1. An antibacterial peptide LL37 active center derived peptide diploid antibacterial peptide LG is characterized in that the sequence is shown in a sequence table SEQ ID No. 1.

2. The preparation method of the antimicrobial peptide LL37 diploid antimicrobial peptide LG derived from the active center of the antimicrobial peptide according to claim 1, wherein the method comprises the following steps:

(1) selecting an active center FD of antibacterial peptide LL37, and replacing aspartic acid D in the antibacterial peptide FD with arginine R to obtain a derivative peptide FR;

(2) design by direct ligation, rigid linker peptide AEAAAKA ligation and Flexible linker peptide GGGGS ligationObtaining three diploids (FR)₂LG and LA, and screening by taking the 'selection index' as an evaluation index;

(3) synthesizing the diploid LG with the highest selection index by a polypeptide synthesizer by adopting a solid-phase chemical synthesis method to complete the preparation of the antibacterial peptide LG.

3. The application of the antibacterial peptide LL37 active center derived peptide diploid antibacterial peptide LG in the preparation of a medicament for treating infectious diseases caused by gram-positive bacteria or gram-negative bacteria according to claim 1.

Background

In recent years, serious residues of antibiotics in animal products have had an adverse effect on human health. Since the first prohibition of adding antibiotics into feed in 2006, related policies have been introduced in many countries, which also mark the end of the golden age of antibiotics.

The Antimicrobial peptides (AMPs) are used as a novel feed additive, have excellent development prospect, do not have negative influence generated by the application of the traditional antibiotics in the animal husbandry, and have the characteristics of small molecular weight, high heat stability, broad-spectrum antibiosis, unique action mechanism and the like. LL37 is the only member of the human cathelicidin family, is produced by leukocytes (mainly neutrophils) and various epithelial cells and is widely present in various human tissues and fluids. In recent years, researchers have kept a high interest in LL37 because of its outstanding advantages in immune regulation, inducing immunocyte chemotaxis and promoting wound healing. In addition, it has a defense against many types of pathogens, including bacteria, fungi, viruses, parasites, and even cancer cells. Serial studies have shown that amino acid residues 17-29 (FD) of LL37 are the active center. However, FD has not high antibacterial activity against gram-negative and gram-positive bacteria, and has a problem of causing a large decrease in antibacterial activity in an environment where salt ions exist. The FR derived peptide has small molecular weight (3.82KDa) and is easy to be degraded by microbial protease.

Disclosure of Invention

Based on the defects, the invention aims to provide the diploid antibacterial peptide LG derived from the active center of the antibacterial peptide LL37, so as to solve the problems that the FD antibacterial activity is not high and the antibacterial activity is greatly reduced in the presence of salt ions, thereby improving the antibacterial activity and the stability of the salt ions.

The invention is realized by the following technology: an antibacterial peptide LL37 active center derived peptide diploid antibacterial peptide LG, the sequence of which is shown in a sequence table SEQ ID No. 1.

The invention also discloses a preparation method of the diploid antibacterial peptide LG derived from the active center of the antibacterial peptide LL37, which comprises the following steps:

(1) selecting an active center FD of antibacterial peptide LL37, and replacing aspartic acid D in the antibacterial peptide FD with arginine R to obtain a derivative peptide FR;

(2) three diploids (FR) were obtained by design of direct linkage, rigid linker peptide AEAAAKA linkage and flexible linker peptide GGGGS linkage₂LG and LA, and screening by taking the 'selection index' as an evaluation index;

(3) synthesizing the diploid LG with the highest selection index by a polypeptide synthesizer by adopting a solid-phase chemical synthesis method to complete the preparation of the antibacterial peptide LG.

The invention also aims to disclose an application of the antibacterial peptide LL37 active center derived peptide diploid antibacterial peptide LG in preparation of a medicament for treating infectious diseases caused by gram-positive bacteria or gram-negative bacteria.

The invention has the advantages that: compared with the original active center fragment FD, the diploid antibacterial peptide LG has the antibacterial activity improved by about 20 times; compared with the derivative peptide FR, the antibacterial activity of the antibacterial peptide LG is improved by about 3 times, and the antibacterial peptide LG has better salt ion stability and has the potential of being developed into a novel antibacterial drug.

Drawings

FIG. 1 is (FR)₂And hemolytic activity patterns of LG and LA.

FIG. 2 is a 6 XHis-SUMO-LG recombinant expression vector construction diagram.

