CN106674335A - Antibacterial peptide - Google Patents

Abstract

The invention relates to the field of biomedicine, particularly a novel antibacterial peptide of which the amino acid sequence is KYYGNGVHCGKHSCTVDILGKIWEGIKSLF. The pharmacodynamical test indicates that the antibacterial peptide has inhibiting actions on Staphylococcus aureus, Bacillus subtilis and the like, can not cause erythrocyte hemolysis, and can be used for preparing antibacterial drugs.

Description

A kind of antibacterial peptide

Technical field

The present invention relates to a kind of antibacterial peptide, the peptide is the linear peptides containing 30 amino acid residues of engineer's synthesis.

Background technology

Antibacterial peptide is the micromolecule polypeptide that a class has biologic activity, is typically made up of 12~60 aminoacid.It is rich in The basic amino acids such as lysine, arginine, and usually contain the hydrophobic amino acid higher than 30%.Antibacterial peptide is mostly amphiphilic Property and with a certain amount of positive charge, is cationic polypeptide.

Since finding penicillin from nineteen twenty-eight Fleming, people progress into control and treat bacterial infection disease Epoch.But the use of a large amount of broad ectrum antibiotic accelerates the evolution of pathogenic bacterium so that a large amount of drug-resistant bacterias and multiple in recent years Drug-resistant bacteria continuously emerges.At present, all of Conventional antibiotic all occurs in that corresponding drug resistance causes a disease strain, pathogenic bacterium it is anti- Property of medicine problem has increasingly seriously threaten the health of people.Antibacterial peptide is because antibacterial activity is high, and has a broad antifungal spectrum, species is more, Alternative scope is wide, and target bacterial strain is not likely to produce the reasons such as resistant mutation, and is considered as will having wide on medical industry Wealthy application prospect.But antibacterial peptide there is also some shortcomings, the hemolytic activity of mainly some antibacterial peptides is too high.Also some face Bed application problem, the such as high cost of antibacterial peptide, internal limited stability, microbiological resistance (film variation), heterogeneous, unknown poison Reason and pharmacokineticss etc..

The content of the invention

The present invention devises a kind of new antibacterial peptide, and its hemolytic activity is little, is difficult to cause side effect.

The present invention antibacterial peptide sequence be：

KYYGNGVHCGKHSCTVDILGKIWEGIKSLF

Polypeptide involved in the present invention is prepared with solid-phase synthesis, and technology maturation, product quality is easily controlled, and can meet big The needs that technical scale metaplasia is produced.

Pharmacodynamics test proves that the polypeptide of the present invention can suppress or kill.

Toxic and side effects test shows that the polypeptide of the present invention does not observe hemolytic activity to sheep red blood cell (SRBC), illustrates that its haemolysis is lived Low, the Small side effects of property.

The polypeptide of the present invention can also be combined with pharmaceutically acceptable salt, equally with its drug effect.These salt include (but It is not limited to) and alkali metal or the salt of alkaline-earth metal (such as sodium, potassium, calcium or magnesium) formation.Other salt include and following mineral acid shape Into salt：Such as hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, and the salt formed with organic acid, and organic acid then refers to acetic acid, oxalic acid, fourth two Acid, tartaric acid, methanesulfonic acid and maleic acid.Pharmaceutically acceptable salt particular certain cancers.

The invention discloses preparation made by a kind of antibacterial peptide, wherein containing the polypeptide or its pharmaceutically acceptable salt and Pharmaceutically acceptable carrier.

Aforementioned polypeptides can make injection, tablet, injectable sterile powder, powder, granule, capsule, oral liquid, Various dosage forms such as unguentum, cream.Above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.Can be by note Penetrate, orally, collunarium, eye drip, the method that physically or chemically mediates import muscle, endothelium, subcutaneous, vein, mucosal tissue, or quilt Human body is imported after other material mixings or parcel.

Usually, using dosage of the invention is at 0.001~10 gram/day, it is also possible to deviate this according to the situation of disease Dosage range.

