CN111333700B - Pseudosciaena crocea whey acidic protein antibacterial peptide and application thereof - Google Patents
Pseudosciaena crocea whey acidic protein antibacterial peptide and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A23L3/3526—Organic compounds containing nitrogen
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- A—HUMAN NECESSITIES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses large yellow croaker whey acidic protein antibacterial peptide (LCWAP), the amino acid sequence of which is FTKPGVCPRRRWGAG. The molecular weight of the antibacterial peptide is 1688 Da. The invention also discloses application of the large yellow croaker whey acidic protein antibacterial peptide in preventing staphylococcus aureus, vibrio alginolyticus, aeromonas hydrophila and other bacteria. The invention lays a foundation for further researching the large yellow croaker whey acidic protein antibacterial peptide as a food preservative and developing a feed additive for preventing fish diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to large yellow croaker whey acidic protein antibacterial peptide and application thereof.
Background
Food-borne diseases caused by pathogenic microorganisms are a major problem facing consumers, industries and regulatory agencies. It is estimated that about 6 million people (about one tenth of the world's population) suffer from illness after eating contaminated food, causing 42 million deaths each year. The use of chemically synthesized preservatives has led to various health-related problems over the past few decades. Meanwhile, the breeding industry in China has the problems of serious diseases, drug abuse and the like in the rapid development process. The antibacterial peptide is a plurality of immune factors produced by organisms, has good inhibition effect on bacteria, and is expected to become a substitute of chemical preservatives and breeding drugs.
Staphylococcus aureus and Escherichia coli are the most interesting pathogenic microorganisms in food products. Staphylococcus aureus is widely distributed on the skin and mucous membranes of many warm-blooded animals, including humans, and contaminates food during manufacture and processing; coli is also one of the most common food-borne pathogens in food products and is considered a major public health risk. The streptococcus iniae has the characteristics of wide infected hosts, strong infectivity, high mortality and the like, is mostly in acute neurotropic tissue diseases, and causes huge loss to aquaculture industry.
Large yellow croaker (Larimichthys crocea) is the largest fish in Chinese cage culture. Several families of antimicrobial peptides have been identified in large yellow croakers, including piscidin, LEAP-2 and NK-lysin. Whey acidic protein is an important component of the immune system of marine organisms, and is found to be related to the immunoregulation of large yellow croakers at present, but most of researches are concentrated on whey acidic protein with a large molecular weight, and the peptide of the whey acidic protein has the defects of long and complex chain, difficult synthesis by a chemical method, difficult purification, unstable structure, easy inactivation and the like, so that the further research and application of the whey acidic protein become extremely difficult.
Disclosure of Invention
The invention aims to provide large yellow croaker whey acidic protein antibacterial peptide and application thereof, and provides experimental basis for searching a new food preservative and an aquatic feed additive for preventing fish diseases through the research on the antibacterial activity of the large yellow croaker whey acidic protein antibacterial peptide LCWAP on staphylococcus aureus, vibrio alginolyticus and aeromonas hydrophila, thereby promoting the healthy and sustainable development of food and aquaculture industries in China.
In order to solve the above-mentioned purpose, the invention adopts the following technical scheme:
an antibacterial peptide LCWAP of large yellow croaker whey acidic protein has an amino acid sequence of FTKPGVCPRRRWGAG, which is shown in SEQ ID NO: 1 is shown.
A feasible theoretical basis exists for searching antibacterial peptide in the sequence of the acidic protein of large yellow croaker whey as a target. Therefore, the method firstly utilizes three online servers of AntiBP, APD3 and CAMP to screen and calculate the whey acidic protein sequence of the large yellow croaker, and then predicts the whey acidic protein structure of the large yellow croaker through a Swiss-model server, finds an antibacterial peptide sequence FTKPGVCPRRRWGAG with strong antibacterial effect on staphylococcus aureus, vibrio alginolyticus and aeromonas hydrophila, is named LCWAP and has the molecular weight of 1688 Da. The content of the secondary structure of the product is determined by circular dichroism chromatography: the alpha-helix content was 19.3%; beta-sheet content 26.0%; the content of beta-turn is 24.7 percent, and the content of random coil is 30.0 percent.
