CN101168564B - Human antibiotic peptide and use for derivative thereof - Google Patents

Abstract

The present invention relates to the use of human-derived antimicrobial peptides and their derivatives. The human-derived antimicrobial peptides have an amino acid sequence selected from the sequence table SEQ ID NO: 1, and also include the substitution of individual amino acids in the amino acid sequence polypeptide, ring Functional equivalents obtained by changing B, L-type amino acids into D-type amino acids, deletion or addition. Compared with basic antibacterial peptides from other sources, the human-derived antibacterial peptide has the beneficial characteristics of simple structure, convenient artificial synthesis, and no antigenicity when applied to human body. The human-derived antimicrobial peptide and its derivatives of the present invention have a significant effect of inhibiting the growth of bacteria and fungi, and can be applied to the preparation of antimicrobial infection preparations.

Description

Uses of human-derived antimicrobial peptides and derivatives thereof

technical field

The invention relates to the use of human antibacterial peptides and derivatives thereof, in particular to the amino acid sequence, nucleotide coding sequence and antibacterial use of human semen coagulation protein degradation small peptide SgI-29 and derivatives thereof.

Background technique

Infectious diseases have always been one of the main reasons that threaten human life. In the 1930s, antibiotics were discovered and widely used in clinical treatment, saving the lives of countless patients. Due to the extensive use of traditional antibiotics, the rapid growth of drug-resistant pathogenic microorganisms. Therefore, it is urgent to develop new antibiotics to deal with the increasingly serious drug resistance of pathogenic microorganisms worldwide.

Antimicrobial peptides (also known as peptide antibiotics) are widely distributed in nature. They are endogenous substances produced by organisms to resist exogenous infections. After millions of years, they rarely develop tolerance. Spectrum and other features. Thus when it was discovered, it attracted the attention of the pharmaceutical and high-tech bioindustry (Koczulla AR, Bals R, 2003. Antimicrobial peptides: current status and therapeutic potential. Drugs 63, 389-406).

Antimicrobial peptides are widely distributed in amphibians, insects, plants and mammals. Studies have found that antimicrobial peptides are particularly rich in the skin of amphibians. Over the past 30 years, people have discovered nearly a thousand antimicrobial peptides from nature (available at http://www.bbcm.univ.trieste.it/～tossi/pag1. htm retrieved), more than 800 of them came from amphibian skin (mainly frogs) (Apponyi MA, et al., 2004.Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance.Peptides 25, 1035-1054).

Since the invention of antibiotics, humans have made great achievements in the control and treatment of microbial infections, but with the continuous use of antibiotics, microbial resistance has become a major problem in microbial infection control, so that some microbial bacteria have There is no front-line substance to control the kill. For example, in clinical drug treatment, vancomycin-resistant staphylococci, enterococci and other Gram-negative infectious diseases are currently clinical problems worldwide. The three types of bacteria that mainly cause meningitis also have strong drug resistance clinically. Penicillin-resistant, chloramphenicol-resistant meningococcus, pneumococcus, and pneumococcus resistant to new cephalosporin antibiotics also widely appear. Therefore, the discovery and development of a new class of antimicrobial substances has become an urgent task and a hot spot in the world. Compared with the widely used antibiotics at present, polypeptide antibiotics have many advantages: such as rapid and broad-spectrum killing of microorganisms (including current clinical drug-resistant bacteria) at the minimum effective concentration, and inhibitory effect on fungi, and the ability to generate resistant strains Small, effective for both local infection and systemic infection, it is becoming a new class of antibiotics, and its development is currently receiving extensive attention (Brogden KA, 2005.Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat.Rev.Microbiol.3, 238 -250).

Human antimicrobial peptides have always been one of the hotspots of research. It has no antigenicity, so it is easier to use as a therapeutic drug (DeSmet K, Contreras R, 2005. Human antimicrobial peptides: defensins, cathelicidins and hisstatins. Biotechnol. Lett. 27, 1337 -1347).

