CN116178522A

CN116178522A - Fish source anion antibacterial peptide Lc149 and application thereof

Info

Publication number: CN116178522A
Application number: CN202310304952.1A
Authority: CN
Inventors: 张东玲; 陈美玲; 王志勇
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-05-30

Abstract

The invention belongs to the technical field of biology, and particularly discloses a fish source anion antibacterial peptide Lc149 and application thereof. The amino acid sequence of the fish source anion antibacterial peptide Lc149 is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1. The applicant obtains the fish source anion antibacterial peptide Lc149 by constructing a prokaryotic expression vector, inducing expression, crushing by ultrasonic waves and purifying. The antibacterial peptide Lc149 not only can inhibit the growth of escherichia coli, vibrio harveyi and bacillus subtilis, but also has the effect of killing ciliates of the shield; the antibacterial activity is not affected at 25-100deg.C. In addition, lc149 is non-hemolytic toxic to blood cells of large yellow croaker, shrimp and rabbits. The antibacterial peptide Lc149 can be used for biological control of bacterial and parasitic diseases of aquatic and mammals, or can be used as an antibacterial agent, an insecticide and a feed additive.

Description

Fish source anion antibacterial peptide Lc149 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fish source anion antibacterial peptide Lc149 and application thereof.

Background

In aquaculture, the widespread use and abuse of antibiotics has led to the development of resistance by a variety of pathogenic bacteria, and therefore, there is an urgent need to find alternatives to antibiotics. Antibacterial peptides are widely present in organisms and are the first line of defense in host immune defenses, playing a critical role in innate immunity. The natural antibacterial peptide has antibacterial, antiviral, antiparasitic and antitumor properties, and also has the functions of resisting apoptosis, promoting wound healing, regulating chemical chemotaxis of T cells, promoting angiogenesis, epithelial cell proliferation and regeneration, and being a communication bridge of innate and acquired immune responses in monocytes, macrophages and dendritic cells (ZhangR, xuL, dongC.Antimicrobialpeptides: anoverviewoftheir structure, functional division of protein peptide Lett,2022, 29:641-50.). The bactericidal mechanism of antimicrobial peptides is mainly to destroy the integrity of the cell membrane, act on the negatively charged bacterial cell membrane, form pores in the cell membrane, or inhibit the synthesis of proteins, DNA and RNA, or act in combination with certain specific targets within the cell (Mechanism ofantimicrobial peptides: anti-micro-biological, anti-inflammatory and antibiofilm activites, intjmol Sci,2021, 22:11401.). The antibacterial peptide has various action mechanisms, is not aimed at one or a plurality of targets on cells, but kills the cells in a relatively rough mode, so bacteria are difficult to generate drug resistance to the antibacterial peptide, and research shows that the sterilization efficiency of the antibacterial peptide is not lower than that of the currently used antibiotics, and even the effect is better. In addition, the antibacterial peptide is a small molecular polypeptide which is generally composed of less than 100 amino acids, is easy to biodegrade and cannot be polluted by long-term residual diffusion. In summary, antimicrobial peptides have great potential advantages in the field of novel antimicrobial drug development, and have become the best alternatives for the synthesis of antibiotics.

Antibacterial peptides are widely distributed in bacteria, insects, fish, amphibians, and higher animals, plants and humans. Fish are important aquatic vertebrates, and can also produce large amounts of antimicrobial peptides. To date, more than 100 fish antibacterial peptides have been isolated and purified or expressed by artificial synthesis or genetic engineering techniques, mainly including Hepcidin, defensin, cathelicidin, piscidin, histone-derivep and NK-lysin et al (YulemaV, elenaCP, meseguerJ, et al biological role of fish antimicrobial peptides [ M ] New York: antimicrobial peptides, 2013.). The basic structure of fish antimicrobial peptides is similar to that of mammalian antimicrobial peptides, and there are usually more cationic amino acids to carry positive charges, and more disulfide bonds formed by cysteines and alpha-helical structures. The positive charges, disulfide bonds and alpha-helical structures may interact with the pathogen cell membrane or cell wall components, thereby lysing and entering the interior to kill the pathogen.

