阅读说明：本技术 一种抗菌肽HeHampⅡ-4(63-88)及其应用 (Antibacterial peptide HeHamp II-4 (63-88) and application thereof ) 是由 林强 肖旺红 秦耿 张艳红 于 2021-08-31 设计创作，主要内容包括：本发明公开了一种抗菌肽HeHampⅡ-4(63-88)及其应用,属于生物技术领域。本发明的抗菌肽,其氨基酸序列如SEQ ID NO:1所示。本发明的抗菌肽HeHampⅡ-4(63-88)对革兰氏阴性菌、革兰氏阳性菌和真菌均具有明显的凝集作用,对海马胚胎细胞无毒性,并且能显著抑制副溶血弧菌、鲍曼不动杆菌和大肠杆菌游离内毒素的释放,可用于制备抗菌制剂,从而应用于水产养殖业和医药领域。(The invention discloses an antibacterial peptide HeHamp II-4 (63-88) and application thereof, belonging to the technical field of biology. The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1. The antibacterial peptide HeHamp II-4 (63-88) has obvious agglutination on gram-negative bacteria, gram-positive bacteria and fungi, has no toxicity on hippocampal embryonic cells, can obviously inhibit the release of free endotoxin of vibrio parahaemolyticus, acinetobacter baumannii and escherichia coli, can be used for preparing antibacterial preparations, and is applied to the aquaculture industry and the medicine field.)

1. An antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1.

2. A nucleic acid encoding the antimicrobial peptide of claim 1.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. A genetically engineered cell transduced or transfected with the nucleic acid of claim 2 or the recombinant expression vector of claim 3.

5. Use of the antimicrobial peptide of claim 1, the nucleic acid of claim 2, the recombinant expression vector of claim 3, or the genetically engineered cell of claim 4 in the preparation of an antimicrobial formulation.

6. Use according to claim 5, wherein the antibacterial is against one or more of gram-negative bacteria, gram-positive bacteria and fungi.

7. The use of claim 5, wherein the antibiotic is one or more of anti-Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, multi-drug resistant Escherichia coli, Acinetobacter baumannii-resistant strains, Klebsiella pneumoniae, Salmonella typhimurium, Bacillus thuringiensis, Micrococcus luteus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, enterococcus faecalis, methicillin-resistant Staphylococcus epidermidis, Bacillus subtilis, and Candida albicans.

8. An antibacterial preparation comprising the antibacterial peptide according to claim 1.

9. The antimicrobial formulation of claim 8, wherein the formulation is a feed additive, a veterinary drug, a preservative, or a pharmaceutical inhibitor.

Technical Field

The invention relates to the technical field of biology, and particularly relates to an antibacterial peptide derived from Hippocampus kelloggi.

Background

The sea horse is the only fish with male pregnancy, and has extremely high economic and medicinal values. In recent years, the sea horse cultivation is rapidly developed and gradually enters a mature industrialization stage. With the continuous expansion of the culture scale and the increase of the culture density, the sea horse culture is seriously attacked by diseases, such as enteritis, skin ulcer and the like caused by water quality and pathogenic microorganisms, so that huge economic loss is caused. Meanwhile, a large amount of nutrient substances in the excreted excrement or fish feed can promote the quick growth of bacteria in water, release a large amount of endotoxin and damage the healthy growth of cultured animals. Moreover, the problems of drug residue, pathogenic bacteria drug resistance and the like are increasingly serious due to the large use of antibiotics in mariculture, and the development of the aquaculture and the human health are endangered.

Antimicrobial peptides (AMPs) are important effector factors in the innate system and have a wide range of inhibitory effects on bacteria, fungi, viruses and the like. Aquatic animals live in water rich in microorganisms, face the attack of the microorganisms all the time, and the antibacterial peptide plays an important role in defending the invasion of pathogenic microorganisms. Unlike antibiotics and other chemical drugs, most of the antibacterial peptides have no specificity to microorganisms and are difficult to generate drug resistance, so that the antibacterial peptides become ideal substitutes for antibiotics. And antibiotics kill bacteria, usually cause bacterial death to cause massive release of endotoxin, and many antibacterial peptides have an antibacterial effect and can neutralize endotoxin to reduce bacterial virulence.

