition composition in the congenital system of defense, be divided into following classification according to its structure function characteristics, i.e. brevinin-1 and brevinin-2 family, esculentin-1 and esculentin-2 family, ranalexin family, ranatuerin-1 and ranatuerin-2 family, temporin family, palustrin family, tigerinin family, japonicin-1 and japonicin-2 family, nigrocin-2 family, dybowskin-1 and dybowskin-2 family, melittin related peptides and Caerulein precursor related peptides.
Amphibian animal antibacterial skin peptide generally is comprised of 10 ~ 50 amino-acid residues, and molecular weight is 1 ~ 7kDa approximately, and molecular surface is usually with positive charge, and iso-electric point is much in 7, belongs to the alkaline kation peptide.The signal peptide of the ranid antibacterial skin peptide of having reported and the sequence of acid propetide are very conservative, transcribe with the precursor peptide form, finally secrete with the mature peptide form.Signal peptide has 22 hydrophobic amino acid residue structures, and cleavage site is positioned between halfcystine-glutaminic acid residue (Cys-Glu), and its major function is to impel bioactive peptide from the cell internal secretion to the extracellular.Acid propetide between signal peptide and mature peptide, keep bioactive peptide self Stability Analysis of Structures, prevent enzymolysis, also play the bioactive effect of regulating simultaneously.Mature peptide is positioned at after acid propetide Methionin-arginine (Lys-Arg) residue, in the bioactive peptide ripening process, signal peptide and acid propetide are excised respectively, by alpha-helix, β-pleated sheet structure, the approach such as amino acid modified, finally extremely external by the skin particles glandular secretion, mediate a series of biological effect, can under the prerequisite of injuring normal cell not, play killing action to pathogenic micro-organisms such as bacterium, viruses.
Dongbei forest-frog (
Rana dybowskii) be distributed widely in the Forest wetland of northeast China, comprise Changbai Mountain arteries and veins, Xing'an Mountains region, innerland, Zhang Guangcai mountain range etc.From skin of Northeast China brown frog secretory product, separate and obtained the multiple antibacterial peptide antibacterial, antiviral activity that has, all can form the α-helixstructure of amphiphilic, its mechanism of action is different from traditional microbiotic, it is not to synthesize to come killing bacteria by some important living matter in the anti-bacteria metabolic process, but directly act on cell walls or cytolemma by physics mode, intracellular active substance is leaked cause bacterium dead, therefore such antibacterial peptide can not make bacterium produce resistance, is expected to become antibiotic, the antiviral and cancer therapy drug of a new generation.
Summary of the invention
The object of the present invention is to provide a kind of new skin of Northeast China brown frog antibacterial peptide (ranatuerin-2YJ) with anti-microbial activity, and the encoding sequence of this antibacterial peptide and preparation method and the application in the preparation antibacterials thereof.
The technology used in the present invention is as follows:
A kind of antibacterial peptide of skin of Northeast China brown frog secretion, its aminoacid sequence is that sequence table SEQ ID No.1 is described, its molecular weight is 3000.6 dalton, iso-electric point 9.7.
The present invention also has following feature:
1, a kind of complete genome of antibacterial peptide of the skin of Northeast China brown frog secretion of encoding, it is described that its nucleotides sequence is classified sequence table SEQ ID No.2 as.
2, based on the complete genome of the antibacterial peptide of aforesaid a kind of skin of Northeast China brown frog secretion of encoding, the nucleotides sequence of the antibacterial peptide of coding skin of Northeast China brown frog secretion, it is described that its nucleotides sequence is classified sequence table SEQ ID No.2 124-207 position as.
3, the antibacterial peptide of skin of Northeast China brown frog secretion of the present invention adopts the method for solid state chemistry to be prepared, Fmoc solid-phase peptide synthesis program according to standard carries out synthetic, synthetic peptide is through reversed-phase HPLC Shimadzu Shim – pack VP-ODS reverse-phase chromatographic column 4.6mm * 150mm purifying, adopt acetonitrile/water/trifluoroacetic acid solution wash-out, the synthetic peptide purity that obtains is up to 99%
4, the antibacterial peptide of aforesaid a kind of skin of Northeast China brown frog secretion, the application in preparation treatment infected by microbes disease medicament.
