CN1367177A

CN1367177A - Antibactrial polypeptide separated from biological living body

Info

Publication number: CN1367177A
Application number: CN 02115451
Authority: CN
Inventors: 董先智; 徐辉碧; 黄开勋; 刘琼; 周军
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2002-01-22
Filing date: 2002-01-22
Publication date: 2002-09-04
Anticipated expiration: 2022-01-22
Also published as: CN1176944C

Abstract

The present invention relates to an antibacterial polypeptide separated out from the loach. The antibacterial polypeptide is a monochain polypeptide containing no subunit, its molecular weight is 9300-10300, its isoelectric point PI is 4.68-4.88, and N-end sequence label is CFGWN. This invention also provides its extraction method for the said polypeptide, and the polypeptide can be used as inflammation-relieving medicine having no toxic-side-effect for external application, food preservative and antioxidant.

Description

Isolated a kind of antimicrobial polypeptide from living organisms

Technical field

The invention belongs to field of biological product, adopt biochemical method to isolate a kind of antimicrobial polypeptide from living organisms, specifically is to isolate a kind of antimicrobial polypeptide from loach.

Background technology

Antibacterial peptide (antimicrobialpeptides) be in the organism through inducing a kind of endogenous polypeptide of synthetic, playing an important role aspect the invasion of body opposing cause of disease, more be considered to lack the important defence composition of specific immune function biology.The mechanism of action of antibacterial peptide is obviously different with microbiotic commonly used, it mainly comes killing bacteria by forming ionic channel at surface of cell membrane, obviously strengthen in the present resistance of bacterium, and seek new microbiotic very under the situation of difficult, it is significant to seek new antibacterial peptide.

Loach is the Cobitidae animal, according to documents such as " Chinese medicine voluminous dictionaries " record, and the meat of loach or all be used as medicine, return the spleen channel, have and invigorate the spleen and benefit qi, clearing heat and detoxicating, protect effects such as liver removing jaundice, at folks of china, disturb external application for curing osteomyelitis with fresh loach, the history in existing thousands of years of diseases such as acute mastitis (Jia Yuhai work, blue book on Chinese herbal medicine-Chinese ocean lakes and marhshes pharmacology, Xueyuan Press, Beijing, 1996, p187).But it is less always to the report of the functional component in the loach both at home and abroad.The someone has reported a kind of 21 the amino acid whose antibacterial peptides (Chan Baepark.FEBS Letters 411,173-178,1997) that contain in the loach in recent years, and its anti-microbial activity is 6 times of at present the strongest in the world antibacterial peptide Magainin; Pass people's such as light patent application " functional food for protecting liver made from loach and control hepatitis medicament and preparation method thereof " (patent No. 00114614.9) by the emperor himself, reported isolated a kind of polysaccharide in the loach, experimentation on animals shows to have liver protection effect preferably.It seems that from existing document a kind of molecular weight Mr is about 9800, iso-electric point PI is about 4.78 the polypeptide with anti-microbial activity and also never appears in the newspapers.

Summary of the invention

The present invention has isolated a kind of new polypeptide MAPP with anti-microbial activity again from loach.Its feature is: a kind of single chain polypeptide that does not contain subunit, its molecular weight are 9300～10300, and iso-electric point PI is 4.68～4.88, and N terminal sequence label is CFGWN.This peptide species can show restraining effect to various bacteria such as Bacillus subtilus, intestinal bacteria.It also has good inhibitory effect to the autoxidation of pyrogallol simultaneously.This antibacterial peptide can be used as nontoxic, the externally applied anti-inflammation medicine that has no side effect and food preservatives and antioxidant.The separation method of above-mentioned antibacterial peptide is as follows:

