CN104910265B - A kind of gene and the application of Hejiang spine frog antibacterial peptide and its coded sequence - Google Patents

Abstract

The invention discloses a kind of Hejiang spine frog cathelicidin antibacterial peptide cathelicidin PR1, and it is with SEQ ID NO in sequence table：Nucleotide sequence coded albumen shown in 1, or by SEQ ID NO：2 amino acid residue sequence passes through substitution, missing or the addition of more than one amino acid residue, and has and SEQ ID NO：2 amino acid residue sequence is identical active by SEQ ID NO：Protein derived from 2.Present invention clone obtains Hejiang spine frog antibacterial peptide cathelicidin PR1 encoding gene, and has synthesized cathelicidin PR1 using the method for chemical synthesis.The antibacterial peptide molecular weight is small, and in the amphipathic structure of α spirals, bactericidal action is fast, to Pseudomonas Maltophilia 7404, staphylococcus aureus（Song）, Bacillus cereus, bacillus subtilis, Candida albicans 08030102 and Candida glabrata 08A802 there is strong killing action.In addition, also there is extremely low hemolytic activity and antioxidation activity, without hemagglutination activity.

Description

A kind of gene and the application of Hejiang spine frog antibacterial peptide and its coded sequence

Technical field

The present invention relates to molecular genetics field, the gene of especially a kind of Hejiang spine frog antibacterial peptide and coded sequence is with answering With.

Background technology

With the extensive and incorrect use of the conventional antibiotics such as penicillin, microorganism generates more to conventional antibiotic Carry out stronger tolerance, the microorganism that can be largely resistant to the conventional antibiotics such as penicillin completely clinically occur, Existing antibiotic is helpless to these pathogenic microorganisms.Antibacterial peptide is a kind of new antimicrobial polypeptide, most of anti- Bacterium peptide molecular weight is less than 10000Da, positively charged, rich in hydrophobic bases, can form amphipathic structure.The sterilization machine of antibacterial peptide System is mainly attracted by electrostatic interaction and is attached to electronegative bacterial cell membrane surface, further in bacterial cell membrane The upper hole for forming cross-film, causes leaking for bacterial cell contents, so as to cause the death of bacterial cell.And conventional antibiotic Some enzymes mainly acted in bacterial cell.Just because of the difference of the mode of action, the peptide-mediated bactericidal action of antibacterial Significantly faster than conventional antibiotic, and be not easy to make bacterium produce tolerance.In addition, increasing document report shows to resist Bacterium peptide also has other bactericidal mechanisms, such as suppresses bacteria cell wall synthesis, change bacterial cell plasma membrane suppresses barrier film and formed, swashed Work autolysin, suppress intracellular enzyme activity, the synthesis for suppressing DNA, RNA and protein etc..

Cathelicidin families antibacterial peptide have efficiently, wide spectrum, stably, be not likely to produce drug resistance, tool selective toxicity The features such as, gained the name because its precursor peptide includes one section of highly conserved middle cathelicin region.Cathelicidin families Antibacterial peptide is an important antibacterial peptide family in vertebrate body, is the important component in vertebrate innate immune system, It played an important role in vertebrate resists external microbe invasive procedure.Cathelicidin families are work(more than one The host defense peptide family of energy, played an important role in the first line of defence for resisting external microbe infection.To being at present Only, it is found that in vivo from various vertebrates (including mammality, birds, reptiles, amphibian animal and fish species) Cathelicidins families antibacterial peptide.

Its precursor of Cathelicidin is by N- end signal peptides region (29~30 amino acid residues), conservative centre Cathelin regions (94~114 amino acid residues) and the C- terminal mature peptides region (12~100 residues) of high special Form.Family's antibacterial peptide has the antimicrobial acivity of wide spectrum, to Gram-positive and negative bacteria, fungi, mould, protozoon It is active with part enveloped virus.Cathelicidin antibacterial peptides are rich in positively charged amino acid residue, seldom or are free of Negatively charged residue, therefore all positively charged, cationization degree is higher compared with other families, such as with Defensins, elder brother Worm antibacterial peptide is compared with Ranidae skin-derived antibacterial peptide, and antibacterial activity is stronger, and minimal inhibitory concentration can reach μM, and is sterilized and made With rapid, some can kill the germ being clinically separated in 1 minute.It is most of in vitro antimicrobial test Cathelicidins can quickly kill extensive microorganism under the concentration of micromole and sub-micromolar.Some Cathelicidins, which is clinically separated drug-fast bacteria to many, has extremely strong activity, if the PR-39 in pig source is to methicillin resistance Staphylococcus aureus activity is extremely strong.