The antibacterial peptide LCWAP can cause damage to bacteria from at least the following three aspects: on one hand, the water is adsorbed on the surface of bacterial cell membrane, and the cell membrane is damaged to cause bacterial death; on the other hand, the permeability of the bacterial cell membrane is changed while the cell membrane is damaged, the generation of the cell membrane is inhibited, and the possibility of polluting the apparatus by bacteria is reduced; in yet another aspect, binding to bacterial genomic DNA inhibits bacterial DNA synthesis, thereby causing bacterial death.
The invention also provides a coding gene of the large yellow croaker whey acidic protein antibacterial peptide.
The invention also provides an expression cassette of the coding gene.
The invention also provides a recombinant bacterium of the coding gene.
The invention also provides a recombinant vector of the coding gene.
The invention also provides a transgenic cell line of the coding gene.
The invention also provides the application of the polypeptide, the coding gene, the recombinant vector or the transgenic cell line in inhibiting bacteria.
Preferably, the bacteria include one or more of Staphylococcus aureus, Vibrio alginolyticus, and Aeromonas hydrophila. Preferably, the use of the above-mentioned inhibiting bacteria is for the preparation of food preservatives.
Preferably, the use of the above-mentioned inhibiting bacteria is for preparing feed additives for treating or preventing fish diseases.
The invention has extremely low toxicity to normal human liver cells, can be used as a food preservative and a fish feed additive, particularly as a preservative for preventing the pollution of common pathogenic bacteria of food, such as staphylococcus aureus, vibrio alginolyticus, aeromonas hydrophila and the like, and a fish disease treatment or prevention additive for treating or preventing vibrio alginolyticus and aeromonas hydrophila, and is applied to aquatic feeds.
The antimicrobial peptides of the invention can be synthesized, e.g., by solid phase synthesis, using methods known to those skilled in the art, and purified, e.g., by high performance liquid chromatography, using methods known to those skilled in the art.
The implementation of the invention has the following beneficial effects:
the invention takes whey acidic hemoglobin as a research object and discovers a polypeptide LCWAP with a brand new amino acid sequence by screening and calculating. Researching the antibacterial activity of LCWAP on staphylococcus aureus, vibrio alginolyticus, aeromonas hydrophila and other bacteria; and the damage degree of the LCWAP to the staphylococcus aureus is observed by using a transmission electron microscope as an example; observing whether the LCWAP can be adsorbed on the surface of the bacteria by using a laser confocal microscope; and finally, evaluating the human body safety influence. The experimental result shows that the peptide has strong inhibiting effect on staphylococcus aureus, vibrio alginolyticus, aeromonas hydrophila and other bacteria. Its bacteriostatic mechanism is that it is first adsorbed on the surface of bacteria, then destroys the cell membrane of bacteria and inhibits the generation of membrane, and at the same time it has very low toxicity to normal liver cell of human body. The invention provides experimental basis for LCWAP as food preservative and aquatic feed additive.
Drawings
FIG. 1 is a comparison chart of the determination of the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide LCWAP of the invention to Staphylococcus aureus, wherein A: the concentration of the antibacterial peptide is 0 mug/mL; b: the concentration of the antibacterial peptide is 125 mug/mL; c: the concentration of the antibacterial peptide is 62.5 mu g/mL; d: the concentration of the antibacterial peptide is 31.25 mu g/mL; e: the concentration of the antibacterial peptide is 15.6 mu g/mL; f: the concentration of the antimicrobial peptide was 7.8. mu.g/mL.