Antibacterial peptide performance anti-microbial effect be by itself with the attraction of positive charge and the negative charge of bacterium surface be in contact with one another, then form the cross-film ionic channel by hydrophobic amino acid, cause the entocyte loss, cause necrocytosis.Antibacterial peptide ranatuerin-2YJ aminoacid sequence secondary structure of the present invention shows, it can form alpha-helix, and has a cationic, belong to cationic antibacterial peptide, the C-end shows wetting ability and N-end demonstration hydrophobicity, hydrophilic and hydrophobic amino acid residue is distributed in the helix wheel both sides, and the antibacterial peptide activity is played a crucial role, and these all are the constitutional features nets that antibacterial peptide has.The C-end of mature peptide is Rana box, is that Rana belongs to distinctive structure.
Antibacterial peptide of the present invention (ranatuerin-2YJ) adopts agar diffusion method to carry out the bacteriostatic activity experiment, and its hemolytic activity is detected.The experimental result demonstration, antibacterial peptide ranatuerin-2YJ of the present invention is to G such as Salmonella enteritidis, streptococcus aureus, Pseudomonas aeruginosa, klebsiella pneumoniae, micrococcus scarlatinae, serratia marcescens, Bacillus cereus, enterohemorrhagic Escherichia coli
+Bacterium and G
-Bacterium all has the active (see figure 4) of stronger inhibition, the hemolysis rate of 10mg/mL antibacterial peptide ranatuerin-2YJ is 5.64%, drug level during far below mammalian erythropoietin generation hemolytic action, it has significant anti-microbial activity, this antibacterial peptide ranatuerin-2YJ is described in the situation that do not destroy the mammalian cell integrity, it can suppress part G
+Bacterium and G
-The growth of bacterium demonstrates the application prospect as the new antibiotic medicine.
Description of drawings
Fig. 1 is agarose gel electrophoresis detected result (the M:DNA Marker 2000 of the total RNA extraction effect of skin of Northeast China brown frog; 1-3 is the total RNA of skin of Northeast China brown frog);
Fig. 2 is pcr amplification result (the M:DNA Marker 2000 of skin of Northeast China brown frog antibacterial peptide gene preproranatuerin-2YJ; 1: negative control; The 2:preproranatuerin-2YJ gene);
Fig. 3 is the synthetic HPLC figure of antibacterial peptide ranatuerin-2YJ solid state chemistry;
Fig. 4 is antibacterial peptide ranatuerin-2YJ agar diffusion method bacteriostatic experiment result (A: Salmonella enteritidis; B: Pseudomonas aeruginosa; C: serratia marcescens; D: streptococcus aureus; E: enterohemorrhagic Escherichia coli; F: klepsiella pneumoniae; G: bacillus cereus; H: micrococcus scarlatinae. wherein: 1 is the fungistatic effect of antibacterial peptide ranatuerin-2YJ; 2 negative contrasts).
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment:
The clone of antibacterial peptide ranatuerin-2YJ gene preproranatuerin-2YJ
1, the design of primer
According to the Amphibians batrachia bioactive peptide gene order of including among the GenBank,
Design and synthesize upstream primer XD1:5'-ATGTTCACC (T/A) TGAAGAAA (T/C)-3',
Universal downstream primer XD2:Oligo d (T) 15, primer give birth to worker's biotechnology company limited by Shanghai and synthesize.
2, the cNDA of antibacterial peptide gene preproranatuerin-2YJ clone
2.1 the extraction of the total RNA of skin of Northeast China brown frog: adopt the Dongbei forest-frog skin of back, be ground into powder through superfreeze shredder (U.S. SPEX CertiPrep company, model 6770); Take by weighing the 100mg sample and add 1mL Trizol, abundant mixing, ice bath 5min, centrifugal (12000rpm, 4 ℃) 10min gets supernatant; The chloroform that adds the equal-volume precooling, mixing, ice bath 5min, centrifugal (12000rpm, 4 ℃) 10min gets the upper strata water; The Virahol that adds the equal-volume precooling, mixing, ice bath 10min, centrifugal (12000rpm, 4 ℃) 10min, careful abandoning supernatant; With 1mL 75% washing with alcohol twice, each 4 ℃ of centrifugal 10min of 12000rpm dry up residual ethanol, add 50 μ L DEPC water dissolution precipitation, obtain the total RNA of skin of Northeast China brown frog.Get the total RNA extracting solution of 5 μ L skin of Northeast China brown frogs, detect the integrity of total RNA through 1% agarose gel electrophoresis, and with purity and the concentration of the total RNA of UV spectrophotometer measuring.The high-visible 28SRNA of result, 18SRNA and 5SRNA three bands, and ultraviolet spectrophotometer is measured OD
260/ OD
280Ratio shows the total RNA of the skin of Northeast China brown frog that obtains without degraded between 1.90～2.01, integrity is good, and without obviously protein and DNA pollute.