The multifunctionality of Cathelicidin antibacterial peptides not only shows antimicrobial acivity, participates in exempting from as early warning molecule Epidemic disease regulation reaction, also have and such as suppress tissue damage, promote wound reparation and angiogenesis, acted on reference to endotoxin. These functions of Cathelicidin families antibacterial peptide make be expected to turn into its today emerged in multitude in antibiotic-resistant pathogenic strains The clinical antibacterials of a new generation.

Research finds that Cathelicidin families antibacterial peptide generally carries a large amount of positive charges, and band can be formed when in membrane environment The surface of positive charge, it is attached to using electrostatic interaction on negatively charged bacterial cell membrane.It is most Cathelicidins has amphipathic alpha-helix or β-lamellar structure, and it is thin that hydrophobic side can be attached to negatively charged bacterium rapidly On after birth, insert inside film, water-wet side causes damaged membrane outside film, and permeability becomes big, ultimately results in bacterium content Beyond the region of objective existence lets out death.For this unique mechanism of action is compared with antibiotic, it is difficult to make bacterium production property drug resistance.

Cathelicidins families antibacterial peptide has played important in vertebrate resists external microbe invasive procedure Effect, family's antibacterial peptide have strong antibacterial activity, weak cytotoxicity and are not likely to produce the features such as drug resistance.Its excellent property The mechanism of action with uniqueness is the good candidates molecule and template of novel polypeptide class antibacterials exploitation.At present, foreign countries are to a variety of Cathelicidins antibacterial peptides are researched and developed, and are faced using it as the existing a variety of entrance of anti-infective and anti-inflammation drugs of template Bed experimental stage, as Iseganan (being transformed by the cathelicidin antibacterial peptides protegrin of pig) is used to treat breathing The infection of system and mucous membrane of mouth, Omiganan (being transformed by the cathelicidin antibacterial peptides Indolicidin of ox) are used In treat bacillary and fungal infection, etc..

The food-safe concern of current mankind is increasingly paid attention to, and preservative, the requirement of antistaling agent are more and more tighter in food Lattice, the demand of natural antiseptic agent is caused to surge, cathelicidin antibacterial peptides has a broad antifungal spectrum, activity are strong, the storage for food With it is fresh-keeping, not only safety but also it is healthy, the life to people is brought into more happiness.Feed industry is the crucial production in industrial and agricultural production Industry, conventional antibiotic plays an important role in terms of animals and plants disease is prevented and treated, but its side effect and the increasing of drug resistance Add and gradually show its inferior position, research and develop green and healthy antibacterial peptide, be used for poultry and aquaculture as feed addictive, reduce Dependence of the aquaculture to medicine, its meaning and potential value are huge.Application of the cathelicidin antibacterial peptides in these fields It is great prospect.

The Hejiang spine frog is under the jurisdiction of Amphibia (Amphibia), Anura (Caudata), Ranidae (Ranidae), spine Rana (Paa), be Chinese unique wheat, build is very large, survive in height above sea level 650-1500 rice mountain stream and its near.Pole currently is studied to it It is few, mainly to studying with finding and be distributed.Research about Hejiang's spine frog antibacterial peptide is also without any report.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of Hejiang spine frog antibacterial peptide cathelicidin-PR1.

The present invention also technical problems to be solved are to provide the coded sequence of above-mentioned albumen.

The present invention also technical problems to be solved are to provide above-mentioned albumen and are preparing food additives or animal feed addition The application of agent.

What the present invention was realized in：A kind of Hejiang spine frog (Paa robertingeri) cathelicidin antibacterial peptides Cathelicidin-PR1, it is with SEQ ID NO in sequence table：Amino acid sequences encoded albumen shown in 2.