FIG. 2 is a comparison chart of the measurement of Minimum Inhibitory Concentration (MIC) of antibacterial peptide LCWAP of the present invention to Vibrio alginolyticus, wherein A: the concentration of the antibacterial peptide is 0 mug/mL; b: the concentration of the antibacterial peptide is 125 mg/mL; c: the concentration of the antibacterial peptide is 62.5 mg/mL; d: the concentration of the antibacterial peptide is 31.25 mu g/mL; e: the concentration of the antibacterial peptide is 15.6 mu g/mL; f: the concentration of the antimicrobial peptide was 7.8. mu.g/mL.
FIG. 3 is a comparison chart of the determination of the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide LCWAP of the invention to Aeromonas hydrophila, wherein A: the concentration of the antibacterial peptide is 0 mug/mL; b: the concentration of the antibacterial peptide is 125 mug/mL; c: the concentration of the antibacterial peptide is 62.5 mu g/mL; d: the concentration of the antibacterial peptide is 31.25 mu g/mL; e: the concentration of the antibacterial peptide is 15.6 mu g/mL; f: the concentration of the antimicrobial peptide was 7.8. mu.g/mL.
FIG. 4 is a transmission electron microscope observation image of Staphylococcus aureus with the effect of the antibacterial peptide LCWAP of the present invention, wherein A: a staphylococcus aureus placebo control group; b: staphylococcus aureus after 0.5h of LCWAP treatment; c: staphylococcus aureus after 2h of LCWAP treatment.
FIG. 5 is a confocal microscope observation image of Staphylococcus aureus cells treated by the antibacterial peptide LCWAP of the present invention, wherein A: a fluorescence image; b: a bright field image; c: and combining the images.
Fig. 6 is a bar graph of the inhibitory effect of the antimicrobial peptide LCWAP of the present invention on staphylococcus aureus biofilm formation, wherein p represents significance p < 0.005; indicates significance p < 0.001.
FIG. 7 is the electrophoresis chart of the combination of the antibacterial peptide LCWAP of the present invention and the DNA of Staphylococcus aureus, wherein, the band 1: blank control; 2-7 of the strip: the LCWAP to DNA mass ratios were 100/1, 50/1, 25/1, 25/2, 25/4, 25/8, respectively.
FIG. 8 is a graph showing the UV absorption spectrum of the binding of the antimicrobial peptide of the present invention to DNA of Staphylococcus aureus.
Fig. 9 is a view of a predicted model structure of the antibacterial peptide LCWAP of the present invention.
FIG. 10 is a bar graph showing the effect of LCWAP of the present invention on the viability of normal human hepatocytes (LO2 cells).
Detailed Description
For better understanding of the present invention, the following embodiments and the accompanying drawings are used to describe the present invention in further detail, but those skilled in the art will appreciate that the following embodiments are not intended to limit the scope of the present invention, and any changes and modifications based on the present invention are within the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: screening of large yellow croaker whey acidic protein derived antibacterial peptide
Predicting an antibacterial sequence possibly existing in the acidic protein sequence of the large yellow croaker whey by using antibacterial peptide prediction online servers AntiBP, APD3 and CAMP, analyzing the charge, hydrophobicity and reliability of the antibacterial sequence possibly existing, finally screening out an amino acid sequence FTKPGVCPRRRWGAG for chemical synthesis, and verifying the antibacterial activity.
Example 2: minimum Inhibitory Concentration (MIC) assay
Culturing Staphylococcus aureus, Vibrio alginolyticus and Aeromonas hydrophila at 37 deg.C for 12h to logarithmic phase, and diluting to 10 in 0.01M phosphate buffer solution with pH of 7.26-7CFU/mL. The peptide was dissolved in phosphate buffer and mixed with the bacteria at equal volume at 37 ℃ for 2 h. The Minimum Inhibitory Concentration (MIC) is the lowest concentration of the antimicrobial peptide at which no bacterial growth is visible from the microtiter plate after incubation at 37 ℃ overnight. As shown in FIG. 1, FIG. 2 and FIG. 3, the Minimum Inhibitory Concentration (MIC) of LCWAP to Staphylococcus aureus was 62.5. mu.g/mL, the Minimum Inhibitory Concentration (MIC) to Vibrio alginolyticus was 15.6. mu.g/mL, and the Minimum Inhibitory Concentration (MIC) to Aeromonas hydrophila was 31.2. mu.g/mL (FIG. 1, FIG. 2 and FIG. 3).