2.2 RT-PCR: get the total RNA of skin of Northeast China brown frog of 3 μ L extraction through the synthetic cNDA of reverse transcription, the reverse transcription system is as follows:
| 10×RT mix | 2μL |
| dNTP (2.5mmol/L each) | 2μL |
| Primer XD2 :Oligo d(T) 15 (10mmol/L) | 2μL |
| Total RNA template | 3μL |
| Quant Reverse Transcriptase | 1μL |
| RNase-free H 2O | 10μL |
Reverse transcription condition: hatch 60min for 36 ℃, obtain cDNA solution.Take the cDNA that obtains as template, carry out the PCR reaction, amplification antibacterial peptide ranatuerin-2YJ gene preproranatuerin-2YJ, (50 μ L) is as follows for the PCR reaction system:
| 10×PCR buffer | 5μL |
| dNTP (2.5mmol/L each) | 4μL |
| Primer XD1 (10mmol/L) | 2μL |
| Primer XD2 (10mmol/L) | 2μL |
| The cDNA template | 2μL |
| Ex Taq DNA Polymerase (8U/μL) | 0.5μL |
| ddH 2O | 34.5μL |
The PCR reaction conditions is: 94 ℃ of 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s carry out 30 circulations; 72 ℃ are extended 5min.The PCR product is through 3% agarose gel electrophoresis separation and purification and reclaim the goal gene fragment, and the purpose PCR product of recovery is connected with cloning vector pMD-19-T vector, the transformed competence colibacillus e. coli jm109, and 37 ℃ of incubated overnight are identified positive recombinant bacterium.
2.3 the sequential analysis of antibacterial peptide gene: the positive recombinant bacterium of picking increases bacterium and cultivates, extract plasmid DNA and carry out dna sequencing (examining order is given birth to worker's biotechnology company limited by Shanghai and synthesized), obtained the complete nucleotide sequence of skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ gene preproranatuerin-2YJ.This antibacterial peptide ranatuerin-2YJ gene preproranatuerin-2YJ nucleotide sequence total length 291bp(sees sequence table), on the structure of mRNA, it has the structures such as complete and effective signal peptide district, mature peptide district, 3 ' non-translational region and PolyA, is the mRNA that a class can normally be translated.
The solid phase synthesis of skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ
According to standard Fmoc solid-phase peptide synthesis program synthetic skin of Northeast China brown frog antibacterial peptide:
Gly Leu Met Asp Ile Phe Lys Val Ala Val Asn Lys Leu Leu Ala
1 5 10 15
Ala Gly Met Asn Lys Pro Arg Cys Lys Ala Ala His Cys-NH2
20 25
Synthetic peptide is through the reversed-phase HPLC (purifying of Shimadzu Shim – pack VP-ODS reverse-phase chromatographic column 4.6mm * 150mm), adopt acetonitrile/water/trifluoroacetic acid solution wash-out, the synthetic peptide purity that obtains is up to 99%, and antibacterial peptide is synthetic synthetic by the living worker's biotechnology in Shanghai company limited.