The part pharmacodynamics test and result of the polypeptide of the present invention are presented herein below, polypeptide used is according to following sequence, commission The great biological company limited's synthesis of Shanghai sunrise, using the Fmoc solid-state chemical reaction method methods of standard, i.e., by deprotection and activation crosslinking Two are reacted iterative cycles and most prepare peptide symthesis at last.Post is prepared using C18, using high performance liquid chromatography (HPLC) method purification Polypeptide, purity reaches more than 95%, and carries out peptide identification by mass spectrum (MS).

The sequences of Peptide 1 are (this patent protection sequence)：

KYYGNGVHCGKHSCTVDILGKIWEGIKSLF

The sequences of Peptide 2 are

KYYGNGVHCGKHSCTVDILGKIWEGIKSLFWATGGNAGWNK

The sequences of Peptide 3 are

KYYGNGVHCGKHSCTVDILGKIWEGIKSLF-NH2

First, micro meat soup method determines MIC：

1. single bacterium colony being selected in 10ml liquid medium test tubes, putting 37 DEG C, 200 turns, shaking table culture allows bacterium solution to grow To exponential phase (OD600 values about 0.5).Incubation time is：Staphylococcus aureuses (5-6 hours), bacillus subtilises (10-12 hours), Listeria monocytogenes (10-12 hours).

2. the seed liquor of staphylococcus aureuses, bacillus subtilises, Listeria monocytogenes is divided with culture medium OD600 values are not diluted to for the bacteria suspension between 0.08-0.1.

3. add the bacteria suspension of 180 μ l dilutions, 2-11 hole to add the bacterium of 100 μ l dilutions in first hole of 96 orifice plates Liquid, 12 holes add sterile LB medium or PYG culture medium as control.

4. 20 μ l mother liquid medicines are added in first hole, mixed, take 100 μ l to second holes, diluted successively, make polypeptide female Doubling dilution of liquid is 256,128,64,32,16,8,4 μ g/ml.It is parallel that every plant of bacterium is cooked 3 rows.Replace polypeptide with cefuroxime Mother solution, as positive control；Replace polypeptide mother solution with culture medium, as negative control.

5. be placed in 37 DEG C culture 4 hours after, with microplate reader determine OD595 values, observation thalli growth situation.

6. not grow the minimum drug level of antibacterial as MIC value.

Bacteriostatic test result as shown in Table 1 to Table 3, there is bacteria growing in+number expression hole, No. one represents in hole without bacterium life It is long.As can be seen from the test results Peptide 1 has one to staphylococcus aureuses, bacillus subtilises, listeria spp Fixed fungistatic effect, MIC is in 32~64 μ g/ml；Peptide 2 has weak suppression to staphylococcus aureuses and bacillus subtilises Effect processed, MIC does not have inhibition in 64~256 μ g/ml to listeria spp；Peptide 3 is to Staphylococcus aureus Bacterium, bacillus subtilises, listeria spp fungistatic effect it is of a relatively high, MIC is in 16 μ g/ml；

Fungistatic effect of the table 1 to staphylococcus aureuses

Fungistatic effect of the table 2 to bacillus subtilises

Fungistatic effect of the table 3 to listeria spp

2nd, hemolytic toxicity test

1st, the preparation of red cell suspension：The sheep erythrocyte 10ml of de- fiber is taken, in adding centrifuge tube, 10ml lifes is added It is centrifuged after reason salt water washing, centrifugal force is set as 3000g, 3min is centrifuged, supernatant is abandoned after taking-up, portion's cell of keeping on file, cyclic washing Until supernatant does not show abandoning supernatant after redness, 2ml lower sediment things are drawn, add normal saline dilution to 100ml, matched somebody with somebody Into 2% red blood cell suspension.

2nd, medicinal liquid prepares：Sample configuration concentration is called the polypeptide mother solution 500ml of 2560 μ g/ml, is diluted successively, finally obtains dense Degree is respectively 256 μ g/ml, 128 μ g/ml, 64 μ g/ml, 32 μ g/ml, 16 μ g/ml, the polypeptide solution of 8 μ g/ml.It is another to prepare 3 EP is managed, and one adds 600 μ l normal saline to make background color pipe, and one plus 300 μ l distilled water do positive control pipe, and another adds 300 μ l Normal saline makees negative control pipe.