Anionic antimicrobial peptides are very rare, and are generally considered to be supplements to cationic antimicrobial peptides, with a different mechanism of action than cationic antimicrobial peptides. Existing experimental evidence suggests that membrane interactions are key steps in the antimicrobial mechanism of anionic antimicrobial peptides, including circular pore (i.e., worm hole) formation and Shai-Huang Matsazuki membrane interaction model, and bacterial cell membrane breakdown by tilting of the peptide steric structure (Dennison SR, harris F, mura M, et al, an atlas ofanionic antimicrobial peptides from amp proteins, curr Protein peptide Sci,2018, 19:823-38.). For example, DCD-1L is a more thorough investigation of anionic antimicrobial peptides that are capable of binding to negatively charged bacterial surfaces in the form of amphiphilic α -helices, which then assemble into an oligomeric state. The oligomerized DCD-1L has the ability to form ion channels in bacterial membranes leading to cell death (Paulmann M, arnold T, linke D, et al Structure-activity analysis of the dermcidin-derived peptide DCD-1L,an anionic antimicrobial peptide present in human sweat.J Biol Chem,2012,287:8434-43.).

Disclosure of Invention

The invention aims to provide a fish-derived anionic antibacterial peptide Lc149, wherein the amino acid sequence of the fish-derived anionic antibacterial peptide Lc149 is shown as SEQ ID NO.2, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.

It is a further object of the present invention to provide the use of a fish-derived anionic antimicrobial peptide Lc149, which fish-derived anionic antimicrobial peptide Lc149 can be used for the preparation of, but is not limited to, bacteriostats, or pesticides, or feed additives.

In order to achieve the above object, the present invention adopts the following technical scheme:

the applicant constructs a prokaryotic expression vector pET-32a-Lc149 by connecting a coding gene of the fish-derived anionic antibacterial peptide Lc149 with a pET-32a plasmid, converts the pET-32a-Lc149 into E.coli DH5 alpha competent cells by heat shock to be efficiently amplified, extracts the plasmid, transfers the plasmid into E.coli BL21 cells to obtain a pET-32a-Lc149 fusion expression strain, induces expression, ultrasonic disruption and purification, and finally obtains the fish-derived anionic antibacterial peptide Lc149. The nucleotide sequence SEQ ID NO.1 of the antibacterial peptide Lc149 and the sequence of the corresponding amino acid is shown as SEQ ID NO. 2. The antimicrobial peptide Lc149 may be obtained by any means for preparing a protein, including but not limited to prokaryotic, eukaryotic expression, synthesis, and the like.

The nucleotide sequence SEQ ID NO.1 and the amino acid sequence SEQ ID NO.2 of the fish source anion antibacterial peptide Lc149 belong to the protection scope of the invention.

The application of the fish source anionic antibacterial peptide Lc149 comprises the step of preparing a bacteriostatic agent or an insecticide or a feed additive from the fish source anionic antibacterial peptide Lc149 provided by the invention.

In the above applications, the anionic antimicrobial peptide Lc149 of fish origin inhibits or kills bacteria and parasites including but not limited to: coli, vibrio harveyi, bacillus subtilis, and ciliates.

The invention has the remarkable advantages that:

the fish source anionic antibacterial peptide Lc149 provided by the invention can effectively inhibit the growth of gram negative bacteria, and particularly has a strong bactericidal effect on escherichia coli and vibrio harveyi which are difficult to control by chemical medicines. Meanwhile, the antibacterial peptide has a strong insecticidal effect on ciliates in a short time. The hemolysis experiment proves that the antibacterial peptide Lc149 is harmless to blood cells of fish, shrimp and mammal rabbits. The antibacterial peptide Lc149 is expected to be used for biological control of diseases of aquatic animals and mammals in later research, or can be used as a drug or used as a feed additive for development and utilization.

Drawings

FIG. 1 shows the results of purification of recombinant protein expression of antibacterial peptide Lc149.

FIG. 2 shows the antibacterial peptide Lc149 and the control against the inhibition/sterilization of E.coli (A), vibrio harveyi (B) and Bacillus subtilis (C).

