CN106589136A

CN106589136A - Hybrid antibacterial peptide based on FV7 anti-bio-membrane, preparation method and application thereof

Info

Publication number: CN106589136A
Application number: CN201611054376.6A
Authority: CN
Inventors: 单安山; 谭婷婷; 吴迪
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2016-11-25
Filing date: 2016-11-25
Publication date: 2017-04-26
Anticipated expiration: 2036-11-25
Also published as: CN106589136B

Abstract

The invention relates to a hybrid antibacterial peptide based on an FV7 anti-bio-membrane, and a preparation method and an application thereof. The sequence of the hybrid antibacterial peptide is represented as the SEQ ID No.1. According to main physical and chemical parameters and structural and functional relationship of an original peptide and a hybrid peptide, an anti-bio-membrane fragment FV7 and an active centre fragment LL of a natural antibacterial peptide LL-37 are synthesized through a chemical method to obtain the hybrid peptide FV-LL. Through a series of in-vitro test, the antibacterial activities of the original peptides FV7 and LL and the hybrid antibacterial peptide FV-LL are verified, and through comparison, it is found that the FV-LL is greatly improved in antibacterial activity and also has anti-bio-membrane activity, so that through functional fragment hybridization, the antibacterial activity of the antibacterial peptide can be improved, thereby improving cell selectivity. Furthermore, the antibacterial mechanism of the hybrid antibacterial peptide is verified, thereby providing theoretical basis for application of the antibacterial peptide.

Description

Heterozygous antibacterial peptide and preparation method and application based on FV7 antibiont films

Technical field

This invention belongs to agriculture animal and veterinary application, and in particular to a kind of heterozygosis based on FV7 antibiont films Antibacterial peptide and preparation method and application.

Background technology

As the abuse of feeding antibiotic causes the generation of environmental pollution, drug residue and bacterial drug resistance, this is to agricultural Greatly harm is brought with animal husbandry, the maximum of animal productiong potentiality has been had a strong impact on and has been played and green safety animal products Supply.Therefore find the new of alternative antibiotic, noresidue, efficiently immunity catalyst become asking for current urgent need to resolve Topic.The small molecule polypeptide of external microbe infringement is resisted as animal body, antibacterial peptide has broad spectrum antibacterial, without drug resistance Property, avirulence, pollution-free and noresidue the advantages of, and heat stability is generally preferably, and additive capacity is little, complies fully with livestock products Product security needs, is adapted to used in feed preparation, has the potential quality as feed additive of new generation.

In nature, antibacterial peptide species is various, widely distributed, but is seldom applied to medicine and herding field.Limit anti- The factor of bacterium peptide application is a lot, wherein for some antibacterial peptides antibacterial activity it is low be limit the main impact of its application because Element.It is one of effective way of high-efficiency antimicrobial preparation development that molecular modification is carried out to existing natural antibacterial peptide.Antibacterial peptide changes Great majority are made with natural antibacterial peptide as female peptide, after reaching design requirement, will have therapeutic or preventative fragments of peptides to be used in peptide Source body, to keep body health.Hybrid design is a kind of good strategy for optimizing these antibacterial peptide molecules.This method Can be good at retaining the biologic activity of required fragment, and do not affected by the change of a certain parameter.Further, since only protecting Active center is stayed, therefore this method can be significantly shorter peptide chain length, reduce chemosynthesis cost.

The content of the invention

Based on above weak point, it is an object of the invention to provide a kind of heterozygous antibacterial peptide based on FV7 antibiont films and Preparation method and application, improve its antibacterial activity.

The present invention is realized by following technology：A kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, its sequence is such as Shown in sequence table SEQ ID No.1.

The present invention also has following technical characteristic：

1st, a kind of preparation method of the heterozygous antibacterial peptide FV-LL based on FV7 antibiont films is as follows：

(1) choosing polypeptide fragment FV7 and LL carries out heterozygosis, obtains T1249 FV-LL；

(2) physicochemical property of FV7, LL and FV-LL, spiral wheel construction are analyzed by peptide database analysis software and Prediction；

(3) antibacterial peptide for obtaining design is synthesized using solid-state chemical reaction method method, that is, is completed by Peptide synthesizer The preparation of polypeptide, sequence is as shown in sequence table SEQ ID No.1.

2nd, a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films as above, is preparing treatment Gram-positive Application in bacterium or gram positive bacterial infection disease medicament.

The heterozygous antibacterial peptide FV7 that the present invention is obtained only remains the active center fragment of former peptide FV7, not because of the change of parameter And its activity is affected, peptide chain length is shortened, the cost of chemosynthesis is reduced.The heterozygous antibacterial peptide for obtaining simultaneously has wide spectrum Antibiotic property, and with developing into the potentiality of antibacterium biomembrane medicine.Illustrate to strengthen antibacterial peptide by functional fragment heterozygosis Antibacterial activity, improve cell selective, further demonstrate its Antibacterial Mechanism, the application for antibacterial peptide provides theoretical base Plinth.

Description of the drawings

Fig. 1 is the helical wheel prognostic chart of former peptide LL and T1249 FV-LL.

Fig. 2 is the antibiont film activity comparison diagram of former peptide FV7 and T1249 FV-LL.