Sepn process A. gets the healthy loach that lives and is prepared into homogenate, and homogenate vacuum-drying obtains faint yellow protein example after ethyl acetate and acetone are handled respectively.This sample is crossed Sephadex G-50 post collect the maximum elution peak, freeze-drying is after DEAE-Cellulose 52 anion-exchange chromatographies are collected first elution peak, and freeze-drying is after the non-sex change electrophoretic separation of preparation property.The last groove damping fluid that uses is 0.01～0.2M Tris-base, 0.05～0.2MTricine, and 1～2mM SDS, electrophoresis to tetrabromophenol sulfonphthalein leading edge arrives the bottom under the 150V constant-pressure conditions.Take out laminar gel, longitudinally downcut the wide adhesive tape of 0.5cm at its edge, with coomassie brilliant blue staining and decolouring.Downcut the gel band of corresponding position on the laminar gel according to the position of second band on the adhesive tape, this band is carried out electroelution under non-sex change condition, elutriant freeze-drying after fully dialysing can obtain purified MAPP.MAPP is identified that (N-terminal sequence label, mass spectrum fingerprint image or amino acid composition analysis) carries out the information biology retrieval according to qualification result.The result shows that MAPP is a new polypeptide.

In addition, also can obtain MAPP by sepn process B.Method is as follows: loach protein group sample is reduced foreign matter content through the acetone pre-treatment, the sample of handling is carried out isoelectrofocusing (IEF) under non-sex change condition, downcut two IEF adhesive tape that width is identical from gel after the IEF, containing 0.05～0.2M Tris-base, 0.05～0.2M Tricine, 2～4mM sodium lauryl sulphate (SDS), pH be in 9.0 the buffer A balance carry out after 20～30 minutes second to polyacrylamide gel electrophoresis (PAGE), the acrylamide of use 8～16% and 3.3% methylene diacrylamide prepare native gel, and its pH is 9.0.Last groove damping fluid is 0.05～0.2M Tris-base, 0.1M Tricine, 1～2mM SDS.150V constant voltage electrophoresis to tetrabromophenol sulfonphthalein leading edge arrives the bottom; Subsequently two glue of PAGE gained are handled respectively, a glue dyes with Xylene Brilliant Cyanine G (CBB), the decolouring back is with backuping, another piece glue is then through electroblotting, working voltage is 50V, and the time is 1～2 hour, and protein spot is transferred on the nitrocellulose filter (NC film), use not contain SDS, pH is 9.0 buffer A; The NC film is through 0.05～0.2M Tris-Cl, and pH is that SDS final vacuum drying is removed in 7.5 damping fluid rinsing; The bacterium that distributes equably on the NC film makes its density reach 1 * 10 ³～1 * 10 ⁵CFU/mm ²After, with film place 1mm thick contain 0.00025～0.025% bromothymol blue, 0.00025～0.025% is phenol red, cultivated 24 hours for 37 ℃ on the extractum carnis solid medium of 0.5～3% N.F,USP MANNITOL.The bacterium of normal growth on the NC film and has the local bacterial growth of antibacterial peptide spot to be suppressed because its meta-bolites is acid thereby is glassy yellow around it, so and present aberration on every side, be the spot of dead color; Spot on spot on the NC film and the resulting gel of two-dimensional electrophoresis is contrasted, the spot corresponding to color spot on the NC film on the gel is downcut, the polypeptide in the gel is MAPP.The resulting MAPP of method as mentioned above has following physicochemical characteristics:

MAPP is an a kind of single chain polypeptide that does not contain subunit, and its relative molecular weight Mr is about 9800, and iso-electric point PI is about 4.78, and N terminal sequence label is CFGWN, and its amino acid composition sees Table 1.