Contents of the invention

One object of the present invention is to provide human-derived antimicrobial peptides and their derivatives, which have the beneficial characteristics of simple structure, convenient artificial synthesis, and no antigenicity when applied to human bodies. Another object of the present invention is to provide human-derived antimicrobial peptides and their derivatives. The use of the derivative, which has a significant effect of inhibiting the growth of bacteria and fungi.

The purpose of the present invention is achieved like this:

The present invention relates to a human-derived antimicrobial peptide, which is isolated from the liquefied semen of healthy men, and its amino acid sequence is as follows (SEQ ID NO: 1):

Histidine-Asparagine-Lysine-Glutamine-Glutamic Acid-Glycine-Arginine-Aspartic Acid-Histidine-Aspartic Acid-Lysine-Serine-Lysine- Glycine-Histidine-Phenylalanine-Histidine-Arginine-Valine-Valine-Isoleucine-Histidine-Histidine-Lysine-Glycine-Glycine-Lysine Acid-Alanine-Histidine (His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val IleHis His Lys Gly Gly Lys Ala His).

The human-derived antimicrobial peptides of the present invention include functional equivalent polypeptides obtained by substitution, cyclization, L-type amino acid into D-type amino acid, deletion or addition of individual amino acids in the above polypeptides.

That is, an antimicrobial peptide derived from the amino acid residues of the isolated human antimicrobial peptide substituted by the above-mentioned human antimicrobial peptide and having antibacterial activity, the antimicrobial peptide has an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 2 in the sequence table: His Asn Lys Gln Glu GlyLys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His.

It can also be an antimicrobial peptide derived from the above-mentioned human-derived antimicrobial peptide with multiple amino acid deletions and antibacterial activity. The antimicrobial peptide has an amino acid sequence selected from the sequence table SEQ ID NO: 3: Lys Gln Glu Gly Arg Asp His Asp LysSer Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys.

The preparation method of the human-derived antimicrobial peptide provided by the present invention can be a solid-phase chemical method, or can be obtained by cloning the gene encoding the antimicrobial peptide into a vector, and then expressing it in a host cell. The expression vector can be one of plasmid or virus. The host cell can be a prokaryotic cell, including Escherichia coli or Bacillus subtilis, and the host cell can also be a eukaryotic cell, including yeast cells, plant cells, insect cells, or mammalian cells. The prepared antimicrobial peptides can be identified by mass spectrometry.

In the method for isolating human-derived antimicrobial peptides of the present invention, the isolated antimicrobial peptides have the amino acid sequence shown in SEQ ID NO: 1.

The preparation method of the human-derived antimicrobial peptide and its derivatives of the present invention is synthesized by a solid-phase chemical method.

An isolated nucleotide sequence or its complementary sequence, said nucleotide sequence encoding the above-mentioned antimicrobial peptide.

A construct, the construct contains a nucleotide sequence selected from the amino acid sequence shown in the sequence listing SEQ ID NO: 3.

A cell containing the above-mentioned peptide, the above-mentioned nucleotide sequence or the above-mentioned construct.

A method for preparing the above-mentioned antimicrobial peptides, the method comprising:

(1) cultivating the above-mentioned cells under conditions suitable for the expression of the above-mentioned antimicrobial peptides;

(2) Isolate the above-mentioned antimicrobial peptides.

The use of the human-derived antimicrobial peptide and its derivatives of the present invention, and the application of the antimicrobial peptide and its derivatives in the preparation of antimicrobial infection drugs.

In order to further study the structure and function relationship of the human antimicrobial peptide and its derivatives of the present invention, a group of polypeptides were synthesized by an automatic polypeptide synthesizer for research.

The SgI-29, SgI-29R7K and SgI-25 antimicrobial peptides prepared with the following amino acid sequences are described below as an example:

SgI-29 (SEQ ID NO: 1):

His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

SgI-29R7K (SEQ ID NO: 2):

His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

SgI-25 (SEQ ID NO: 3):

Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His LysGly Gly Lys

The antibacterial activity of the polypeptide was detected by agar diffusion test. The result shows that the synthetic antimicrobial peptide of the present invention has strong bactericidal activity.