Example 3: transmission electron microscopy analysis
At 106-7CFU/mL of bacteria were treated with 2 × MIC of LCWAP at 37 ℃ for 2h, then centrifuged at 2700g for 10min and washed twice with phosphate buffer (pH 7.2). After fixation with 1% osmic acid, dehydration with 95% ethanol followed by acetone treatment for 20 min. Samples were baked at 70 ℃ for 24h, and 70-90nm thin slices were prepared on a copper grid and then stained with lead citrate and uranium acetate. The ultrastructure was observed and captured by H-7650 transmission electron microscope.
By taking staphylococcus aureus as an example, as shown in fig. 4, for untreated bacteria, the intracellular structure of the bacteria is compact, the tissues are intact, and the cell surfaces are not damaged. However, after 0.5h of treatment with the peptide LCWAP, it was clearly observed that bacterial cells were cleared and the edges of the cell membranes began to blur; after 2h of treatment with the peptide LCWAP, severe damage to the bacterial cell membrane, cytoplasmic efflux and cell death were observed. The transmission electron microscope result shows that the LCWAP peptide has destructive effect on the cell membrane and the internal structure of the bacteria.
Example 4: LCWAP aggregation on bacterial cell surface
In the case of Staphylococcus aureus, after incubation to log phase, the bacterial concentration was adjusted to 106-7CFU/mL, was incubated with the fluorescently labeled peptide LCWAP at 37 deg.C, 2 × MIC concentration for 60min, then centrifuged, cells washed, resuspended in phosphate buffer, and then analyzed with confocal laser scanning microscopy.
As shown in fig. 5, the results indicate that LCWAP can accumulate on the surface of s.aureus cells, and that during the interaction of the antimicrobial peptide with the cell membrane, the antimicrobial peptide covers the cell surface until a threshold concentration is reached, resulting in cell membrane disruption and cell death.
Example 5: effect of LCWAP on bacterial cell membrane formation
To investigate the effect of the peptide LCWAP on biofilm formation, the bacteria were incubated at low concentrations of LCWAP, as exemplified by staphylococcus aureus, and their effect on bacterial cell membrane formation was observed. The specific operation is as follows: staphylococcus aureus cells were collected by centrifugation, resuspended in Luria Bertani (LB) broth, and then mixed with equal volumes of corresponding dilutions of LCWAP at different concentrations. The mixture was added to a 96-well flat bottom plate, incubated at 37 ℃ for 72h, then gently washed 3 times with PBS (pH 7.2) to remove non-adherent cells, and then 200. mu.L of 0.1% crystal violet was added. The plates were incubated at 25 ℃ for 30min and then washed with PBS to remove crystal violet. After drying the plate at room temperature, 200mg of 95% ethanol was added to each well. Nisin and PBS served as positive and negative controls, respectively. Biofilm formation ability was inversely correlated with LCWAP concentration. However, when the concentration of the LCWAP peptide was below 1/2MIC, there was little effect on biofilm formation, and when the concentration of the LCWAP peptide was 1 × MIC, biofilm formation did not occur, as shown in fig. 6. Biofilm formation represents a protected mode of growth, providing protection for bacterial cells to survive in harsh environments, which is considered a major health risk in the food processing industry. Thus, the biofilm-formation inhibiting activity of LCWAP is a good feature of the peptide, suggesting that it may be used as a preservative.