Described Hejiang spine frog antibacterial peptide cathelicidin-PR1 has SEQ ID NO in sequence table：Amino shown in 2 Acid sequence；Hejiang spine frog antibacterial peptide cathelicidin-PR1 is α-helixstructure polypeptide, containing 29 amino acid residues, is divided Son amount is 3195.88Da, isoelectric point 10.59, has an intramolecular disulfide bond.

Hejiang spine frog antibacterial peptide cathelicidin-PR1 encoding gene, it is one of following nucleotides：

(1) SEQ ID NO in sequence table：Nucleotide sequence shown in 1；

(2) SEQ ID NO in polynucleotide：The polynucleotides of amino acid sequence shown in 2.

Applications of the Hejiang spine frog antibacterial peptide cathelicidin-PR1 in antimicrobial agents are prepared.

Beneficial effect

Present invention clone obtains Hejiang spine frog antibacterial peptide cathelicidin-PR1 encoding gene, and utilizes chemical synthesis Method synthesized cathelicidin-PR1.The antibacterial peptide molecular weight is small, to the Pseudomonas Maltophilia, golden yellow being clinically separated Color staphylococcus, Bacillus cereus, bacillus subtilis, Candida albicans 08030102 and Candida glabrata have strong kill The effect of going out.Additionally there is low hemolytic activity, antioxidation activity and hemagglutination activity.

Embodiment

According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real Apply and be merely to illustrate the present invention described by example, without should be also without limitation on this hair described in detail in claims It is bright.

Embodiments of the invention 1：Hejiang's spine frog antibacterial peptide cathelicidin-PR1 gene clonings.

1) Hejiang spine frog skin Total RNAs extraction：

1. with scissors clip adult Hejiang spine frog skin of back, to be grown up, nail cover size is advisable, and rapid input is equipped with RNA In the centrifuge tube for protecting liquid (Sample Protector for RNA/DNA, TaKaRa), protected from RNA and frog skin is taken out in liquid Sample, it is put into rapidly in the mortar for filling liquid nitrogen after cleaning one time with DEPC water, adds liquid nitrogen grinding into powder, be transferred to EP pipes In, 1m1 Total RNAs extractions buffer solution (Trizol, U.S.'s Invitrogen Products) is added, is fully mixed, and after 4 DEG C, 12000rpm centrifuges 10min.

2. centrifuging and taking supernatant, adding 0.2ml chloroformic solutions, acutely mixing, room temperature is placed 10 minutes, then with 4 DEG C, 12000rpm is centrifuged 10 minutes, reject precipitation.

3. supernatant adds isometric isopropanol, room temperature is placed 10 minutes, and with 4 DEG C, 12000rpm is centrifuged 10 minutes, is collected Precipitation is washed once with 75% (V/V) ethanol, is dried, ttom of pipe sediment is rana margaretae skin total serum IgE.

2) Hejiang spine frog skin cDNA library is built：Using Clontech companies In-Fusion SMARTer TM Directional cDNA Library Construction Kit。

(1) chains of cDNA first synthesis (mRNA reverse transcriptions)：

3'In-fusion SMARTer CDs Primer/3 ' Primer and SMARTTM IV provided using kit Oligonucleotide, add the component reverse transcriptions such as the PowerScriptTM reverse transcriptases synthesis chains of cDNA mono-.

1. the preparation of component 2：

Reagent	Dosage
		5xFirst-stand Buffer	1μl
DTT(100mM)	0.125μl
		dNTP Mix(10mM)	0.5μl
Smarter V oligonucleotide(12Um)	0.5μl
		RNase Inhibitor	0.125μl

After preparing, mix, be positioned on ice, after the completion for the treatment of that component 1 is handled, before the mixing of component 1,2, add into system 2 Enzyme

2. formulation components 1：

A：Prepared in PCR pipe (no DNase and RNase)：

B：Component 1 mixes, if PCR pipe wall has solution, can be centrifuged for a moment on micro centrifuge.

c：72 DEG C, 3min, latter 42 DEG C, 2min, it is put into after having handled in ice chest.

3. component 2 is added in component 1, can be centrifuged on centrifuge, 42 DEG C, 90min, 68 DEG C, 10min in PCR instrument, Synthesize cDNA1 chains.