Similarly, the antimicrobial peptide functional equivalents of the amino acid deletion derivatives in the polypeptides were also prepared and tested for their biological activity, and the results showed that these polypeptides also had good bactericidal activity.

The beneficial effect of the present invention is that: the human antimicrobial peptide and its derivatives have a significant effect of inhibiting the growth of bacteria and fungi. Compared with basic antibacterial peptides from other sources, the human antibacterial peptide has the beneficial characteristics of simple structure, convenient artificial synthesis, and no antigenicity when applied to human body.

Description of drawings

Fig. 1 is the SP-Sepharose ion-exchange chromatogram of the human-derived antimicrobial peptide of the present invention.

Fig. 2 is a G-25 molecular sieve chromatogram of the human-derived antimicrobial peptide of the present invention.

Fig. 3 is the Mono-S ion-exchange chromatogram of the human-derived antimicrobial peptide of the present invention.

Fig. 4 is a HPLC reverse phase C18 column chromatogram of the human-derived antimicrobial peptide of the present invention.

Fig. 5 is the mass spectrogram of peak III of the HPLC reverse phase C18 column chromatography of the human-derived antimicrobial peptide of the present invention.

Fig. 6 is the nucleotide sequence of the human-derived antimicrobial peptide SgI-29 gene of the present invention.

Fig. 7 is the amino acid sequence of the human antimicrobial peptide SgI-29 of the present invention.

Fig. 8 is the mass spectrum of the artificially synthesized human-derived antimicrobial peptide SgI-29 of the present invention.

Detailed ways

Example 1

1. Separation and purification of human antimicrobial peptides:

Normal people masturbate after 3-7 days of abstinence, take the semen in a dry and sterilized measuring cup, and after liquefaction at room temperature, the sperm microbiological dynamic and static computer-aided automatic analysis system (model: CASAS-QH-III, produced by Beijing Tsinghua Tongfang) confirms that the sperm indicators are normal The samples were centrifuged at 10,000 rpm for 10 minutes to separate the sperm and semen, and the supernatant was carefully collected and stored at -20°C for later use.

The first step: SP-Sepharose ion exchange: The normal human semen (n=40) obtained by the above method is loaded on the SP-Sepharose (Pharmacia product) cation exchange column equilibrated with 50mM Na2HPO4-NaH2PO4, PH=7.4 buffer in advance (130mm long, 20mm diameter). After loading the samples were linearly eluted with 0-0.5M NaCl. Collect 3ml per tube. Antibacterial activity test showed that 58-131 tube had strong bactericidal activity. Combined 58-131 tubes were freeze-dried and concentrated, see Figure 1.

HiPrepTM G-25脱盐柱(26/10，Pharmacia产品)，所用仪器为AKTA

Explore 100(Amersham Biosciences产品)。收集第II峰并进行冰冻干燥浓缩，参见附图2。The second step: molecular sieve Sephadex G-25 chromatography: the first step is to freeze-dry and concentrate the sample, dissolve it in Milli-Q ultrapure water, and load the sample on AKTA pre-balanced with 50mM Tris-HCl, pH8.8 buffer

HiPrepTM G-25 desalting column (26/10, Pharmacia product), the instrument used is AKTA

Explore 100 (product of Amersham Biosciences). The peak II was collected and concentrated by lyophilization, see accompanying drawing 2.

The third step: Mono-S ion exchange: the second step is to freeze-dry and concentrate the sample, dissolve it in Milli-Q ultrapure water, and load the sample on the Mono-S cation exchange column pre-equilibrated with 50mM Tris-HCl, pH8.8 buffer (1ml, HR5/5, Pharmacia product), used instrument is AKTAExplore 100 (Amersham Biosciences product). Collect the VII peak, see accompanying drawing 3.