Embodiment 3
The pharmacologically active of antibacterial peptide
1, the test experience of anti-microbial activity
Adopt agar diffusion method to measure the bacteriostatic activity of skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ, its specific operation process is as follows: get respectively 8 kinds of bacterium Salmonella enteritidis that are in logarithmic phase, streptococcus aureus, Pseudomonas aeruginosa, klebsiella pneumoniae, serratia marcescens, Bacillus cereus, enterohemorrhagic Escherichia coli and micrococcus scarlatinae are cultivated each 100 μ L of bacterium liquid, respectively with nutrient agar (about the 60 ℃) mixing of 10mL 1.1%, after culture medium solidifying, punch, the antibacterial peptide ranatuerin-2YJ that in the hole, adds 100 μ g/mL purifying, with liquid nutrient medium as negative control, paraxin is as positive control, cultivate 24-36h for 37 ℃, observe fungistatic effect.The mensuration of antibacterial peptide ranatuerin-2YJ minimum inhibitory concentration (MIC): adopt agar dilution to detect, in the nutrient agar that the soon Dongbei forest-frog antibacterial peptide ranatuerin-2YJ of different concns adding is about 50 ℃, make the agar plate that contains the different concns antibacterial peptide, inoculate tested bacterium, hatch rear observation bacterial growth situation, antibacterial peptide minimum concentration contained in the bacteria growing inhibiting agar plate is as minimum inhibitory concentration.
The minimum inhibitory concentration (MIC) of table 1 skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ
| Bacterium | Minimum inhibitory concentration (μ g/mL) |
| Salmonella enteritidis | 42.5 |
| Streptococcus aureus | 22.5 |
| Pseudomonas aeruginosa | 65 |
| Klepsiella pneumoniae | 35 |
| Serratia marcescens | 25 |
| Bacillus cereus | 55 |
| Enterohemorrhagic Escherichia coli | 25 |
| Micrococcus scarlatinae | 50 |
2, the test experience of hemolytic activity
Get the blood of 1mL Healthy Rabbits, wash to supernatant with 0.9% physiological saline colourless, the centrifugal 2min of 3000rpm, abandoning supernatant; It is 2 * 10 that the red corpuscle that obtains is mixed with concentration with 0.9% physiological saline
7Cell suspension; Adding concentration in red cell suspension is the skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ of 10mg/mL, hatches 60min for 37 ℃; The centrifugal 5min of 10000rpm gets supernatant, detects the 540nm light absorption value, and red corpuscle and 1% (v/v) Tween-20 is hatched and is considered as 100% haemolysis, as positive control, and the negative contrast of 0.9% physiological saline, each sample is established 3 parallel holes.Hemolysis rate=(the negative OD of sample OD-)/(the negative OD of positive OD-) * 100%.The result shows, the hemolysis rate of the skin of Northeast China brown frog antibacterial peptide ranatuerin-2YJ of 10mg/mL only is 5.64%, drug level during far below mammalian erythropoietin generation hemolytic action, this antibacterial peptide ranatuerin-2YJ is described in the situation that do not destroy the mammalian cell integrity, can suppress the growth of part bacterium, demonstrate the application prospect as the new antibiotic medicine.
＜110〉Heilungkiang Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center
＜120〉antibacterial peptide and encoding sequence and the application of skin of Northeast China brown frog secretion
<160>2
<210>1
<211>28
<212>PRT
＜213〉Dongbei forest-frog
<400>1
Gly Leu Met Asp Ile Phe Lys Val Ala Val Asn Lys Leu Leu Ala
1 5 10 15
Ala Gly Met Asn Lys Pro Arg Cys Lys Ala Ala His Cys
20 25
<210>2
<211>291
<212>cDNA
＜213〉Dongbei forest-frog
<400>2
ATGTTCACCT TGAAGAAATC CATGTTACTC CTTTTCTTTC TTGGGACCAT CTCCTTATCT 60
CTCTGTCAGG ACGAAGGTGC CGATGAAGAC CATGGAGGGG AAATGACCAC GGAAGAAAAA 120
AGAGGTCTCA TGGATATTTT TAAGGTTGCA GTCAATAAAT TGTTAGCAGC TGGTATGAAT 180
AAGCCAAGAT GTAAAGCTGC TCACTGTTAA ATCCTGAATG GAAAGTCTCC TGATGTGGGA 240
TATCATTAAG CTACAAAAAT GCTAAATGTC TAATAAAAAA AAAAAAAAAA A 291
Claims (5)
1. the antibacterial peptide of skin of Northeast China brown frog secretion, it is characterized in that: its aminoacid sequence is that sequence table SEQ ID No.1 is described, and its molecular weight is 3000.6 dalton, iso-electric point 9.7.
