CN105801669A - Hybrid anti-biology type antibacterial peptide and preparation method and application thereof - Google Patents

Abstract

The invention relates to a hybrid anti-biology type antibacterial peptide and a preparation method and application thereof.The sequence of the hybrid anti-biology type antibacterial peptide R-FV-I16 is shown as SEQ No.1.A fragment RR7, with a poor effect, in antibacterial peptide is speculated according to the primary structure of the porcine antibacterial peptide RI16, an anti-biological film fragment FV7 is inserted in a specific position according to the structure and function relation of the antibacterial peptide, and the hybrid peptide R-FV-I16 is synthesized with a chemical design method.Compared with the original peptide RI16, the antibacterial activity of the newly synthesized hybrid antibacterial peptide R-FV-I16 is greatly improved, and the hybrid antibacterial peptide R-FV-I16 also has the anti-biological film activity; meanwhile, the hybrid antibacterial peptide R-FV-I16 is good in anti-heat effect and suitable for large-scale industrial production.

Description

A kind of heterozygosis formula antibiont type antibacterial peptide and preparation method and application

Technical field

The invention belongs to biological engineering peptide art and antibacterials design field, be specifically related to a kind of heterozygosis formula and resist Biotype antibacterial peptide and preparation method and application.

Background technology

In the last few years, along with the continuous expansion of antibiotic usage scale, the irregular use to it also became day by day Distinct issues.The cause a disease appearance of strain of Drug resistance makes existing no longer can effectively kill these with antibiotic and cause Sick microorganism.Therefore, for fundamentally solving this problem, the strict supervision of antibiotic usage is become gesture and exists One of means that must go.But the antibacterials meanwhile, finding brand new class are also effective solution ways Footpath.Have antibacterial activity height, has a broad antifungal spectrum, kind is many, be not likely to produce the plurality of advantages such as resistant mutation, antibacterial Peptide is considered to have broad application prospects in medicine, food and animal husbandry.Researcher is from certainly The number of ways such as right screening, synthetic have understood thoroughly the mechanism of antibacterial peptide and corresponding physicochemical property.On a little bases On existing antibacterial peptide be optimized become one of effective design.

Biomembranous formation creates huge threat to clinical treatment, and the high resistance of biomembrane form makes Therapeutic effect unsatisfactory, and also at numerous areas such as medical treatment, food, herding, military affairs to human society Bring serious harm, cause huge economic loss.Then, research in recent years finds, part is antibacterial Peptide, while having antibacterial activity, also has the characteristic that antibiont film is formed, is proved tool as compared LL-37 There is good antibiont film activity.Therefore, if can be antibacterial in one in conjunction with the function of the different fragment of antibacterial peptide On peptide, this will research and propose important guiding suggestion for later, and our this brand-new model is called heterozygosis Antibacterial peptide.

Summary of the invention

Present invention aim at a kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16 and preparation method being provided and answering With.

The purpose of the present invention is realized by following technology: a kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16, its Sequence is as shown in sequence table SEQ No.1.

The present invention also has a following technical characteristic:

1, a kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16 as above, preparation method is as follows:

(1) in pig derived antimicrobial peptide PMAP36, a short chain antibacterial peptide RI16, its sequence such as sequence table are intercepted Shown in SEQ No.2；

(2) substitute the center sequence RR7 of RI16 fragment by antibiont film activity fragment FV7, thus obtain Brand-new antibacterial peptide R-FV-I16；FV7 sequence as shown in sequence table SEQ No.3, RR7 sequence such as SEQ No.4 Shown in；

(3) solid-state chemical reaction method method synthetic antibacterial peptide R-FV-I16 is used.

2, a kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16 as above is in preparation treatment Gram-positive Application in bacterium or gram positive bacterial infection disease medicament.

It is an advantage of the current invention that heterozygous antibacterial peptide R-FV-I16 is thin to multiple Gram-negative and Gram-positive Bacterium has stronger antibacterial activity, and while bactericidal effect raises, haemolysis, toxicity do not raise significantly, And heat treatment is had preferable resistance.R-FV-I16 inherits the antibiont of antibiont film sequence FV7 simultaneously Film activity.Safety of the present invention is high, has no side effect, without bad flavor, protects intestinal health, improves dynamic The production performance of thing.

Accompanying drawing explanation

Fig. 1 is antibacterial peptide helical wheel prognostic chart,

Wherein, the helical wheel prognostic chart of A: heterozygous antibacterial peptide R-FV-I16；The helical wheel prognostic chart of B:RI16.

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention It is not limited to this.

The structure prediction of embodiment 1 heterozygous antibacterial peptide R-FV-I16

1, the aminoacid sequence of heterozygous antibacterial peptide

Antibacterial peptide RI16 is the short chain antibacterial peptide intercepted from the antibacterial peptide PMAP36 of Medulla Sus domestica, has perfection Amphipathic, relatively low bactericidal activity and cytotoxicity.Secondary structure is α-helixstructure.RR7 is antibacterial One section of sequence in the middle part of peptide RI16 is it is considered to be affect the fragment of the bactericidal activity of RI16, and experiment proof does not has Bacteriostatic activity.FV7 mono-section has the sequence of antibiont film activity, shows certain beta sheet conformation.