Example 6: interaction of LCWAP with bacterial DNA
The interaction of LCWAP and staphylococcus aureus genome DNA was studied by DNA gel retardation. Staphylococcus aureus was cultured in 50mL nutrient broth at 37 ℃ for 12h, and bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The purity of the extracted genomic DNA was evaluated at an optical density ratio of 260 and 280nm (OD260/OD 280. gtoreq.1.90). Next, 3. mu.L of DNA (100 ng/. mu.L) was mixed with a continuous amount of the peptide LCWAP at 25 ℃ for 10min, and the mixture was subjected to electrophoresis on a 0.8% agarose gel. Gel retardation was observed under UV irradiation using a GelDoc XR gel imaging system (Bio-Rad, USA), as shown in FIG. 7.
In addition, the interaction of LCWAP with bacterial DNA was further analyzed using uv spectroscopy. Different volumes of S.aureus DNA (100 ng/. mu.L) were mixed with 200. mu.L (1000 ng/. mu.L) of peptide LCWAP for 10min at 37 ℃. Then, the UV absorption spectrum was measured at 250-330nm using a Thermo scientific Microplate reader, as shown in FIG. 8.
Example 7: 3D structure prediction for LCWAP
The structures of the large yellow croaker whey acidic protein and the antibacterial peptide LCWAP derived from the large yellow croaker whey acidic protein are predicted by using an online structure prediction server Swiss-model, and are edited and modified by using Pymol software to obtain the structure of the antibacterial peptide LCWAP and the spatial position of the antibacterial peptide LCWAP in the large yellow croaker Whey Acidic Protein (WAP), as shown in FIG. 9.
Example 8: effect of LCWAP on human Normal hepatocytes
The MTT (3- (4-5-dimethylthiazol-2-yl) -2-5-diphenyl-2H-tetrazolium bromide) method is the classical method for assessing cytotoxicity. The specific operation is as follows: normal human hepatocytes (LO2) 10mg/mL were added to 96-well plates at 5% CO2And incubation in a 37 ℃ carbon dioxide incubator until the cells adhere, then adding different concentration gradients of LCWAP to each well. 5% CO at 37 deg.C2And (4) performing medium incubation for 24 h. After the incubation was completed, 5mg/mL was added to each wellMTT solution with concentration of 20 μm is incubated at 37 deg.C for 4h, the supernatant of the well plate is discarded, 150 μ L of dimethyl sulfoxide is added to each well, the crystals are completely dissolved in a low speed shaker for 10min, and the absorbance value is measured at 490 nm. Nisin and PBS served as positive and negative controls, respectively. The results showed that different LCWAP concentrations did not have adverse cytotoxic effects on LO2 cells, and cell survival rates of greater than 90.3% were found, as shown in figure 10. The cytotoxic effect of LCWAP is similar to that of NISIN, which shows that LCWAP has potential application prospect in food industry.
SEQUENCE LISTING
<110> college university
<120> large yellow croaker whey acidic protein antimicrobial peptide and application thereof
<130> do not
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213> Artificial Synthesis
<400> 1
Phe Thr Lys Pro Gly Val Cys Pro Arg Arg Arg Trp Gly Ala Gly
1 5 10 15
Claims (9)
1. The amino acid sequence of the large yellow croaker whey acidic protein antimicrobial peptide is shown as SEQ ID NO: 1 is shown.
2. A gene encoding the large yellow croaker whey acidic protein antimicrobial peptide of claim 1.
3. An expression cassette comprising the gene encoding the gene of claim 2.
4. A recombinant bacterium comprising the gene according to claim 2.
5. A recombinant vector comprising the coding gene of claim 2.
6. A transgenic cell line comprising the gene encoding claim 2.
7. Use of the large yellow croaker whey acidic protein antimicrobial peptide of claim 1, the encoding gene of claim 2, the recombinant vector of claim 5, or the transgenic cell line of claim 6 for the preparation of a preparation for inhibiting bacteria; the bacteria include one or more of Staphylococcus aureus, Vibrio alginolyticus, and Aeromonas hydrophila.
8. Use of the inhibitor of bacteria according to claim 7 for the preparation of a food preservative.
9. The use of the inhibitor of bacteria according to claim 7 for the preparation of a feed additive.
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