The fourth step: reversed-phase high-pressure liquid chromatography: the VII peak obtained by Mono-S cation exchange column chromatography is eluted with water (containing 0.1% trifluoroacetic acid): acetonitrile (containing 0.1% trifluoroacetic acid) The system performs gradient elution, and the third peak collected is the human semen coagulation protein I-derived peptide containing antibacterial activity, as shown in Figure 4.

Identification of human semen coagulation protein I-derived peptides containing antimicrobial activity:

Step 1: Determination of the amino acid sequence of the third peak of reversed-phase high-pressure liquid chromatography: The amino acid sequence structure is determined by using an automatic amino acid sequencer (model: 491, product of ABI Company). The results showed that the amino acid sequence of peak III in reversed-phase high-pressure liquid chromatography was: His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser.

The second step: fast atom bombardment mass spectrometry (Fast atom bombardment mass spectrometry, FAB-MS) determination of molecular weight: glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1:1:1, V:V:V, volume ratio) as the substrate, Cs+ as the bombardment particles, the current is 1mA, and the emission voltage is 25Kv. The results show that the third peak of reversed-phase high-pressure liquid chromatography contains 4 molecules, and their molecular weights are 3377.17 Daltons, 3850.67 Daltons, 5117.64 Daltons and 5174.18 Daltons respectively, see accompanying drawing 5.

Step 3: Identification of peptide derived from human semen coagulation protein I: The amino acid sequence of peak III of reversed-phase high-pressure liquid chromatography His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser The results of BLAST analysis reveal that it is derived from human Degradation of semen coagulation protein I, a derivative product of human semen coagulation protein I. The precise molecular weight determination of FAB-MS mass spectrometry proves that the polypeptide with a molecular weight of 3377.17 Daltons is derived from mature human semen coagulation protein I, and its cleavage sites are the 85th and 113th positions of mature human semen coagulation protein I. The obtained derivative fragment is named SgI-29 in the present invention, and its full sequence is: His AsnLys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Phe His Arg Val Val Ile His His Lys Gly Gly GlyLys Ala His. The theoretical average molecular weight [M+H]+ of SgI-29 is 3377.74, and the measured molecular weight [M+H]+ is 3377.17. The experimental results fully prove that SgI-29 is produced by the hydrolysis of human semen coagulation protein I.

Example 2

1. Preparation, separation and purification of human antimicrobial peptides:

This embodiment is carried out by solid-phase chemical synthesis, using a fully automatic peptide synthesizer to synthesize their full sequences respectively. The names of the synthetic antimicrobial peptides and their full-sequence amino acid primary structures are as follows:

SgI-29 (SEQ ID NO: 1):

His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

SgI-29R7K (SEQ ID NO: 2):

His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

SgI-25 (SEQ ID NO: 3):

Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His LysGly Gly Lys.

Desalted and purified by HPLC reverse phase C18 column chromatography.

2. Identification of human antimicrobial peptides:

The purity of the purified human-derived antimicrobial peptide was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

Example 3

1. Expression of human antimicrobial peptide SgI-29 gene in Escherichia coli

According to the known gene sequence encoding mature human semen coagulation protein I from position 86 to position 112, it was cloned into the expression vector pMAL-p2X plasmid, and then transformed into Escherichia coli DH5α, which was induced to express MBP-SgI-29 by IPTG The fusion protein was placed in the periplasm of Escherichia coli, and part of the protein in the periplasm of the bacteria was treated with hypotonicity and then passed through the MBP affinity column to obtain the purified MBP-SgI-29 fusion protein, which was then cleaved by FXa to obtain the antimicrobial peptide SgI-29.

The high-fidelity pfu enzyme used in the examples, T4DNA ligase was purchased from Promega Company of the United States, and restriction enzymes Xmn I, Hind III, affinity resin (Amylose resin) and expression vector pMAL-p2X were purchased from New England BioLabs Company of the United Kingdom , tryptone and yeast extract were purchased from Oxoid Company, and acrylamide, methylene bisacrylamide, ammonium persulfate, isopropyl-β-D-thiogalactoside (IPTG), etc. were purchased from Sigma Company. FXa was purchased from Novagen.