(2) the second chain is expanded using long end polymeric PCR (LD-PCR) method (agents useful for same is CLONTECH companies In-Fusion SMARTer TM Directional cDNA Library Construction Kit are built It is equipped with the kit of storehouse)

Reaction system：

Reagent	Dosage
		Deionized H2O	20μl
10x Advantage 2 PCR Buffer	2.5μl
		First-strand cDNA	0.5μl
50x dNTP Mix(10mM)	0.5μl
		5’PCR Primer IIA(12μM)	0.5μl
3’IF SMARTer PCR Primer(12μM)	0.5μl
		50x Advantage 2 Polymerase Mix	0.5μl
Total Volume per reaction	25μl

PCR conditions:95℃ 1min

20cycles:

95℃ 15sec

65℃ 30sec

68℃ 6min

After circulation terminates, -80 DEG C of the cDNA double-strands that will be synthesized in centrifuge tube preserve.

(3) Hejiang spine frog antibacterial peptide cathelicidin-PR1 gene clonings are screened：

Two forward primers are designed according to Ranidae antibacterial peptide signal peptide area conserved sequence and enter performing PCR amplification, its sequence is：

P1(SEQ ID No：3)：5'-AGATGAAGGTCTGGCAGTGTGTG-3'

P2(SEQ ID No：4)：5'-GTGTGCTATGGATCTCCGCTCTC-3'

PCR amplification reverse primers are CLONTECH companies In-Fusion SMARTer TM Directional cDNA 3 '-PCR primer in Library Construction Kit, its sequence are 5'-CTAGAGGCCGAGGCGGCCGACATG- 3'.With Hejiang's spine frog cDNA library (10 times of dilution) for template, first PCR is matched with P1 and 3'-PCR primers.Produced again with P1 PCR Thing is template, and half Chao Shi PCR clones are carried out with P2.

1PCR is expanded：Using the chains of cDNA bis- for diluting 10 times as template, enter performing PCR using forward primer and reverse primer and expand Increase：

PCR system：

Reagent	Dosage
		PCR water	38μl
10x Buffer	5μl
		dNTP	4μl
The PCR of reverse primer 3 '	1μl
		Forward primer P1	1μl
The chains of cDNA bis- (10x)	1μl
		RTaq enzymes	0.25μl
Total Volume per reaction	50μl

To make PCR reactions more abundant, effect is more preferable, and 50 μ l system is distributed into the reaction of the μ l systems of two pipe 25.

To make PCR reactions more abundant, effect is more preferable, and 50 μ l system is distributed into the reaction of the μ l systems of two pipe 25.

PCR conditions:①94℃ 5min

②30cycles:

94℃ 30sec

57.4℃ 30sec

72℃ 1min

③72℃ 10min

After reaction terminates, 5 μ l samples, 1% agarose gel electrophoresis testing goal band are taken.

2 half Chao Shi PCR are expanded：

Using P1 PCR primer as template, matched with P2 and 3 '-PCR Primer primers, reaction condition ibid walks with method PCR is expanded, and after reaction terminates, takes 40 μ l samples, 1% agarose gel electrophoresis testing goal band.

Tiangeng company glue reclaim kit (the common fine jades of TIANgel Midi Purification Kit are used after the completion of amplification Sepharose DNA QIAquick Gel Extraction Kits (centrifugation column type)) carry out purpose fragment recovery.The purpose fragment of recovery is connected to pMD19- Carrier T (Takara, Dalian), it is transformed into the DH5 α competent cells that CaCl2-MgCl2 methods prepare.Coated plate simultaneously carries out ammonia benzyl green grass or young crops Mycin and blue hickie Double Selection, picking single bacterium colony detect Insert Fragment size with M13 primer PCRs.Picking positive bacterium colony, shakes bacterium Plasmid is extracted, nucleotides survey is carried out using AppliedBiosystems DNA sequencer, modelABI PRISM 377 Sequence.

Measurement result：

The gene for encoding Hejiang's spine frog antibacterial peptide cathelicidin-PR1 precursors is held to 3 ' terminal sequence SEQ ID from 5 ' No：Shown in 1.