Although antimicrobial peptides have many advantages, they have certain disadvantages, for example, a significant proportion of natural antimicrobial peptides are highly toxic and can even cause hemolysis in eukaryotic cells; furthermore, many antimicrobial peptides have a narrow antimicrobial spectrum and exhibit antimicrobial activity against only a specific group of microorganisms, such as only gram-negative bacteria, only gram-positive bacteria, only fungi, or only certain microorganism or microorganisms. To solve the above problems, the present inventors have made an effort to develop novel antimicrobial peptides having a broad spectrum, no eukaryotic cytotoxicity and capable of inhibiting the release of bacterial endotoxin, to meet the needs of practical applications.

Disclosure of Invention

The first aspect of the invention provides an antibacterial peptide which has a wide microorganism agglutination spectrum, has NO toxicity to eukaryotic cells and can effectively inhibit the release of bacterial endotoxin, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1, and specifically, the amino acid sequence of the antibacterial peptide is as follows: HSGPCKFCCNCCGRMHFCGFCCEWRF are provided.

In a second aspect, the invention provides a nucleic acid encoding the antimicrobial peptide of the first aspect.

In a third aspect, the present invention provides a recombinant expression vector comprising the nucleic acid of the second aspect.

In a fourth aspect, the invention provides a genetically engineered cell transduced or transfected with the nucleic acid of the second aspect or the recombinant expression vector of the third aspect.

The fifth aspect of the present invention provides the use of the antimicrobial peptide of the first aspect, the nucleic acid of the second aspect, the recombinant expression vector of the third aspect, or the genetically engineered cell of the fourth aspect, in the preparation of an antimicrobial agent;

preferably, the antimicrobial is against one or more of gram negative bacteria, gram positive bacteria and fungi;

preferably, the antibiotic is one or more of vibrio parahaemolyticus, vibrio harveyi, escherichia coli, multi-drug resistant escherichia coli, acinetobacter baumannii drug resistant strains, klebsiella pneumoniae, salmonella typhimurium, bacillus thuringiensis, micrococcus luteus, staphylococcus aureus, methicillin-resistant staphylococcus aureus, enterococcus faecalis, methicillin-resistant staphylococcus epidermidis, bacillus subtilis and candida albicans.

In a sixth aspect of the present invention, there is provided an antibacterial preparation comprising the antibacterial peptide of the first aspect;

preferably, the formulation is a feed additive, veterinary drug, preservative or pharmaceutical inhibitor.

Compared with the prior art, the invention has the following beneficial effects: compared with other antibacterial peptides, the antibacterial peptide has a wider agglutination spectrum on microorganisms, has a better inhibition effect on free endotoxin of bacteria, has no toxicity on eukaryotic cells, and can be applied to the fields of aquaculture industry and medicines.

Drawings

FIG. 1 shows the effect of the synthetic peptide HeHamp II-4 (63-88) on the release of free endotoxin from Acinetobacter baumannii, with the different letters on the fold lines indicating significant differences between the different fold lines;

FIG. 2 is a graph showing the effect of the synthetic peptide HeHamp II-4 (63-88) on the release of free endotoxin from Vibrio parahaemolyticus, where the letters on the fold lines are different, indicating that there is a significant difference between the different fold lines;

FIG. 3 shows the effect of the synthetic peptide HeHamp II-4 (63-88) on the release of free endotoxin from E.coli, the letters on the fold lines being different, indicating that there is a significant difference between the different fold lines;

FIG. 4 shows the effect of the synthetic peptide HeHamp II-4 (63-88) on hippocampal embryonic cell activity.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

EXAMPLE 1 determination of the sequence of Hippocampus kelloggi antimicrobial peptide hepcidin (HeHamp II-4)

The invention screens and obtains a cDNA sequence of HeHamp II-4 based on a hippocampus kelloggi genome and transcriptome database, clones and obtains the antibacterial peptide sequence from hippocampus kelloggi tissues, and comprises the following specific steps:

