阅读说明：本技术 脂肽Lin-Lf4NH2和Lin-Lf5NH2及其应用 (Lipopeptide Lin-Lf4NH2And Lin-Lf5NH2And uses thereof ) 是由 王建华 刘鹤 毛若雨 滕达 王秀敏 杨娜 郝娅 于 2020-05-06 设计创作，主要内容包括：本发明提供新型的脂肽Lin-Lf4NH<Sub>2</Sub>和Lin-Lf5NH<Sub>2</Sub>及其应用。脂肽Lin-Lf4NH<Sub>2</Sub>/Lin-Lf5NH<Sub>2</Sub>是在抗菌肽LfcinB4/LfcinB5的N端偶联亚油酸,C端经过酰胺化修饰得到的。实验表明,脂肽Lin-Lf4NH<Sub>2</Sub>和Lin-Lf5NH<Sub>2</Sub>对革兰氏阳性菌和阴性菌均具有较好的抑制作用,比母体肽的MIC值小,热稳定性好,在小鼠皮肤脓肿模型试验中能够显著降低皮肤荷菌量,减轻皮肤组织损伤,具有比Lf4NH<Sub>2</Sub>和Lf5NH<Sub>2</Sub>更好的治疗效果,是极具应用价值的小分子脂肽,可用于制备新型抗菌抗感染药物等,应用前景广阔。(The invention provides a novel lipopeptide Lin-Lf4NH 2 And Lin-Lf5NH 2 And applications thereof. Lipopeptide Lin-Lf4NH 2 /Lin‑Lf5NH 2 Is obtained by coupling linoleic acid at the N end of antibacterial peptide LfcinB4/LfcinB5 and amidating modification at the C end. Experiments show that the lipopeptide Lin-Lf4NH 2 And Lin-Lf5NH 2 Has better inhibiting effect on gram-positive bacteria and gram-negative bacteria, has smaller MIC value than parent peptide, has good thermal stability, can obviously reduce the skin bacterial load and lighten the skin tissue damage in a mouse skin abscess model test, and has the inhibiting effect on gram-positive bacteria and gram-negative bacteria compared with Lf4NH 2 And Lf5NH 2 Better treatment effect, is the micromolecular lipopeptide with great application value, can be used for preparing novel antibacterial and anti-infective medicaments and the like, and has wide application prospect.)

1. Lipopeptide Lin-Lf4NH₂Characterized in that the lipopeptide Lin-Lf4NH₂Is obtained by coupling linoleic acid at the N end of antibacterial peptide LfcinB4 and amidating and modifying the C end.

2. Lipopeptide Lin-Lf5NH₂Characterized in that the lipopeptide Lin-Lf5NH₂Is obtained by coupling linoleic acid at the N end of antibacterial peptide LfcinB5 and amidating and modifying the C end.

3. A broad spectrum antibacterial medicament or composition comprising a lipopeptide according to claim 1 and/or claim 2.

4. A preservative, antiseptic or surfactant comprising a lipopeptide according to claim 1 and/or claim 2.

5. The lipopeptide Lin-Lf4NH of claim 1₂Or the lipopeptide Lin-Lf5NH of claim 2₂Any of the following uses:

1) for the preparation of an antibacterial medicament or composition;

2) for the preparation of preservatives;

3) is used for preparing bactericide;

4) is used for preparing the surfactant.

6. Use according to claim 5, wherein the bacteria comprise gram-positive and gram-negative bacteria.

7. Use according to claim 6, characterized in that said bacteria comprise bacteria of the genus Staphylococcus (Staphylococcus), Escherichia (Escherichia), Salmonella (Salmonella).

8. Use according to claim 7, wherein the bacteria comprise Staphylococcus suis (Staphyloccocus aureus), Staphylococcus aureus (Staphyloccocus aureus), Escherichia coli (Escherichia coli), Salmonella pullorum (Salmonella pullorum), Salmonella enteritidis (Salmonella enteritidis).

Technical Field

The invention relates to the technical field of biology, in particular to lipopeptide Lin-Lf4NH₂And Lin-Lf5NH₂And applications thereof.

Background

Lactoferricin (Lfcin for short) is a segment of multifunctional antibacterial peptide released from the N-terminal of lactoferrin under the action of pepsin in an acidic environment (Gifford et al, 2005), and Lfcin is closely related to the function of lactoferrin, contains most functional domains of lactoferrin, and has strong antibacterial, anticancer, antiviral, antiparasitic and anti-inflammatory activities (Hao et al, 2018). In many cases, Lfcin not only retains the activity of lactoferrin, but is even more potent than the parent protein (Arias et al, 2014). LfcinB, which consists of amino acid residues 17-41 of bovine lactoferrin, is considered to be the most effective of the various Lfcins in cows, mice and goats (Bruni et al, 2016; Ulvatne et al, 2001). The rapid increase in bacterial resistance poses a serious challenge to antibiotics, and compared to traditional antibiotics, because antibacterial peptides have stronger antibacterial activity and different antibacterial mechanisms, are not easy to form resistance, and have the potential to become future antibacterial drugs (Andersson et al, 2016; Seo et al, 2012). However, clinical treatment and application of antibacterial peptides still have problems, and in order to improve the antibacterial activity of Lfcins, develop new functions and resist the emergence of drug-resistant bacteria, various molecular design strategies including amino acid substitution, chemical modification, cyclization, chimerization, polymerization and the like are needed (Hao et al, 2018; Yin et al, 2014).

Disclosure of Invention

The invention aims to provide a novel lipopeptide Lin-Lf4NH₂And Lin-Lf5NH₂And applications thereof.