The sequence table of Hejiang's spine frog antibacterial peptide cathelicidin-PR1 gene nucleotides is：Sequence length is 587 alkali Base, sequence type：Nucleic acid, chain number：It is single-stranded, topology：Straight-chain, sequence species：CDNA, source：Hejiang's spine frog skin.

Embodiment 2：Hejiang spine frog antibacterial peptide cathelicidin-PR1 chemical synthesis.

Ith, Hejiang spine frog antibacterial peptide cathelicidin-PR1 chemical synthesis process：The mature peptide ammonia derived according to gene Base acid sequence, its complete sequence is synthesized with automatic Peptide synthesizer (433A, Applied Biosystems), passes through HPLC reversed-phase columns Chromatograph desalination.

IIth, the measure of molecular weight and isoelectric point (pI) uses ProParam tool (http://web.expasy.org/ Protparam/) online website tools prediction.

IIIth, the Hejiang spine frog antibacterial peptide cathelicidin-PR1 of purifying identifies that its is pure with high-efficient liquid phase chromatogram HPLC method Degree, amino acid sequence structure is determined with automatic Protein Sequencer.

Hejiang spine frog antibacterial peptide cathelicidin-PR1 is that Hejiang's spine frog antibacterial peptide cathelicidin-PR1 genes are compiled A kind of α-helixstructure polypeptide of code, contains the electricity such as 29 amino acid residues, theoretical molecular 3195.88Da, theory Point is 10.59, has an intramolecular disulfide bond (C3-C7).

Hejiang spine frog antibacterial peptide cathelicidin-PR1 complete sequences such as SEQ ID No：Shown in 2.Embodiment 3：Hejiang spine Frog antibacterial peptide cathelicidin-PR1 pharmacological evaluations：

1st, Hejiang spine frog antibacterial peptide cathelicidin-PR1 antibacterial activities detect：

Respectively picking be stored on inclined-plane test strain (Escherichia coli ATCC25922, it is lung gram bacillus 08B343, motionless Bacillus 2178, Pseudomonas Maltophilia 7404, pseudomonas aeruginosa ATCC27853, staphylococcus aureus ATCC27853, Staphylococcus aureus (Song), Bacillus cereus, bacillus subtilis, Candida albicans 08022710, Candida albicans 08030102nd, Candida glabrata 08A802 and Salmonella paratyphi A) it is spread evenly across LB solid mediums and (is purchased from Qingdao Hai Bo Bioisystech Co., Ltd) on flat board, the filter paper of the 0.5cm diameters by sterilizing is placed in media surface, is added dropwise The 2mg/ml of the sterile deionized water μ l of Hejiang's spine frog antibacterial peptide cathelicidin-PR1 sample solutions 10 are dissolved in, in 37 DEG C It is inverted culture 16-24 hours, whether observation inhibition zone is formed.If sample has antibacterial activity, can be formed around filter paper Clear ＆ Transparent inhibition zone, inhibition zone show that more greatly sample antibacterial activity is stronger.

2nd, Hejiang spine frog antibacterial peptide cathelicidin-PR1 minimal inhibitory concentrations (Minimum Inhibitory Concentration) determine：

It will appear from test strain (acinetobacter calcoaceticus 2178, Pseudomonas Maltophilia 7404, the staphylococcus aureus of inhibition zone (Song), Bacillus cereus, bacillus subtilis, Candida albicans 08030102 and Candida glabrata 08A802) it is inoculated into LB In fluid nutrient medium (Qingdao Hai Bo Bioisystech Co., Ltd), 37 DEG C of shaken cultivations to exponential phase, then with fresh LB Fluid nutrient medium will be cultivated to the nutrient solution of exponential phase and be diluted to 2 × 10⁵Cfu/ml is stand-by.

The inoculum of the above-mentioned dilutions of 1.9mL is taken, the Hejiang spine frog for adding the 2mg/ml through 0.22m apertures membrane filtration resists As the first pipe, the first pipe takes out 1mL after mixing and added in the 2nd pipe bacterium peptide cathelicidin-PR1 sample solutions 0.1mL, according to Secondary doubling dilution (referring to table 1), suction out 1mL from the 9th pipe and discard, the 10th piping control tube.