Specific embodiment

The design synthesis of 1 antibacterial peptide of embodiment

LL：The 17-29 aminoacid functional sequences of people derived antimicrobial peptide LL-37, this fragment is rich in the amphipathic of cation The peptide chain of helical structure.Be identified as the most short sequence with antibacterial and active anticancer, can effectively with bacterial cell membrane Anionic phospholipid combines and presents selective cytotoxicity, does not have toxicity to human cell.

By one section of sequence FV7 with antibiont film activity and polypeptide fragment LL heterozygosis, heterozygous antibacterial peptide FV-LL is obtained. The helical wheel prognostic chart of T1249 FV-LL is as shown in Figure 1.Using Peptide synthesizer, synthesize above-mentioned three using solid-phase synthesis Antibacterial peptide.The sequence and physical and chemical parameter of three peptides is as shown in table 1.

The sequence and physical and chemical parameter of 1 three peptides of table

A molecular weight：Molecular weight is measured by mass spectrum.

The physical and chemical index analysis of 2 antibacterial peptide of embodiment

The secondary structure of antibacterial peptide is analyzed by CD spectrum, is as a result shown, find in water environment T1249 with Former peptide is presented random coil structure, and in the hydrophobic environment (50%TFE) and negatively charged protokaryon of simulation microbial film In cell-membrane environment (30mM SDS), former peptide LL and T1249 FV-LL show certain α-helixstructure, and FV7 then tables Reveal certain beta sheet conformation.

Shown by the stability analyses to antibacterial peptide, T1249 FV-LL has preferable resistance to heat treatment.However, Under the physiological concentration of different salt ions, there is reduction by a small margin in the antibacterial activity of peptide FV-LL.

The antibacterial and antibiont film activity of 3 antibacterial peptide of embodiment

1 bacteriostatic activity

Peptide is configured as into 2.56mM/L storing liquids standby.Using the minimum of several antibacterial peptides of micro-broth dilution method Mlc.Graded series, as diluent, are configured successively using doubling dilution using 0.01% acetic acid (containing 0.2%BSA) Antibacterial peptide solution.Take 100 μ L of above-mentioned solution to be placed in 96 porocyte culture plates, then add isopyknic bacterium solution to be measured respectively (～10⁵Individual/mL) in each hole.It is respectively provided with positive control (antibacterial peptide is not contained containing bacterium solution) and negative control is (neither Peptide is not contained containing bacterium solution) yet.With naked eyes, 37 DEG C of constant temperature culture 20h, have no that there is the as minimal inhibitory concentration of research of chaotic phenomenon in bottom hole portion. As a result it is as shown in table 2.

The antibacterial activity of 2 FV7 of table and FV-LL

Note:^aMinimum inhibitory concentration is that antibacterial peptide can be with the least concentration of bacteria growing inhibiting.Test at least carry out three times it is heavy It is multiple.

2 antibiont film activities

To determine the effect that low concentration antibacterial peptide suppresses to biomembrane, we are from static biosolids surface method (crystallization Purple staining).Take frozen in -20 DEG C of Pseudomonas aeruginosa PAO1, be inoculated in TSB culture medium, at 37 DEG C, under the conditions of 220r/m Culture.Thalline after overnight is forwarded in brand-new culture medium, cultivates 1-2h, is in exponential phase to antibacterial.Spectrophotometric The lower brand-new TSB culture medium corrected concentrations to 0.4 Maxwell of meter make the clump count of gained thalline be adjusted to 10 than turbid standard⁶CUF/mL Left and right；500 μ L are added separately in 12 orifice plates with the antibacterial peptide that TSB culture medium contains variable concentrations gradient.Tune is taken with liquid-transfering gun Whole good 500 μ L of thalline are sequentially added in 12 orifice plates, and in mixed liquor, the concentration of antibacterial is 5 × 10⁵CUF/mL or so, sealed membrane will 12 orifice plates are sealed and are incubated 20-24h under the conditions of 37 DEG C.Positive controls are set wherein to be not added with antibacterial peptide.Carefully discard and carry The culture medium of planktonic bacteria, is then rinsed 2-3 time with PBS, it is ensured that established biomembrane is not destroyed.Hereafter, 12 are attached to Biomembrane violet staining 20min of orifice plate bottom.After dyeing 20min, reject crystal violet solution is rinsed repeatedly with sterilized water The bottom in hole and side wall, until the purple in sterilized water is invisible.Plus 100 μ L dehydrated alcohol shaking table vibration, be transferred to 96 holes 570nm ultraviolet light measured values are used in plate.As a result it is as shown in Figure 2.Show T1249 FV-LL with the antibiont with FV7 similarity degrees Film activity.

<110>Northeast Agricultural University

<120>Heterozygous antibacterial peptide and preparation method and application based on FV7 antibiont films

<160>1

<210>1

<211>20

<212>PRT

<213>Artificial sequence

<400>1

Phe Arg Ile Arg Val Arg Val Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg-NH2

1 5 10 15 20

Claims

1. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, it is characterised in that its sequence such as sequence table SEQ ID Shown in No.1.

2. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, it is characterised in that preparation method is as follows：

(2) physicochemical property of FV7, LL and FV-LL, spiral wheel construction are analyzed by polypeptide database analysises software and in advance Survey；

(3) antibacterial peptide for obtaining design is synthesized using solid-state chemical reaction method method, that is, is completed polypeptide by Peptide synthesizer Preparation, its sequence is as shown in sequence table SEQ ID No.1.

3. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films according to claim 1, is preparing treatment leather orchid Application in family name's positive bacteria or gram positive bacterial infection disease medicament.