The invention has the following conception: in order to further improve the antibacterial activity of polypeptides LfcinB4 and LfcinB5, the N ends of LfcinB4 and LfcinB5 are coupled with linoleic acid, and the C ends of the LfcinB4 and the LfcinB5 are subjected to amidation modification to obtain two lipopeptides Lin-Lf4NH₂And Lin-Lf5NH₂. Then, Lin-Lf4NH₂And Lin-Lf5NH₂The antibacterial activity, toxicity and in-vivo efficacy are evaluated, and a theoretical basis is provided for the creation of novel antibacterial drugs.

In order to implement the inventionIn a first aspect, the present invention provides a lipopeptide Lin-Lf4NH₂The antibacterial peptide is obtained by coupling linoleic acid at the N end of antibacterial peptide LfcinB4 and modifying the C end through amidation. The sequence is as follows: (Linoleic acid) -FKAWRWAWRWKKLAAPS-NH₂(SEQ ID NO:1)。

Lipopeptide Lin-Lf4NH₂The structure of (A) is shown as formula (I):

in a second aspect, the invention provides the lipopeptide Lin-Lf5NH₂Lipopeptide Lin-Lf5NH₂Is obtained by coupling linoleic acid at the N end of antibacterial peptide LfcinB5 and amidating and modifying the C end. The sequence is as follows: (Linoleic acid) -FKAFRWAWRWKKLAAPS-NH₂(SEQ ID NO:2)。

Lipopeptide Lin-Lf5NH₂The structure of (A) is shown as formula (II):

in a third aspect, the present invention provides a lipopeptide-containing Lin-Lf4NH₂And/or the lipopeptide Lin-Lf5NH₂The broad spectrum antibacterial drug or composition of (1).

In a fourth aspect, the present invention provides a polypeptide comprising the lipopeptide Lin-Lf4NH₂And/or the lipopeptide Lin-Lf5NH₂A preservative, bactericide or surfactant.

In a fifth aspect, the invention provides the lipopeptide Lin-Lf4NH₂And/or the lipopeptide Lin-Lf5NH₂Any of the following uses:

1) for the preparation of an antibacterial medicament or composition;

2) for the preparation of preservatives;

3) is used for preparing bactericide;

4) is used for preparing the surfactant.

The bacteria include gram positive bacteria and gram negative bacteria. Preferably including Staphylococcus (Staphylococcus), Escherichia (Escherichia), Salmonella (Salmonella) bacteria. More preferably, Staphylococcus suis (Staphylococcus hyicus), Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Salmonella pullorum (Salmonella pullorum), and Salmonella enteritidis (Salmonella enteritidis).

By the technical scheme, the invention at least has the following advantages and beneficial effects:

lipopeptide Lin-Lf4NH of the invention₂And Lin-Lf5NH₂Is artificially designed and synthesized active polypeptide, which comprises 17 amino acid residues and has 5 positive charges, the N end is coupled with linoleic acid, the C end is amidated and modified, and the molecular weights are 2450.09Da and 2411.05Da respectively. With the parent peptide Lf4NH₂And Lf5NH₂In contrast, Lin-Lf4NH₂And Lin-Lf5NH₂Has higher antibacterial activity and thermal stability, and has little cytotoxicity in a low concentration range.

Experiments show that the lipopeptide Lin-Lf4NH₂And Lin-Lf5NH₂The peptide has better inhibition effect on gram-positive bacteria and gram-negative bacteria, has a smaller MIC value than that of parent peptide, has good thermal stability, can obviously reduce the skin bacterial load and relieve skin tissue damage in a mouse skin abscess model test, has better effect than that of the antibiotic mupirocin, is a micromolecular lipopeptide with high application value, can be used for preparing novel antibacterial anti-infective drugs and the like, and has wide application prospect.

Drawings

FIG. 1 shows the lipopeptide Lin-Lf4NH of the present invention₂And Lin-Lf5NH₂Mass spectrum of (2).

FIG. 2 shows the lipopeptide Lin-Lf4NH in example 3 of the present invention₂And Lin-Lf5NH₂The cytotoxicity of (a).

FIG. 3 shows the lipopeptide Lin-Lf4NH in example 5 of the present invention₂And Lin-Lf5NH₂The mouse skin charge after treatment of (1).

FIG. 4 shows the lipopeptide Lin-Lf4NH in example 5 of the present invention₂And Lin-Lf5NH₂After treatment mice skin tissue sections.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Experimental materials used in the following examples: MEM high-sugar cell culture fluid, trypsin digestion fluid, PBS for cells, fetal bovine serum and streptomycin mixture were purchased from Gibco, USA, ELISA plate and sterile 96-well culture plate were purchased from Thermo Scientific, antibiotic 2% mupirocin ointment was purchased from China veterinary medicine institute, human immortalized epidermal cells (Hacat) were purchased from Beijing collaborating Hospital, and six-week-old SPF-grade BALB/c mouse Beijing Wintolite laboratory animals Co. Other conventional reagents are imported and subpackaged or domestic analytically pure.

Media and buffer formulations referred to in the following examples:

MH liquid medium: dissolving 2.4g in 80mL of distilled water, dissolving completely, diluting to 100mL, and autoclaving at 121 deg.C for 20 min.

MH solid medium: dissolving 3.65g in 80mL of distilled water, dissolving completely, diluting to 100mL, and autoclaving at 121 deg.C for 20 min.

Cell complete culture solution: 10% fetal bovine serum, 100U/mL streptomycin (diabody), 90% MEM high sugar base medium.

PBS phosphate buffer: 4.25g of NaCl, KH₂PO₄0.12g，Na₂HPO₄Dissolving 1.825g and KCl 0.1g in distilled water, dissolving completely, diluting to 500mL, and autoclaving at 121 deg.C for 20 min.

The species referred to in the following examples are shown in table 1:

TABLE 1 strains and sources