FIG. 3 is a scanning electron microscope observation result of antibacterial peptide Lc149 and control after inhibiting/sterilizing effect on Escherichia coli, vibrio harveyi and Bacillus subtilis. A, E.coli in control group; b, antibacterial peptide Lc149 group escherichia coli; c, vibrio harveyi in a control group; d, antibacterial peptide Lc149 group Vibrio harveyi; e, control group bacillus subtilis; and F, antibacterial peptide Lc149 group bacillus subtilis.

Fig. 4 shows the killing effect of the antibacterial peptide Lc149 and the control on ciliates. A, controlling the ciliates of the group; b, the antibacterial peptide Lc149 acts with the ciliates for 15min; c, the antibacterial peptide Lc149 and the ciliates act for 30min; d, the antibacterial peptide Lc149 acts on the ciliates for 1h; e, the antibacterial peptide Lc149 acts on the ciliates for 2h.

FIG. 5 is a graph showing the results of the heat stability test of the antibacterial peptide Lc149.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

The technical scheme of the invention is conventional in the art unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available.

Example 1:

through computer simulation, large yellow croaker genome is compared to obtain antibacterial peptide Lc149, and the nucleotide sequence of the corresponding coding gene is as follows:

AACATCAACATCGACATTACCGACGAGGAGCTCACAACTCACGCTATTTATCTGATGACC

AGGGAGACGTCCGTGAAGGACTCGGACCTGGAACAATTCAAGCAGCAGGCGAGCTGC

CTCGGCTTCACCGGAGAACCACACTTCAGCTACGACCCCAAAAATGATTTCTGTGCTGAAGGCGAGGGCTCTCATCTGTCACTC。

the amino acid sequence corresponding to the antibacterial peptide Lc149 is:

NINIDITDEELTTHAIYLMTRETSVKDSDLEQFKQQASCLGFTGEPHFSYDPKNDFCAEGEGS HLSL。

analysis in the antimicrobial peptide library (Antimicrobial Peptide Calculator and Predictor (un. Edu)) found that the antimicrobial peptide Lc149 had a net charge of-8.25 and a 31% proportion of hydrophobic residues, which was a negative antimicrobial peptide.

Preparation of antibacterial peptide Lc149:

(1) Constructing an expression vector pET-32a-Lc149: and (3) recombining and connecting an antibacterial peptide Lc149 encoding gene fragment containing EcoR I and Xho I enzyme cutting sites with the pET-32a vector to obtain a fusion expression vector pET-32a-Lc149 containing the target gene.

(2) And transforming the fusion expression vector pET-32a-Lc149 plasmid with correct sequencing into E.coli DH5 alpha competent cells by a heat shock method to efficiently amplify.

(3) And (3) extracting plasmids, and transferring the plasmids into escherichia coli BL21 cells to obtain pET-32a-Lc149 fusion expression strains.

(4) Expression was induced with 0.1mol/L IPTG inducer for 16h at 20 ℃.

(5) The induced solution was centrifuged at 4℃at 7000r/min, and after washing 3 times with 1mol/LPBS buffer, the cells were harvested, and the pellet was resuspended in pre-chilled buffer A (1 mol/L PBS,20mmol/L imidazole, pH 7.4) and the mixture sonicated with a cell disruptor.

(6) Centrifugation was performed at 12000r/min for 20 minutes at 4℃and the supernatant and pellet were separated and analyzed by SDS-PAGE, respectively.

(7) The supernatant was filtered through a 0.22 μm filter and purified by His-tag column. The non-target proteins were washed with buffer B (1 mol/L PBS,35mmol/L imidazole, pH 7.4) and then the target proteins were collected with elution buffer C (1 mol/L PBS buffer, 500mmol/L imidazole, pH 7.4).

(8) Removing high concentration imidazole with dialysis bag, and freeze drying the eluate.

FIG. 1 shows the result of purification of antibacterial peptide Lc149 expression, resulting in a soluble purified protein.