Human seminal vesicles were obtained from a 56-year-old male patient who underwent total bladder, prostate, and seminal vesicle resection for bladder urothelial carcinoma. Pathological examination confirmed that the seminal vesicles were not invaded by tumors. The excised seminal vesicles were immediately stored in liquid nitrogen until total RNA was extracted.

2. Total RNA extraction and cDNA synthesis

Total RNA was extracted using Qiangen Total RNA Extraction Kit. The cDNA was synthesized using a kit from TAKALA Company, and the specific operation was carried out according to the instructions.

3. Primer design and synthesis

Primers were designed according to the sequence of the SgI gene, SgI-1 5′GGT TTT CCA AGC AAG ATG AAG 3′; SgI-1R: 5′AGG TGG TGT CAT CCA TGG ACC AAG 3′; SgI-EX1 5′CAT AAT AAA CAAGAA GGC AGA GAC C 3′; SgI52-EXR 5′CCT AAC AAG CTT TTA ATA TTG ACT GGATAT TCC 3′ was synthesized by Beijing Saibaisheng Company. Wherein AAG CTT is the enzyme cutting site of HindIII, and the base CCTAAC in front of it is the protection sequence.

4. SgI52 gene amplification and cloning

Use the obtained cDNA as a template, and use SgI-1 and SgI-1R as primers for PCR amplification. Conditions: cDNA 1 μl, 10 X buffer 5 μl, dNTP (10mM) 1 μl, SgI-1 (20p/μl) 1 μl, SgI -1R (20p/μl) 1 μl, pfu 0.25 μl, ddH2O 40.75 μl. PCR amplification conditions: 94°C for 2min; 92°C for 15s, 50°C for 30s, 72°C for 1min, 35 cycles; 72°C for 10min. PCR products were subjected to agarose gel electrophoresis, and the obvious band at 1.3Kb was recovered with Tiangen DNA Gel Recovery Kit. The recovered DNA was used as a substrate, and SgI-EX1 and SgI52-EXR were used as primers to amplify the SgI-52 gene fragment by PCR. Conditions: recovered SgI DNA 0.5 μl, 10 X buffer 5 μl, dNTP (2.5mM) 4 μl, SgI-EX1 (20p/μl) 1 μl, SgI52-EXR (20p/μl) 1 μl, pfu 0.25 μl, H2O 38.25 μl. PCR amplification conditions: 94°C for 2min; 92°C for 15s, 50°C for 30s, 72°C for 20s, 35 cycles; 72°C for 2min. The PCR product was electrophoresed and found that there were obvious bands around 150bp. The PCR product was digested with HindIII, and the gene fragment was gel-cut and purified. The pMAL-p2X plasmid in Escherichia coli JM109 was extracted with a kit, cut with endonucleases HindIII and XmnI, and confirmed by electrophoresis. The plasmid was ligated with the SgI-52 gene cut with endonuclease HindIII with T4 ligase. overnight at 4°C. Transform competent Escherichia coli DH5α. Colonies were picked for identification, and plasmids were extracted for sequencing.

5. Induced expression of SgI-52 polypeptide

The pMAL-p2X plasmid Escherichia coli DH5α carrying the SgI-52 gene was cultured overnight. Add 1/100 overnight cultured Escherichia coli DH5α into 1000ml of RB medium, shake the bacteria at 200rpm at 37°C for about 2-3 hours until the O.D.600 is 0.5. Add IPTG (final concentration: 0.3mM) and continue culturing until induced for 3 hours, and the bacterial culture transformed with each recombinant plasmid induced without adding IPTG is used as a control.