The amino acid composition analysis of table 1 loach polypeptide MAPP

Total number of atnino acid/mol mol%

CYS 19 20.2

GLY 17 18

MET 11 11.7

VAL 9 9.5

TYR 9 9.5

ALA 8 8.5

SER 6 6.3

GLU 6 6.3

LEU 5 5.3

ASP 4 4.2

∑ 94 100

Description of drawings

MAPP is for Bacillus subtilus, and intestinal bacteria all have good inhibitory effect.Fig. 1 measures the bacteriostatic activity of MAPP to different bacterium for the plate bacteriostatic method, has represented the dose-effect relationship of MAPP bacteriostatic action; X-coordinate is: the concentration of MAPP (μ g/ml); Ordinate zou is: the diameter of observed inhibition zone (cm) on plate; Symbol zero is represented Bacillus subtilus among the figure, and symbol △ represents intestinal bacteria.MAPP has good inhibitory effect to the autoxidation of pyrogallol.Fig. 2 is the inhibiting rate of the MAPP of different concns to the autoxidation speed of pyrogallol.X-coordinate is: the percentage concentration of MAPP (%); Ordinate zou is: MAPP is to the inhibiting rate of the autoxidation speed of pyrogallol.The autoxidation speed of pyrogallol in the autoxidation speed of measuring pyrogallol under the 325nm condition respectively and after having added the MAPP of different concns, the calculation formula of inhibiting rate is:

Embodiment

The preparation of example 1.MAPP and purifying

A. roughing out: get healthy fresh loach (Misgurnus Anguillicaudatus Hubei product) 2000g, in clear water, raised 5～7 days, change water purification every day once.Pull loach elimination moisture content out, send into refiner 15,000g * 60min is prepared into homogenate, and homogenate obtains the 340g powder 4 ℃ of following vacuum-dryings after the pulverizing.Add 2000ml ,-10 ℃ ethyl acetate is separated supernatant-10 ℃ of extractions after 12 hours, is deposited in 0 ℃ of condition downstream drying, obtains 300g brown dry powder.The 0.1M Na that adds pH7.0 ₂HPO ₄～KH ₂PO ₄Damping fluid 1500ml, 4 ℃ are stirred extraction 24 hours.Extracting solution 10,000g * 10min centrifugation supernatant obtains the 900ml brown solution, the acetone that adds 3000ml-20 ℃ discards precipitation, collects supernatant, adds 5000ml-20 ℃ acetone again, freeze-drying after the collecting precipitation usefulness secondary water dissolution obtains the 3.0244g pale yellow powder;

B. gel permeation chromatography: taking by weighing 0.5g lyophilized powder crude product, to be dissolved in 15ml pH be in 8.0 the 0.05M Tris-Cl solution, high speed frozen centrifugation 15,000g * 10min, get supernatant and cross Sephadex G-50 post (2.6cm * 150cm),, control flow velocity 0.5ml/min with 0.05M Tris-Cl solution equilibria and wash-out, every pipe is collected 10ml, detect elution peak with 280nm, collect main elution peak, immediately freeze-drying;

C.DEAE-Cellulose 52 anion-exchange chromatographies.Earlier DEAE-Cellulose52 (DE-52) resin is handled with 0.4M HCl and 0.4M NaOH, used the 0.05M Tris-Cl damping fluid balance of pH8.2 again.Sample that previous step obtains is dissolved in the 0.05M Tris-Cl damping fluid, 4 ℃ of high speed frozen centrifugations 15,000g * 10min, get supernatant and cross DE-52 post (2.6cm * 25cm), use the NaCl linear gradient elution of 0.05～0.5M again, control flow velocity 0.3ml/min, every pipe is collected 3ml, 280nm detects elution peak, collects first elution peak, immediately freeze-drying;

D. non-sex change electrophoretic separation: the acrylamide of use 12.5% and 3.3% methylene diacrylamide prepare native gel, and its pH is 9.0.Last groove damping fluid is 0.1M Tris-base, 0.1M Tricine, and 1.5mM SDS, following groove damping fluid is 0.2M Tris-Cl, 150V constant voltage electrophoresis to tetrabromophenol sulfonphthalein leading edge arrives the bottom.Take out shallow layer gel, longitudinally downcut the wide adhesive tape of 0.5cm at its edge, with coomassie brilliant blue staining and decolouring.Downcut the gel band of corresponding position on the shallow layer gel according to the position of second band on the adhesive tape, this band is carried out electroelution under non-sex change condition, elutriant freeze-drying after fully dialysing can obtain purified MAPP;

E. MAPP is identified that (N-terminal sequence label, mass spectrum fingerprint image or amino acid composition analysis) carries out the information biology retrieval according to qualification result.The result shows that MAPP is a new polypeptide.