Example 2: antibacterial peptide Lc149 inhibiting/sterilizing effect

Preparation of indicator bacteria: selecting Escherichia coli, vibrio harveyi and Bacillus subtilis (Bacillus subtilis) as indicator bacteria, inoculating the indicator bacteria into LB liquid medium at 1% -2% v/v inoculum size, shake culturing at 28deg.C or 37deg.C (Escherichia coli, vibrio harveyi and Bacillus subtilis) for 8-10 hr at 120-150r/min to obtain bacterial OD ₆₀₀ Adjust to 1.0.

Preparing a double-layer plate: firstly, pouring a solid LB culture medium as a lower plate, and standing for 3-5min; and then taking 300 mu L of indicator bacteria and 3mL of semisolid culture medium, mixing uniformly, pouring the mixture on the lower layer plate rapidly, shaking gently and uniformly, dividing the culture dish into a plurality of areas after the semisolid culture medium is solidified, and placing a sterile oxford cup in each area.

Antibacterial activity assay: the concentration of purified Lc149 protein was adjusted to 500. Mu.g/mL, 50. Mu.L was slowly added to an oxford cup, incubated at 28℃or 37℃for 18 hours, and the size of the inhibition zone was measured and photographed. pET-32a empty-load protein was used as a control.

FIG. 2 shows the inhibitory/bactericidal effect of antibacterial peptide Lc149 and empty carrier protein control on E.coli, vibrio harveyi and Bacillus subtilis (3 replicates), and the size of the zone of inhibition is shown in Table 1.

TABLE 1 antibacterial/bactericidal Activity of antibacterial peptide Lc149

Example 3: antibacterial peptide Lc149 sterilization mechanism

E.coli, vibrio harveyi and bacillus subtilis are selected as indicator bacteria, and the bacteria are regulated to 1 multiplied by 10 by using a working medium ⁸ CUF/mL(OD ₆₀₀ =1.0). Antibacterial peptide Lc149 was added to the bacterial solution at a final concentration of 500 μg/mL and the mixture was incubated at 37 ℃ in 24-well plates for 2h. Sample 3 was fixed with 2.5% glutaraldehyde at 4 ℃After h, dehydration was performed in a series of graded ethanol. Finally, the sample is dehydrated fully by 100% ethanol, then is air-dried in an ultra-clean workbench, and the thalli are observed under a scanning electron microscope. pET-32a empty-load protein was used as a control.

FIG. 3 shows the results of scanning electron microscope observations after the antibacterial peptide Lc149 and empty carrier protein controls have been subjected to the inhibition/sterilization of E.coli, vibrio harveyi and Bacillus subtilis. FIG. 4 shows that the empty carrier protein control group E.coli (FIG. 3A), vibrio harveyi (FIG. 3C) and Bacillus subtilis (FIG. 3E) are in round bar shape, normal bar shape, have complete cell wall and complete round surface; after 2h co-culture of the antibacterial peptide Lc149 and the indicator bacteria, the cell wall is perforated and broken by escherichia coli (fig. 3B), vibrio harveyi (fig. 3D) and bacillus subtilis (fig. 3F).

Example 4: insecticidal effect of antibacterial peptide Lc149

Culturing the collected yellow croaker ciliate in a 16 deg.C incubator, and replacing fresh seawater subjected to sterilization and filtration for 1-2 times a day. Culturing until the 3 rd to 4 th days, and enabling the ciliates to stably amplify when the ciliates adapt to a culture environment, thus being applicable to experiments. Approximately 300 ciliate larvae were added to a 48-well plate containing 500 μg/mL Lc149 protein solution in a total volume of 1.5mL. 200. Mu.L of the mixture was fixed with 5.0% glutaraldehyde at 4℃for 3h. The sterilized seawater was used as a control instead of the antibacterial peptide Lc149. The insect bodies were observed under an optical microscope.

Fig. 4 shows the killing effect of the antibacterial peptide Lc149 and the control on ciliates. Fig. 4A shows that the control group ciliates had full bodies, free swimming, tidy and intact cilia, and the internal storage particles were light black. After 15min of action of the antibacterial peptide Lc149 with ciliates, the worms slowly swim and ciliates become disordered (fig. 4B); after 30min, the cilia of the larvae had shrunken and the somatic cells of the larvae began to appear ruptured (fig. 4C); after 1h, the insect body is obviously contracted, takes a spherical shape, is seriously broken, and the content is largely leaked, so that most cilia disappear (fig. 4D); after 2 hours, the whole ciliate body disintegrated.