6. Recombinant protein detection and identification

Centrifuge 1 liter of the bacterial culture at 4000g for 10 minutes to collect the bacteria, and resuspend the bacteria with 30mM Tris-HCl+20% Sucrose pH8.0 (80ml per g of bacteria). Add 0.5M EDTA to make the final concentration 1mM, shake at room temperature for 5-10 minutes. Centrifuge at 8000 g for 10 minutes at 4°C. Resuspend with 2.5ml of ice-cold 5mM MgSO4. Shake in an ice bath for 10 minutes. Centrifuge at 8000 g for 10 minutes at 4°C. The supernatant is the protein expressed in the periplasm. Add appropriate protein loading buffer, boil for 5min, conduct SDS-PAGE electrophoresis, and stain with Coomassie brilliant blue to determine whether the recombinant protein is expressed. The recombinant protein was excised from the fusion protein with Factor X for mass spectrometry and sequence determination.

Example 4

Antibacterial activity detection, referring to the literature method (Lehrer et al., 1991.Ultrasensitive assays for endogenous antimicrobial polypeptides.J.Immunol.Methods 137:167-173), using agarose double-layer punching method to determine the minimum effective concentration MEC (Minimal Effective Concentration ). The samples in this example were chemically synthesized by the solid-phase method, and the names of the synthetic antimicrobial peptides and the primary structure of the complete sequence of amino acids are shown in Example 2.

Among them, the nucleotide sequence of SgI-29 is shown in Figure 6, the amino acid sequence is shown in Figure 7, and the mass spectrum of the synthesized human-derived antimicrobial peptide SgI-29 is shown in Figure 8.

The bottom medium formula is: 1% low melting point agarose (SigmaA6013), 0.3mg/ml trypsin jelly (Oxoid product) is dissolved in 10mM pH7.4 Na2HPO4-NaH2PO4 buffer. Add 20ml of the bottom culture medium at 42°C to culture logarithmic growth phase bacteria (about 105-106 CFU) overnight, shake well and spread it evenly in a 76mm diameter petri dish as the bottom layer. After solidification, 3 mm circular holes were drilled on the bottom medium, and 5 μl of antimicrobial peptide aqueous solutions of different concentrations were added to each hole. After incubating at 37°C for 3 hours, cover the bottom layer with a layer of nutrient agar (top layer medium: 1% Sigma A6013 low-melting point agarose, 0.6mg/ml Oxoid trypsin dissolved in 10mM pH7.4 Na2HPO4-NaH2PO4 buffer) , and continue to incubate at 37°C for 12-16 hours. Measure the diameter of the transparent ring of sterile growth. Calculation of antibacterial activity: antibacterial activity unit U=(diameter mm-3 of antibacterial ring)×10. The logarithm value of the antibacterial peptide concentration is used as the abscissa, and the antibacterial activity is used as the ordinate to make a regression equation, and the minimum concentration MEC of the antibacterial peptide that can inhibit the growth of bacteria is calculated. The bacterial strains were obtained from the First Affiliated Hospital of Kunming Medical College. The test was repeated four times and the average value was taken. The results are shown in Table 1.

Table 1. Bacteriostatic activity of human-derived antimicrobial peptide SgI-29

Microorganisms   Minimum Effective Concentration MEC (μg/ml) SgI-29 SgI-29R7K SgI-25 Escherichia coli ATCC 25922Escherichia coli ML-3 5PEscherichia coli clinic strain 1Escherichia coli clinic strain 2Bacillus pyocyaneus ATCC 27853Bacillus pyocyaneus CMCCB 10104Bacillus pyocyaneus PA 01Bacillus pyocyaneus clinic strainStaphylococcus aureus ATCC 25923Staphylococcus aureus ATCC 43300 2.8317.4333.5563.9375.2927.33412.86410.405＞100＞100   3.0626.8824.0793.6384.9966.37810.63811.909 not determined not determined 5.36110.2316.8237.23111.73415.63418.88525.362 not determined not determined Candida albicans ATCC 10231Pichia pastoris GS 115 20.52318.244 25.76821.224 30.66524.315

Explanation of the bacteria and fungi strains used in the experiment:

Staphylococcus aureus (Staphylococcus aureus) ATCC 43300 is a methicillin-resistant Staphylococcus aureus ATCC standard strain, resistant to penicillins and β-lactams, and sensitive to vancomycin.

Escherichia coli (Escherichia coli) ML-35P is an ampicillin-resistant standard strain.