total RNA was extracted from liver tissue of adult male hippocampus using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the instructions attached thereto. Using the Reverace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) kit, 1. mu.g of total RNA was taken for the first strand cDNA synthesis. Primers F, R (F: 5'-ATGAAGCCCTTCAGTTTGTC-3'; R:5'-TTAGAATCTCCATTCGCAGC-3') specific for this gene were designed using Primer 5.00(Palo Alto, CA) for amplification of the Open Reading Frame (ORF) of HeHamp II-4. In this study, the PCR reaction follows the following cycle parameters of 95 ℃ for 5 min; 36 cycles of 95 ℃ for 30s,60 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃. The DNA product of the corresponding molecular mass was purified using the E.Z.N.AGEL Extraction Kit (Omega Bio Tek, Norcross, GA, USA). The 3 positive clones were selected and sequenced by Biotechnology Limited (Shanghai, China). The amplified fragment of the open reading frame of the cDNA of the hippocampus kelhamp II-4 is 267bp and encodes 88 amino acid residues (the amino acid sequence is shown as SEQ ID NO: 2). The amino acid sequence structure of the polypeptide consists of a signal peptide, a leader peptide and a mature peptide, and the polypeptide has obvious similarity with the reported antimicrobial peptide hepcidin family, so that the hippocampus kelloggi Hehamp II-4 is a new member of the hepcidin family.

EXAMPLE 2 detection of biological Activity of Hippocampus kelloggi antimicrobial peptide hepcidin (HeHamp II-4) mature peptide

Synthesis of Hehamp II-4 mature peptide

The amino acid sequence of the mature peptide HeHamp II-4 (63-88) is as follows: HSGPCKFCCNCCGRMHFCGFCCEWRF, or as shown in SEQ ID NO: 1. Synthesized by GenScript Biotech Inc. (Nanjing, China) using a solid phase peptide synthesis method. The molecular mass and purity of the synthetic peptide were 2.747kD and 96.1%, respectively. The synthetic peptide was stored at-80 ℃ in the form of a dry powder prior to use.

Microbiological agglutination test

Gram-negative bacteria used for agglutination experiments: vibrio parahaemolyticus, vibrio harveyi, escherichia coli, multi-drug resistant escherichia coli, acinetobacter baumannii drug resistant strains, klebsiella pneumoniae and salmonella typhimurium; gram-positive bacteria: bacillus thuringiensis, Micrococcus luteus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, enterococcus faecalis, methicillin-resistant Staphylococcus epidermidis, and Bacillus subtilis; fungi: candida albicans. Bacteria (non-vibrio) were cultured in Nutrient Broth (NB), MH broth, vibrio in 2216E medium, and fungi in YPD medium. Culturing at optimum temperature to logarithmic phase, centrifuging microbial cells at 4000 Xg for 5min at room temperature, washing with TBS buffer (10mM Tris-HCl, 150mM sodium chloride, pH 7.4) three times, and resuspending to OD with TBS buffer₆₀₀0.6. Dissolving the synthetic peptide in sterile water, adding into solution containing microorganism, and adding 10mM CaCl₂After mixing, the bacterial solution was OD-treated₆₀₀The value was 0.3, and the final protein concentrations in the microorganism agglutination test were 3. mu.M, 6. mu.M, and 12. mu.M, respectively. While BSA (bovine serum albumin, 2mg/mL) was used as a control, three replicates were set up for each group. The agglutination reaction was observed under an optical microscope (LEICA dmi4000b, germany) and the results are shown in table 1.

TABLE 1 microbial agglutination activity of HeHamp II-4 (63-88)

Protein concentration	12μM	6μM	3μM	Control
					Bacillus thuringiensis	+++	++	+	-
Bacillus subtilis	+++	++	+	-
					Micrococcus luteus	+++	++	+	-
Staphylococcus aureus	+++	++	+	-
					Methicillin-resistant staphylococcus aureus	+++	++	+	-
Enterococcus faecalis	+++	++	+	-
					Methicillin-resistant staphylococcus epidermidis	+++	++	+	-
Vibrio parahaemolyticus	+++	++	+	-
					Vibrio harveyi	+++	++	+	-
Acinetobacter baumannii	+++	++	+	-
					Acinetobacter baumannii drug-resistant strain	+++	++	+	-
Escherichia coli	+++	++	+	-
					Multi-drug resistant escherichia coli	+++	++	+	-
Salmonella typhimurium	+++	++	+	-
					Klebsiella pneumoniae	+++	++	+	-
Candida albicans	+++	++	+	-

Wherein "+++" means 95% -100% of the bacteria agglutinate; "+ +" means 70% -95% of the cells agglutinated; "+" means 30% -70% of the cells agglutinated; "-" indicates the presence or absence of visible cell aggregation.