2. the complete genome of the antibacterial peptide of the skin of Northeast China brown frog secretion of encoding, it is characterized in that: it is described that its nucleotides sequence is classified sequence table SEQ ID No.2 as.
3. the complete genome of the antibacterial peptide of a kind of skin of Northeast China brown frog secretion of encoding according to claim 2, it is characterized in that: the nucleotides sequence of the antibacterial peptide of coding skin of Northeast China brown frog secretion, it is described that its nucleotides sequence is classified sequence table SEQ ID No.2 124-207 position as.
4. the preparation method of the antibacterial peptide of a skin of Northeast China brown frog secretion as claimed in claim 1 is as follows: adopt the method for solid state chemistry to be prepared, Fmoc solid-phase peptide synthesis program according to standard carries out synthetic, synthetic peptide is through reversed-phase HPLC Shimadzu Shim – pack VP-ODS reverse-phase chromatographic column 4.6mm * 150mm purifying, adopt acetonitrile/water/trifluoroacetic acid solution wash-out, the synthetic peptide purity that obtains is up to 99%.
5. such as the antibacterial peptide of the described a kind of skin of Northeast China brown frog secretion of claim 1, it is characterized in that: the application in preparation treatment infected by microbes disease medicament.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015174855A (en) * | 2014-03-18 | 2015-10-05 | 国立研究開発法人産業技術総合研究所 | Amphibian xenopus tropicalis skin-derived functional peptides |
| CN110585482A (en) * | 2019-08-02 | 2019-12-20 | 大连理工大学 | Heteronaphthalene biphenyl poly (arylene ether nitrile) with antibacterial property and surface modification method thereof |
| CN111334544A (en) * | 2020-03-26 | 2020-06-26 | 吉林大学 | Preparation method of wood frog bone and meat oligopeptide with immunocompetence |
| CN114045258A (en) * | 2021-10-21 | 2022-02-15 | 辽宁盛京干细胞科技有限公司 | Serum-free medium for mesenchymal stem cell culture and application thereof |
| CN115246877A (en) * | 2021-12-27 | 2022-10-28 | 昆明理工大学 | Rana nigromaculata source antibacterial peptide Brevinin-1EG and application |
-
2012
- 2012-10-10 CN CN2012103816046A patent/CN102875659A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| SHU-JING YANG ET AL.: "Introduction of antimicrobial peptides from Rana dybowskii under Rana grylio virus stress, and bioactivity analysis", 《CAN. J. MICROBIOL.》 * |
| 金莉莉: "东北林蛙皮抗菌肽及其生物学特性", 《中国博士学位论文全文数据库》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015174855A (en) * | 2014-03-18 | 2015-10-05 | 国立研究開発法人産業技術総合研究所 | Amphibian xenopus tropicalis skin-derived functional peptides |
| CN110585482A (en) * | 2019-08-02 | 2019-12-20 | 大连理工大学 | Heteronaphthalene biphenyl poly (arylene ether nitrile) with antibacterial property and surface modification method thereof |
| CN110585482B (en) * | 2019-08-02 | 2022-03-01 | 大连理工大学 | Heteronaphthalene biphenyl poly (arylene ether nitrile) with antibacterial property and surface modification method thereof |
| CN111334544A (en) * | 2020-03-26 | 2020-06-26 | 吉林大学 | Preparation method of wood frog bone and meat oligopeptide with immunocompetence |
| CN111334544B (en) * | 2020-03-26 | 2023-05-23 | 吉林大学 | Preparation method of wood frog bone and meat oligopeptide with immune activity |
| CN114045258A (en) * | 2021-10-21 | 2022-02-15 | 辽宁盛京干细胞科技有限公司 | Serum-free medium for mesenchymal stem cell culture and application thereof |
| CN114045258B (en) * | 2021-10-21 | 2024-04-16 | 辽宁盛京干细胞科技有限公司 | Serum-free culture medium for mesenchymal stem cell culture and application |
| CN115246877A (en) * | 2021-12-27 | 2022-10-28 | 昆明理工大学 | Rana nigromaculata source antibacterial peptide Brevinin-1EG and application |
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