On the basis of RI16, substituting by antibiont film activity fragment FV7 is considered as to affect its RI16 fragment Center sequence RR7, on the basis of not changing aminoacid sum, improve RI16 bactericidal activity control simultaneously Its cytotoxicity within the acceptable range, thus obtains the peptide R-FV-I16 of brand-new design.Above-mentioned four kinds antibacterial The sequence of peptide is as shown in table 1.

The structural parameters of table 1RI16, FV7, RR7 and R-FV-I16

^aMolecular weight (in Da) is recorded by mass spectrography.

2, the prediction to antibacterial peptide helical wheel structure

Having scientific research to report, antibacterial peptide helical wheel structure has certain shadow to its antibacterial activity and cytotoxicity Ring, destroy under certain condition and the most amphipathic can improve while bactericidal activity, reduce its molten activity. As shown in Figure 1B, RI16 has perfect amphipathic, and after transformation, the amphipathic of R-FV-I16 is broken, As shown in Figure 1A.

The synthesis of embodiment 2 antibacterial peptide

Being synthesized by the biochemical company limited of Shanghai gill (GL) by above-mentioned antibacterial peptide, method uses solid state chemistry Synthetic method.The preparation of antibacterial peptide is carried out to N end one by one from C end, uses FMOC (9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl) protection synthetic method, is come by Peptide synthesizer Complete.Antibacterial peptide after synthesis utilizes high performance liquid chromatography (HPLC) purification, then recycling electron spray matter Antibacterial peptide is identified by spectrometry.

The minimal inhibitory concentration of embodiment 3 antibacterial peptide and hemolytic activity

Utilize the minimal inhibitory concentration of several antibacterial peptide of micro-broth dilution method, and gather fresh blood mensuration Its hemolytic activity.Result is as shown in table 2.

The minimal inhibitory concentration of table 2 antibacterial peptide and hemolytic activity

^aMinimum inhibitory concentration is that antibacterial peptide can be with the least concentration of bacteria growing inhibiting.Test at least carries out three times and repeats.

^bPseudomonas aeruginosa mutation I type, II type are obtained by ciprofloxacin and gentamycin progressively culture method respectively,

^cMinimum hemolytic concentration is that antibacterial peptide can cause 5% to be the least concentration of human erythrocyte's haemolysis (hRBC).Do not observe that 5% is molten at 256 μMs During blood, concentration 512 μMs is used for calculating therapeutic index.

^dGM represents that MIC value geometric mean is to microbial strains all in this table.

^eTherapeutic index (TI) refers to that the value of MHC and the ratio of MIC geometric mean (GM), bigger value explanation have preferable cell selective.

As shown in table 2, newly synthesized T1249 R-FV-I16 is for multiple Gram-negative and Gram-positive Antibacterial has stronger antibacterial activity, and former peptide RI16, FV7 show more weak bacteriostatic activity, and RR7 does not has There is bacteriostatic activity.T1249 R-FV-I16 shows relatively low hemolytic activity and cytotoxicity.This is with former peptide The performance of RI16, FV7 is similar, illustrate R-FV-I16 bactericidal effect raise while, haemolysis, toxicity Do not raise significantly.

The physics and chemistry activity analysis of embodiment 4 heterozygous antibacterial peptide

By showing the stability analysis of antibacterial peptide, heat treatment is had the most anti-by T1249 R-FV-I16 Property.But, under the physiological concentration of different salt ions, the antibacterial activity of peptide R-FV-I16 occurs by a small margin Reducing, under serum human and different Protease Treatment, the loss of its antibacterial activity declines about 50%.

By showing the antibiont film analysis of antibacterial peptide, T1249 R-FV-I16 has and FV7 similarity degree Antibiont film activity.The result of scanning electron microscope also indicates that, R-FV-I16 inherits antibiont film sequence The antibiont film activity of row FV7.

The embodiment 5 detection to heterozygous antibacterial peptide antifungal mechanism

Being found by the antifungal mechanism of detection T1249 R-FV-I16 further, T1249 R-FV-I16 can make carefully Bacterium has inner membrance and outer membrane depolarization, scanning electron microscope and transmission electron microscope then to further demonstrate that, heterozygosis Peptide R-FV-I16 can destroy the cell membrane of cell, the integrity of infringement film, thus kills antibacterial.

Claims (3)

1. a heterozygosis formula antibiont type antibacterial peptide R-FV-I16, it is characterised in that its sequence such as sequence table SEQ Shown in No.1.

A kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16 the most according to claim 1, its feature exists In, preparation method is as follows:

(1) in pig derived antimicrobial peptide PMAP36, a short chain antibacterial peptide RI16, its sequence such as sequence table are intercepted Shown in SEQ No.2；

(2) substitute the center sequence RR7 of RI16 fragment by antibiont film activity fragment FV7, thus obtain Brand-new antibacterial peptide R-FV-I16；FV7 sequence as shown in SEQ No.3, RR7 sequence such as sequence table SEQ No.4 Shown in；

(3) solid-state chemical reaction method method synthetic antibacterial peptide R-FV-I16 is used.

A kind of heterozygosis formula antibiont type antibacterial peptide R-FV-I16 the most according to claim 1 is at preparation treatment leather Application in Lan Shi positive bacteria or gram positive bacterial infection disease medicament.