FIG. 3 is a diagram showing PCR verification of Pichia pastoris positive transformants,

wherein, 1 is Marker, 2 is PCR product of pPICZ alpha A empty plasmid, and 3-7 is PCR product of recombinant yeast X33/6 XHis-SUMO-LG.

FIG. 4 is a diagram showing the results of the detection of Tricine-SDS-PAGE as a fusion protein,

wherein, 1 is empty vector contrast, 2 is an ultra-low molecular weight pre-staining protein Marker, and 3-6 is an expression product of fusion protein 6 × His-SUMO-LG.

FIG. 5 is a Tricine-SDS-PAGE analysis of 6 XHis-SUMO-LG cleaved by SUMO protease,

wherein 1 is Marker; 2 is purified 6 XHis-SUMO-LG; 3 is the 6 XHis-SUMO tag and rLG after cleavage by SUMO protease.

FIG. 6 is a diagram showing the purification and identification of recombinant LG (rLG),

(A) Tricine-SDS-PAGE analysis of rLG. Wherein 1 is Marker; and 2 is purified rLG.

(B) MALDI-TOF mass spectrum of purified rLG.

Detailed Description

Example 1 design Synthesis of antimicrobial peptides

The derived peptide FR has small molecular weight (3.82KDa) and is easily degraded by microbial protease, so that the recombinant expression of the derived peptide FR in a microbial expression system is more challenging. Fusion or tandem expression of AMPs provides a solution to this problem. In addition, the connecting peptide tandem expression strategy also has the advantages of improving biological activity, enlarging expression yield and the like. Therefore, rational design of the linker peptide is of great importance. In general, linker peptides are divided into two classes, flexible linker peptides and rigid linker peptides. In this example, arginine (R) was substituted for aspartic acid (D) in the active center of the antimicrobial peptide LL37 to obtain the derivative peptide FR with high activity.

FD: the 17 th to 29 th amino acid sequences of human antibacterial peptide LL37, which is a peptide chain of a cation-rich amphipathic helical structure, are structural regions of LL37 which exert antimicrobial, anticancer and antiviral activities.

FR: aspartic acid (D) in antimicrobial peptide FD was replaced with arginine (R) to obtain derivative peptide FR.

(FR)₂: direct concatenation of the derived peptide FRs to give the diploid (FR)₂。

LG: connecting derivative peptide FR together in series through a connector GGGGS to obtain diploid LG,

LA: the derived peptides FR were concatenated together via linker AEAAAKA to give diploid LA.

The antibacterial peptide is synthesized by a solid phase synthesis method by using a polypeptide synthesizer. The amino acid sequence and physicochemical parameters of the peptide are shown in Table 1.

TABLE 1 FD, FR, (FR)₂Amino acid sequences and physicochemical parameters of LG, LA

Note:^amolecular mass detected by mass spectrometer

^bHydrophobicity by on-line tool calculationhttp://heliquest.ipmc.cnrs.fr/cgi-bin/ ComputParams.py

EXAMPLE 2 bacteriostatic Activity of antimicrobial peptides

Peptides were prepared as 1.28mM/L stock solutions for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined using the broth dilution method. The bacteria were incubated overnight in Mueller-Hilton broth (MHB) at 37 ℃ until the logarithmic growth phase was reached. The bacteria were then diluted to 10⁵CFU/mL. mu.L of the highest concentration peptide was mixed with 95. mu.L of 0.01% (v/v) acetic acid and 0.2% (w/v) bovine serum albumin, mixed well and then diluted twice in series, and 50. mu.L of the bacterial solution was added to each well. Positive control (containing bacterial solution and no antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) were set separately. The mixture was incubated at 37 ℃ for 24 hours. The minimum inhibitory concentration is the turbidity phenomenon at the bottom of the invisible hole. The results are shown in Table 2.

TABLE 2 FD, FR, (FR)₂Minimum inhibitory concentrations of LG and LA

Note:¹the minimum inhibitory concentration is the lowest concentration of antimicrobial peptide that can inhibit bacterial growth. The test was performed in at least three replicates. Data are presented as mean ± standard deviation.

EXAMPLE 3 hemolytic Activity

The collected human healthy blood was centrifuged at 1000 Xg for 5 minutes to collect erythrocytes. The red blood cells were then washed and resuspended in 10mM PBS (pH 7.4). 50 μ L of red blood cell dilutions and equal volumes of peptide solution (2-128 μ M) were incubated in 96-well plates for 1 hour at 37 ℃. A suspension of erythrocytes treated with 0.1% Triton X-100 was used as positive control (100% hemolysis) and an untreated suspension of blood cells was used as negative control. The 96-well plate was centrifuged at 1000 Xg for 5 minutes at 4 ℃ and then the supernatant was transferred to a new 96-well plate. The absorbance was measured with a microplate reader at OD 570. The hemolytic activity of (FR)2, LG and LA is shown in FIG. 1.