The dilution process of table 1

37 DEG C of placement slowly vibrating culture 18 hours, determines light absorbs at 600nm wavelength after above-mentioned each pipe is mixed.Most Small Mlc is the minimum sample concentration of invisible bacterial growth.As a result it is as shown in table 2.

From table 2, Hejiang spine frog antibacterial peptide cathelicidin-PR1 is to staphylococcus aureus (Song), cured sample gemma Bacillus, bacillus subtilis, Candida albicans 08030102 and Candida glabrata 08A802 have stronger antibacterial activity.Show Treatment in terms of it has certain application potential, particularly fungal infection in terms of the treatment of these pathogenic bacterial infections is (common The bacterium for causing fungal infection be mainly Candida albicans and Candida glabrata).

The 2-in-1 river spine frog antibacterial peptide cathelicidin-PR1 antibacterial activities of table

MIC：Minimal inhibitory concentration, result above repeat laboratory mean values to be independent three times.

3rd, Hejiang spine frog antibacterial peptide cathelicidin-PR1 sterilizes dynamic experiment

Detected from bacillus cereus, Negative control Sterile ultra-pure water, positive control ampicillin are set.Choose The bacterial colony newly activated is taken, is seeded to fresh sterile LB liquid medium, 37 DEG C, 200rpm shaken cultivation 10-16h, extremely Exponential phase, OD600 values are surveyed, are diluted to 105CFU/ml；Polypeptide sample, ampicillin are added into bacterium solution, is diluted to each From 5 times of MIC of final concentration, negative control is done with sterilized water.Start timing to add polypeptide, 37 DEG C of cultures, respectively 0min, 10min, 20min, 30min, 45min, 1h, 1.5h and 2h time point respectively take 10 μ l bacterium solutions, dilute 100 times, take 100 μ l to dilute bacterium Liquid applies LB flat boards, and 37 DEG C are inverted culture 10-16h to growing bacterium colony.Repetition three is parallel, and bacterium colony counts, and averages.

As a result show that cathelicidin-PR1 45min under 5 times of MIC concentration can kill the waxy gemma being clinically separated Bacillus, and positive control ampicillin then needs 1.5h to kill the bacterium completely, cathelicidin-PR1 to move faster Mechanics activation plays bactericidal action.

4th, Hejiang spine frog antibacterial peptide cathelicidin-PR1 hemolytic activities determine：

The Healthy People blood of collection is mixed into anti-freezing with Alsever's Solution, brine 2 times is simultaneously resuspended into 10⁷-10⁸cell/ml Suspension.Hejiang's spine frog antibacterial peptide of various concentrations of the good red cell suspension of above-mentioned dilution with being dissolved in physiological saline Cathelicidin-PR1 samples mix, 37 DEG C of insulation 30min, centrifuge 5min then at 1000rpm, supernatant is surveyed in 540nm to be inhaled Receipts value.Negative control uses physiological saline, and positive control uses Triton X-100, and percent hemolysis calculates as follows： Percent hemolysis H%=A_Sample-A_{Negative control}/A_{Positive control}× 100%.As a result when to show sample concentration be 100 μ g/ml, Cathelicidin-PR1 percent hemolysis is 3.87%；In 200 μ g/ml, hemolysis rate is only 1.78%.Explanation Cathelicidin-PR1 has extremely low hemolytic activity, will not cause human erythrocyte rupture dissolving and produce wound to human body Evil, therefore it is very beneficial in the further development and application of field of medicaments.

5 Hejiang's spine frog antibacterial peptide cathelicidin-PR1 hemagglutination activities

Prepare Alsever's Solution：Weigh 0.8g sodium citrates, 0.055g citric acids, 2.05g glucose, 0.42g NaCl is molten In 80ml deionized waters, pH to 6.1 is adjusted, adds deionized water to be settled to 100ml, autoclaving, 4 DEG C of refrigerators preserve.

Prepare TBS-buffer：0.606g Tris base, 0.584g NaCl are weighed, are dissolved in 80ml deionized waters, are adjusted PH to 7.5, adds deionized water to be settled to 100ml.

Prepare TBS+Ca2+-buffer：Weigh 0.606g Tris base, 0.584g NaCl, 0.112g CaCl₂It is dissolved in In 80ml deionized waters, pH to 7.5 is adjusted, adds deionized water to be settled to 100ml.