3rd, each EP pipes are put in 37 DEG C of constant temperature pots and are incubated after 30min, take the μ l of polypeptide medicinal liquid 300 of variable concentrations respectively and 300 μ l red cell suspensions are mixed, and positive control pipe and negative control pipe are also separately added into the mixing of 300 μ l red cell suspensions.It is put into It is incubated 1 hour in 37 DEG C of constant temperature pots.

4th, EP pipes are taken out, 5min is centrifuged with 6000rpm rotating speeds, perusal supernatant after the completion of centrifugation takes the μ l of supernatant 100 In adding 96 orifice plates, to microplate reader 570nm OD values are surveyed.Independent three repetitions are tested.

5th, haemolysis degree is calculated.Haemolysis degree computing formula：Sample absorbance=style pipe absorbance-background color pipe absorbance；It is molten Blood degree=(sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance) × 100%

6th, concentration of the haemolysis degree more than 5% is labeled as haemolysis

As shown in table 4, an expression does not observe haemolysis to hemolytic toxicity result of the test, and+expression observes haemolysis. Peptide 1 will not cause sheep red blood cell (SRBC) haemolysis, the meetings under 128 μ g/ml concentration of Peptide 2 in the concentration range for determining Cause haemolysis, haemolysis degree is that 7.86%, Peptide 3 is toxic under 32 μ g/ml concentration, and haemolysis degree is 23.82%；

Hemolytic toxicity result of the polypeptide of table 4 to Sheep Blood

Above-mentioned test structure shows, although the bacteriostatic activities of Peptide 3 are higher, but its toxic and side effects are larger, in 32 μ Having under g/ml concentration just can cause sheep red blood cell (SRBC) haemolysis, it is impossible to used as antibacterial medicines are oral or injection.And Peptide 1 will not Cause erythrocyte hemolysis, have preferable fungistatic effect again to staphylococcus aureuses etc., as the polypeptide of this patent protection.

Specific embodiment

Embodiment 1

Preparation containing antibacterial peptide bacteriostatic solution

The sequence according to Peptide 1, the great biological company limited's synthesis polypeptide of commission Shanghai sunrise, weighs 64mg polypeptides, In being dissolved in 500ml deionized waters, 100ml glycerol, 20ml dehydrated alcohol is added after stirring, to supplement deionized water and arrive 1000ml.The bacteriostatic solution can be used for hand, the cleaning of face, and the glycerol of addition may also operate as the antifreeze effect of moisturizing.

Embodiment 2

It is prepared by the collutory containing antibacterial peptide

32mg polypeptides are weighed, in being dissolved in 500ml deionized waters, 10g xylitol, 1 drop clove oil, the anhydrous second of 20ml is added Alcohol, 10ml glycerol after stirring, supplements deionized water to 1000ml.The collutory has the delicate fragrance of Flos Caryophylli, wherein contain Antibacterial peptide can effectively suppress the antibacterial in oral cavity, maintain the pure and fresh of implication.

Embodiment 3

The preparation (capsule) of the pharmaceutical composition containing antibacterial peptide

According to the consumption of 100 capsules, weigh each adjuvant of the above respectively it is finely ground sieve after mix homogeneously, then passed with equivalent Addition adds antibacterial peptide Peptide 1, is fully ground so as to be uniformly dispersed, and after 80 mesh sieves, then records into capsule.

Claims (4)

1. a kind of antibacterial peptide contains the peptide or its pharmaceutically acceptable salt of following aminoacid sequence：

KYYGNGVHCGKHSCTVDILGKIWEGIKSLF。

2. a kind of antibacterials, wherein the peptide containing claim 1 or its pharmaceutically acceptable salt and pharmaceutically acceptable Carrier.

3. the peptide of claim 1 or its pharmaceutically acceptable salt are used to prepare the purposes of antimicrobial drug.

4. the purposes of claim 4, wherein antibacterials are to prevent or treat the infection such as staphylococcus aureuses or escherichia coli The medicine of disease.