Example 4 selection index

To further assess the cell selectivity of the peptides, the peptides of interest were screened for high or low "selection index" (SI) (MHC/GM). The results are shown in Table 3.

Table 3(FR)₂Minimum inhibitory concentration, hemolytic activity and selection index of LG and LA

Note:^aMHC is the minimum antimicrobial peptide concentration that causes 20% hemolysis;

^bGM is the geometric mean of the MIC of the peptide against the bacteria, and when no detectable antibacterial activity is observed at 32 μ M, a value of 64 μ M is used to calculate GM;

^cSI is MHC/GM, and a larger value indicates a higher cell selectivity.

Example 5 salt ion tolerance

Salt ions were added to the BSA solution and configured as salt ion solutions of different final concentrations (150mM NaCl,4.5mM KCl, 6. mu.M NH)₄Cl,8μM ZnCl₂,1mM MgCl₂,2mM CaCl₂,and 4μM FeCl₃). The MIC values of LG against E.coli 25922 in the presence of different concentrations of salt ions were determined as in reference example 2.

TABLE 4 MIC values (μ M) of peptides against E.coli ATCC 25922 in the presence of physiological salts

Physiological concentration of Na⁺、Mg²⁺、Ca²⁺The presence of ions has a greater influence on the antibacterial activity of the monomeric peptide FR, Na⁺、Ca²⁺Even direct deactivation of FR, Mg²⁺The MIC value of FR was increased 4-fold. And diploid LG in Na in the presence of GGGGS⁺、Mg²⁺The antibacterial activity in the ionic solution can be kept almost unchanged. The presence of salt ions resulted in a 2.44-fold increase in the MIC value of FR, while the MIC value of LG was only 1.81-fold.

Example 6 construction of recombinant expression vectors

In order to apply the recombinant diploid antimicrobial peptide LG to livestock production, the recombinant diploid antimicrobial peptide LG with biological activity is successfully obtained by a Pichia pastoris expression system.

Artificially designing a gene fragment for coding LG according to codon preference of yeast, adding a 6 XHis-SUMO fusion tag at the N end of an LG sequence, adding an EcoR I enzyme cutting site at the N end, adding a TAA stop codon and a Kpn I enzyme cutting site at the C end, connecting a target fragment with an expression vector pPICZ alpha A, and constructing a recombinant expression vector pPICZ alpha A-6 XHis-SUMO-LG. FIG. 2 shows a 6 XHis-SUMO-LG recombinant expression vector construction.

The recombinant expression vector plasmid is linearized by Sac I and then is electrically transferred into Pichia pastoris X33. By Zeocin^TMScreening and PCR identification show that the target gene is successfully transformed into Pichia pastoris X33. The PCR identification results are shown in FIG. 3.

Example 7 expression of fusion proteins

The screened positive clones are inoculated into BMGY and BMMY culture media for shake flask expression, P.pastoris X33/6 XHis-SUMO-LG and P.pastoris X33/pPICZ alpha A fermentation supernatant are subjected to TCA concentration, Tricine-SDS-PAGE electrophoresis results show that the target genes are successfully expressed, and the results are shown in figure 4.

Example 8 removal of fusion tag and purification of recombinant LG (rLG)

The fusion protein 6 XHis-SUMO-LG was purified by nickel column and mixed with SUMO protease and digestion buffer and incubated for 16 hours at 4 ℃. The mixed system was again passed through a nickel column, and the results are shown in FIG. 5. Collecting the flow-through, dialyzing with 1KDa MWCO dialysis tube, and lyophilizing. Finally, the purified recombinant LG (rLG) was verified by Tricine-SDS-PAGE. The molecular weight of the peptides was further determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the results are shown in FIG. 6.

Sequence listing

<110> northeast university of agriculture

<120> antimicrobial peptide LL37 active center derived peptide diploid antimicrobial peptide LG and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Phe Lys Arg Ile Val Gln Arg Ile Lys Arg Phe Leu Arg Gly Gly Gly

1 5 10 15

Gly Ser Phe Lys Arg Ile Val Gln Arg Ile Lys Arg Phe Leu Arg

20 25 30