Example 5: determination of the thermal stability of the antibacterial peptide Lc149

Adjusting the concentration of the antibacterial peptide: and (3) respectively placing the antibacterial peptide Lc149 and the pET-32a empty carrier protein into a 2mL centrifuge tube, and adjusting the concentrations of the antibacterial peptide Lc149 and the empty carrier protein to 500 mug/mL by using PBS buffer for later use.

Treatment of the antimicrobial peptides at different temperatures: the antibacterial peptide Lc149 and pET-32a empty carrier protein are respectively heat treated for 30min at 25 ℃,50 ℃, 75 ℃ and 100 ℃ and then are placed at room temperature for standby.

Bacteriostasis experiment: the antibacterial activity was measured by agarose diffusion, and the method was the same as in example 3.

FIG. 5 is a graph showing the results of the heat stability test of the antibacterial peptide Lc149. Antibacterial peptide Lc149 has little effect on the antibacterial activity of Vibrio harveyi as the temperature increases. When the temperature is higher than 75 ℃, the antibacterial activity of the escherichia coli is reduced, but the influence is not great. And for bacillus subtilis, the antibacterial activity is obviously reduced along with the rise of temperature.

Example 6: cytotoxicity of antibacterial peptide Lc149:

fresh large yellow croaker blood, shrimp blood and rabbit blood are respectively taken, centrifuged for 10min at 4 ℃, the supernatant is discarded, the sediment is resuspended 3 times by using 0.25M PBS buffer (PH=7.2), and the blood cells are obtained after centrifugation for 5min at 3000rpm at 4 ℃. Blood cells were diluted to 1% with the same concentration of TBS buffer and the Lc149 protein was adjusted to different concentrations. Mu.l of the blood cell suspension was incubated with 50. Mu.l of the antimicrobial peptide Lc149 at various concentrations in 96-well cell culture plates at 37℃for 1h, with positive control being sterilized ultrapure water (complete hemolysis) and negative control being TBS buffer (non-hemolysis). After incubation for 1h, centrifugation at 4000rpm for 10min, cell debris was removed and 100. Mu.l of the supernatant was taken in a fresh 96-well cell culture plate and absorbance at 540nm was determined. The calculation formula is as follows:

hemolysis ratio (%) = (experimental group a 540-negative control a 540)/(positive control a 540-negative control a 540) ×100%

The results show (Table 2) that the antibacterial peptide Lc149 is not significantly toxic to fish, shrimp and mammalian blood cells and can be used as an antibiotic replacement drug (the concentration of the antibacterial peptide Lc149 in Table 2 refers to the concentration of protein prior to mixing with the blood cell suspension).

TABLE 2 haemolytic properties on blood cells

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A fish-derived anionic antimicrobial peptide Lc149, characterized by: the amino acid sequence of the fish source anion antibacterial peptide Lc149 is shown as SEQ ID NO. 2.

2. The coding gene of the fish-derived anionic antibacterial peptide Lc149 according to claim 1, wherein: the nucleotide sequence of the coding gene of the fish source anion antibacterial peptide Lc149 is shown as SEQ ID NO. 1.

3. Use of an anionic antibacterial peptide Lc149 of fish origin according to claim 1 or of a coding gene according to claim 2 for the preparation of a bacteriostatic agent for inhibiting bacteria.

4. A use according to claim 3, characterized in that: the bacteria include Escherichia coli, vibrio harveyi and Bacillus subtilis.

5. Use of an anionic antibacterial peptide Lc149 of fish origin according to claim 1 or of a coding gene according to claim 2 for the preparation of pesticides.

6. The use according to claim 5, characterized in that: the pesticide is used for killing ciliates.

7. Use of an anionic antibacterial peptide Lc149 of fish origin according to claim 1 or of a coding gene according to claim 2 for the preparation of a feed additive.

8. The use according to claim 7, characterized in that: the feed additive is used for at least one of the following uses 1) to 2):

1) Is used for inhibiting escherichia coli, vibrio harveyi and bacillus subtilis;

2) Is used for killing ciliate.