The separation of example 2.MAPP and screening active ingredients

A. the live loacs with health is prepared into homogenate, extracts total protein with 0.05M Tris-Cl damping fluid from homogenate, reduces foreign matter content through the acetone pre-treatment, and it is standby that the supernatant freeze-drying obtains protein example;

B. the two-dimensional electrophoresis first of non-sex change (carries out among 8cm * 7cm * 0.75mm) at thin layer glue to isoelectrofocusing, acrylamide concentration 5% is used 2.4% Ampholyte (pH3.5～10, Pharmacia company provides), use 5 hole combs, every hole application of sample 65 μ l.Use 0.1M NaOH and 0.01M H ₃PO ₄Respectively as negative electrode and anode buffer liquid.With microsyringe protein example add is gone up bottom, sample hole, behind the 5min beginning first to IEF, 150V constant voltage electrophoresis 30min at first, 400V constant voltage electrophoresis 1.5h in whole process, must controlled temperature be 4 ℃ then;

C. first after IEF finishes, accurately downcut two wide bands of 0.5cm, place 0.1M Tris-base, 0.1M Tricine, balance 20min in the level pad of 3mM SDS (pH is 9.0), the PAGE separation gel for preparing 2 non-sex change is simultaneously carried out mark with marking pen and is made the length of two glue be 7cm (7cm * 7cm * 0.75mm) on sheet glass.The adhesive tape that balance is crossed carefully places the top of Thin-layer separation glue, produces bubble in the middle of noting making the two, and constant voltage electrophoresis 5min under the 50V condition uses 150V voltage electrophoresis edge before tetrabromophenol sulfonphthalein instead and arrives the bottom;

D. after two-dimensional electrophoresis finishes, two glue of PAGE gained are handled respectively: a glue dyes with Xylene Brilliant Cyanine G (CBB), and the decolouring back is with backuping; Another piece glue places 10mMTris-Tricine (pH is 8.9) damping fluid balance 30min, and electroblotting uses the micro-electroblotting device (50V * 1.5h) of Pharmacia company to the NC film in same damping fluid immediately.Second glue obtains being adsorbed with the NC film of protein spots through electroblotting, places level pad (to contain 10mM Tris-cl, pH7.5) soak 10min and change 2 balance liquids to remove remaining SDS, with being about to the vacuum-drying of NC film the NC film;

E. original position is surveyed and is lived.Streptococcus aureus (1 * 10 equably distributes on the NC film ³CFU/mm ²) after, place the thick extractum carnis solid medium of 1mm (to contain 0.0025% bromothymol blue film, 0.0025% is phenol red, 1% N.F,USP MANNITOL) cultivated 24 hours for last 37 ℃, the bacterium of normal growth is acid owing to its meta-bolites thereby is glassy yellow around it on the NC film, and have the local bacterial growth of antibacterial peptide spot to be suppressed, so present dark-coloured spot.More than operation all should be carried out under aseptic condition;

F. can in the first clotting glue, find corresponding spot according to the prompting of spot, it is carried out trace identify that (N-terminal sequence label or mass spectrum fingerprint image) result shows, what we obtained is a new antibacterial peptide, and it can be in the external restraining effect that shows various bacteria.

Claims

1. isolated antimicrobial polypeptide from living organisms, this antimicrobial polypeptide is not for containing the single chain polypeptide of subunit, and its molecular weight is 9300～10300, and iso-electric point PI is 4.68～4.88, and N terminal sequence label is CFGWN.

By claim requirement 1 described from living organisms isolated antimicrobial polypeptide, the living organisms that it is characterized in that indication is a loach.

3. by claim 1 or 2 described antimicrobial polypeptides, it is nontoxic to it is characterized in that this antimicrobial polypeptide can be used as, the externally applied anti-inflammation medicine, food preservatives and the antioxidant that have no side effect.