Healthy People blood is gathered, Healthy People blood and Alsever's Solution are pressed 1:1 ratio mixes.TBS-buffer and TBS+ is used respectively Ca²⁺- buffer washs haemocyte, 1000r centrifugation 5min, untill the no longer aobvious red of supernatant.The haemocyte washed is distinguished Use TBS+Ca²⁺- buffer and TBSCa-buffer are diluted to finite concentration.Then various concentrations testing sample and haemocyte are existed (96 hole V-type plate blood coagulation plate) gently mixes in blood clotting plate hole, is stored at room temperature 45min or so, observes result.Experiment is done three and put down OK, sample dissolving medium is as negative control.

As a result show cathelicidin-PR1 concentration be 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 6.25 μ g/ml and During 6 concentration such as 3.125 μ g/ml, no matter Ca is whether there is²⁺In the presence of, cathelicidin-PR1 does not coagulate to human red blood cells Collection.Human body will not be damaged because causing erythrocyte agglutination when being used for human body as medicine, be advantageous to the exploitation profit of medicine With.

Sequence of the present invention and mark point row are as follows：

(1) SEQ ID NO.1 information

(i) sequence signature：

(A) length：87bp

(B) type：Nucleotides

(C) chain：It is single-stranded

(D) topological structure：Straight-chain

(ii) molecule type：Nucleotides

(iii) sequence description：SEQ ID NO.：1

(2) SEQ ID NO.2 information

(i) sequence signature：

(A) length：29a.a

(B) type：Amino acid

(C) chain：It is single-stranded

(D) topological structure：Straight-chain

(ii) molecule type：Protein

(iii) sequence description：SEQ ID NO.：2

(3) SEQ ID NO.3 information

(i) sequence signature：

(A) length：23bp

(B) type：Nucleotides

(C) chain：It is single-stranded

(D) topological structure：Straight-chain

(ii) molecule type：Nucleotides

(iii) sequence description：SEQ ID NO.3

(4) SEQ ID NO.4 information

(i) sequence signature：

(A) length：23bp

(B) type：Nucleotides

(C) chain：It is single-stranded

(D) topological structure：Straight-chain

(ii) molecule type：Nucleotides

(iii) sequence description：SEQ ID NO.4

(5) SEQ ID NO.5 information

(i) sequence signature：

(A) length：24bp

(B) type：Nucleotides

(C) chain：It is single-stranded

(D) topological structure：Straight-chain

(ii) molecule type：Nucleotides

(iii) sequence description：SEQ ID NO.5.

Claims (4)

1. A kind of 1. Hejiang spine frog cathelicidin antibacterial peptides cathelicidin-PR1, it is characterised in that：The Hejiang spine frog Cathelicidin antibacterial peptides cathelicidin-PR1 is SEQ ID NO in sequence table：It is amino acid sequences encoded shown in 2 Albumen.
2. 2. Hejiang spine frog antibacterial peptide cathelicidin-PR1 according to claim 1, it is characterised in that：Described Hejiang Spine frog antibacterial peptide cathelicidin-PR1 is SEQ ID NO in sequence table：Amino acid sequence shown in 2；The Hejiang spine frog resists Bacterium peptide cathelicidin-PR1 is α-helixstructure polypeptide, containing 29 amino acid residues, molecular weight 3195.88Da, etc. Electricity point is 10.59, has an intramolecular disulfide bond.
3. 3. a kind of Hejiang spine frog antibacterial peptide cathelicidin-PR1 as claimed in claim 1 encoding gene, its feature exist In：It is one of following nucleotides：(1) SEQ ID NO in sequence table：Nucleotide sequence shown in 1；(2) in polynucleotide SEQ ID NO：The polynucleotides of amino acid sequence shown in 2.
4. 4. a kind of Hejiang spine frog antibacterial peptide cathelicidin-PR1 as claimed in claim 1 is preparing anti-thermophilic malt vacation unit cell Bacterium, staphylococcus aureus, Bacillus cereus, bacillus subtilis, Candida albicans 08030102 or Candida glabrata medicine Application in thing.