As can be seen from Table 1, the synthetic peptide HeHamp II-4 (63-88) has good agglutination effect on gram-negative bacteria, gram-positive bacteria and fungi, and particularly has good agglutination activity on drug-resistant strains (methicillin-resistant staphylococcus aureus, methicillin-resistant staphylococcus epidermidis, multi-drug-resistant escherichia coli and acinetobacter baumannii drug-resistant strains), so that the antibacterial broad-spectrum property of the peptide is reflected; at a protein concentration of 3. mu.M, a significant agglutination phenomenon occurred.

Example 3 bacterial endotoxin inhibition assay

The reagents and consumables used in the experiment are sterile and have no endotoxin.

Incubating Escherichia coli, Acinetobacter baumannii and Vibrio parahaemolyticus at optimum temperature overnight, collecting thallus, washing with 1 × PBS for 3 times, and resuspending with 1 × PBS to 1 × 10⁵cfumL^-1. The experimental groups were: the synthetic peptide is mixed with the bacterial liquid in equal volume (when treating escherichia coli and vibrio parahaemolyticus, the final concentration of protein in the mixed liquid of the protein and the bacterial liquid is 6 mu M; when treating acinetobacter baumannii, the final concentration of protein in the mixed liquid of the protein and the bacterial liquid is 6 mu M); the control group was: protein solvent (one thousandth volume of DMSO solution) was mixed with the same volume of bacteria solution, and mature peptide of large yellow croaker hepcidin (PC-hepc) was selected as reference, with three replicates per group. At room temperature, at 0.5h,1.5h and 3h after co-incubation of the polypeptide and the bacteria, the bacteria were removed by filtration through a 0.22 μ M frit and the free endotoxin concentration of the bacteria was measured using Limulus reagent endotoxin detection kit (BIOENDO, China). Escherichia coli,The endotoxin release results of Acinetobacter baumannii and Vibrio parahaemolyticus are shown in FIG. 1, FIG. 2 and FIG. 3, respectively.

As can be seen from FIGS. 1, 2 and 3, the hippocampal hepcidin mature peptide HeHamp II-4 (63-88) can inhibit the release of free endotoxin from bacteria, and the effect is significantly stronger than that of the large yellow croaker hepcidin mature peptide PC-hepc.

Example 4 cytotoxicity assay

When the density of hippocampus kelloggi embryonic cells reached more than 90%, passage was performed. The medium was discarded, washed with 1mL of BSS, and then digested with 1mL of trypsin for 30s to a suspended state. Cell suspensions were diluted with medium (80% DMEM, 20% fetal bovine serum FBS, 1 Xdouble antibody) to a cell concentration of 1X 10⁵Per mL, the diluted cell suspension was added to a 96-well cell culture plate at 100. mu.L per well and incubated overnight at 28 ℃. Prior to the experiment, the medium was aspirated, 100. mu.L of DMEM medium containing Hehamp II-4 (63-88) polypeptide was added to the medium at final protein concentrations of 96. mu.M, 48. mu.M and 24. mu.M, each concentration being set in triplicate, and the medium was incubated at 28 ℃ for 24h with an equal volume of DMEM medium containing no polypeptide added as a control. Sucking out the old culture medium, adding 100 mu of LDMEM fresh culture medium and 10 mu of CCK-8 reagent into each well, culturing for 4h in a dark place, measuring absorbance A450nm, and calculating the survival rate of the cells:

1. the absorbance values of three replicates of A450nm of the control group (final protein concentration of 0. mu.M) were A1, A2, A3, and their mean value was A. A1/A, A2/A, A3/A are cell viability of the control group.

2. The absorbance values of A450nm of three parallel samples of each experimental group (the final protein concentration is 24 mu M, 48 mu M and 96 mu M) are respectively B1, B2 and B3, and the cell survival rates are respectively obtained by dividing A by B1/A, B2/A and B3/A.

Statistical difference analysis is carried out by using Unpaired Student's t test or one-way anova and Tukey's test, and P <0.05 has statistical significance for the difference. The results are shown in FIG. 4.

As can be seen from FIG. 4, the hippocampal hepcidin mature peptide HeHamp II-4 (63-88) was co-incubated with hippocampal embryonic cells without decreasing the survival rate of the cells, indicating that it is not toxic to embryonic cells.

Sequence listing

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