Escherichia coli (Escherichia coli) clinical drug-resistant strains 1 and 2 are clinical isolates producing extended-spectrum β-lactamase, resistant to penicillin, cephalosporins 1st, 2nd, 3rd, and 4th Can, aminoglycoside sensitive.

The clinical resistant strain of Pseudomonas aeruginosa (Bacillus pyocyaneus) is a multi-drug resistant clinical isolate, which is resistant to all kinds of antibiotics currently used clinically, including Taineng and MEM.

The fungi Candida albicans (Candida albicans) ATCC 10231 and Pichia pastoris (Pichia pastoris) GS115 are standard strains.

It can be seen from Table 1 that the synthetic human antimicrobial peptide SgI-29 and its amino acid substitution and amino acid deletion derivatives have significant inhibitory effects on the growth of bacteria and fungi, and can be used as antimicrobial substances for the preparation of antimicrobial infection preparations.

Sequence Listing.SEQ

<110> Kunming Institute of Zoology, Chinese Academy of Sciences

<120> Human antimicrobial peptides and their antimicrobial applications

<160>6

<210>1

<211>29

<212>PRT

<213> Homo sapiens

<400>1

His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe

1 5 10 15

His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

20 25

<210>2

<211>29

<212>PRT

<213> Artificial sequence

<400>2

His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe

1 5 10 15

His Arg Val Val Ile His His Lys Gly Gly Lys Ala His

20 25

<210>3

<211>25

<212>PRT

<213> Artificial sequence

<400>3

Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg

1 5 10 15

Val Val Ile His His Lys Gly Gly Lys

20 25

<210>4

<211>87

<212>DNA

<213> Homo sapiens

<400>4

cataataaac aagaaggcag agaccatgat aaatcaaaag gtcattttca cagggtagtt 60

atacaccata aaggaggcaa agctcat 87

<210>5

<211>87

<212>DNA

<213> Artificial sequence

<400>5

cataataaac aagaaggcaa agaccatgat aaatcaaaag gtcattttca cagggtagtt 60

atacaccata aaggaggcaa agctcat 87

<210>6

<211>75

<212>DNA

<213> Artificial sequence

<400>6

aaacaagaag gcaaagacca tgataaatca aaaggtcatt ttcacagggt agttatacac 60

cataaaggag gcaaa 75

Claims (9)

1. isolating human antibiotic peptide, it is characterized in that said human antibiotic peptide is by the aminoacid sequence shown in the sequence table SEQ ID NO:1: His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His forms.

2. substitute and have the antibacterial peptide of anti-microbial activity derived from the isolating human antibiotic peptide amino-acid residue of claim 1, it is characterized in that said antibacterial peptide is by the aminoacid sequence shown in the sequence table SEQ ID NO:2: His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His forms.

3. derived from a plurality of aminoacid deletion of claim 1 and have the antibacterial peptide of anti-microbial activity, it is characterized in that said antibacterial peptide is by being selected from the aminoacid sequence shown in the sequence table SEQ ID NO:3: Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys forms.

4. the preparation method of claim 1,2 or 3 described antibacterial peptides is characterized in that adopting mechanochemical method synthetic.

5. an isolating nucleic acid molecule is characterized in that any described antibacterial peptide of said nucleotide sequence coded claim 1 to 3.

6. a construction is characterized in that described construction contains the nucleotide sequence of any described antibacterial peptide of coding claim 1 to 3.

7. a cell is characterized in that described cell contains any described antibacterial peptide of claim 1 to 3, the said nucleotide sequence of claim 5 or the described construction of claim 6.

8. method for preparing any said antibacterial peptide of claim 1 to 3 is characterized in that this method comprises:

(1) under the condition that is fit to any described peptide expression of claim 1 to 3, cultivates the described cell of claim 7;

(2) isolate any described antibacterial peptide of claim 1 to 3.

9. the purposes of any described antibacterial peptide of claim 1 to 3 is characterized in that: the application of described antibacterial peptide in the medicine of preparation anti-microbial infection.

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