Iron-ferritin heavy chain antibacterial and antiviral fragment LcFet-H51 of large yellow croaker and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly discloses a large yellow croaker ferritin heavy chain antibacterial and antiparasitic fragment LcFet-H51 and application thereof. The amino acid sequence of the LcFet-H51 is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The applicant obtains the ferritin heavy chain fragment LcFet-H51 by constructing a prokaryotic expression vector, inducing expression, ultrasonic crushing and purification. The LcFet-H51 has a killing effect on Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio vulnificus and Chilodonella sp. The antibacterial activity of the LcFet-H51 is basically not affected by temperature 25-100 DEG C, pH 3-12 and ultraviolet radiation for 15-60 min. In addition, the LcFet-H51 has no hemolytic toxicity to large yellow croaker blood cells. The LcFet-H51 can be applied to biological control of bacterial and parasitic diseases of aquatic animals, and can also be used as an antibacterial agent, an insecticide and a feed additive.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the antibacterial and anti-insect fragment LcFet-H51 of ferritin heavy chain from large yellow croaker and its applications. Background Technology

[0002] High-density, intensive aquaculture practices have led to frequent bacterial and parasitic diseases in large yellow croaker. In the prevention and control of bacterial diseases, the frequent and excessive use of antibiotics not only results in drug resistance in pathogens, making it impossible to effectively control the disease, but also causes drug residues to disrupt the ecological balance of the aquatic environment and seriously threaten human food safety. As for parasites, especially the persistent "scutiformis disease" in juvenile fish in major large yellow croaker farming areas in recent years, the mortality rate is as high as 30%, with tens or even hundreds of millions of fry in many hatcheries dying almost entirely within days. Currently, there are no effective control measures, including no effective drug treatment (Zhang Z, Wang Z, Fang M, et al. Genome-wide association analysis on host resistance against the rottenbody disease in a naturally infected population of large yellow croaker Larimichthys crocea. Aquaculture, 2022, 548:737615.). Therefore, there is an urgent need to develop new, green biological control drugs. Antimicrobial peptides, as an important component of the immune system of aquatic animals, are characterized by being pollution-free, residue-free, having broad-spectrum antibacterial activity, and being less likely to induce drug resistance. They have become a preferred alternative to antibiotics, not only killing pathogenic microorganisms but also improving the immune function and growth performance of farmed aquatic animals. Furthermore, strengthening basic research on the immune defense mechanisms of fish is also a crucial strategy for effectively preventing and controlling diseases and achieving the sustainable and healthy development of aquaculture.

[0003] Antimicrobial peptides, also known as antimicrobial peptides, generally refer to small molecules (less than 100 amino acids) with antimicrobial activity. They are a core component of the innate immune response in all life forms and are widely distributed in bacteria, insects, fish, amphibians, higher plants and animals, and humans. They exhibit strong killing effects not only against Gram-positive bacteria, Gram-negative bacteria, and fungi, but also against viruses, parasites, eukaryotic disease cells, and tumor cells. Based on their targets, antimicrobial peptides can be classified into antimicrobial peptides, antiviral peptides, antifungal peptides, and antiparasitic peptides. The most common mechanism of action for antimicrobial peptides is their direct activity on the bacterial cell membrane. In short, the binding of antimicrobial peptides leads to disruption of membrane potential, alteration of membrane permeability, and leakage of metabolites, ultimately resulting in bacterial cell death. Antimicrobial peptides can also exert their antibacterial effects by targeting key cellular processes, such as DNA and protein synthesis, protein folding, enzyme activity, and cell wall synthesis (Li X, Zuo S, Wang B, et al. Antimicrobial Mechanisms and Clinical Application Prospects of Antimicrobial Peptides. Molecular, 2022, 27:2675.). Furthermore, antimicrobial peptides can exert their effects by modulating adaptive immune responses or by acting as chemokines to recruit other effector cells. It has been demonstrated that antimicrobial peptides can influence the integrity of the upper intestinal barrier by stimulating mucus synthesis and promoting the production of tight junction proteins (Daneshmand A, Kermanshahi H, Sekhavati MH, et al. Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers. Sci Rep. 2019, 9:14176.).

[0004] Ferritin is a globular protein complex composed of 24 subunits with a hollow shell structure capable of mineralizing up to 4,500 iron atoms. In vertebrates, it contains ferritin subunits encoded by two different genes (heavy subunit - H and light subunit - L). During pathogen invasion, ferritin participates in the immune response as an acute-phase reactive protein. The transcriptional levels of these ferritins can be significantly induced under infection. Importantly, they may exhibit resistance to bacterial growth. Zheng et al. demonstrated that ferritin in the ark shell of *Scapharca broughtonii* can inhibit the growth of Gram-negative *Escherichia coli* and Gram-positive *Micrococcus luteus* and *Staphylococcus aureus* (Zheng L, Liu Z, Wu B, et al. Ferritin has an important immune function in theark shell *Scapharca broughtonii*. Dev Comp Immunol, 2016, 59:15-24.). Jung et al. demonstrated that the ferritin subunits (H and M) of mullet possess antibacterial activity against Escherichia coli and iron-binding survival (Jung S, Kim MJ, Lim C, et al. Molecular insights into two ferritin subunits from red-lip mullet (Liza haematocheila): Detectable antibacterial activity with its expressional response against immune stimulants. Gene, 2023, 851:146923.). Ding et al. also showed that the two ferritin subunits (H and M) of blunt snout bream can inhibit the growth of Aeromonas hydrophila (Ding Z, Zhao X, Zhan Q, et al. Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): characterization, expression, iron depriving and bacteriostatic activity. Fish Shellfish Immunol, 2017, 66:411-22.). In addition, ferritin is also involved in the innate defense against viruses.Shrimp infected with silent ferritin had a significantly higher WSSV copy number than the control group (Yang H, Liu Z, Jiang Q, et al. A novel ferritin gene from Procambarus clarkii involved in the immune defense against Aeromonas hydrophila infection and inhibits WSSV replication. Fish Shellfish Immunol, 2019, 86:882-91.). Summary of the Invention

[0005] The purpose of this invention is to provide a large yellow croaker ferritin heavy chain antibacterial and anti-insect fragment LcFet-H51, the amino acid sequence of LcFet-H51 is shown in SEQ ID NO.2, and the nucleotide sequence corresponding to its encoding gene is shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide the application of the large yellow croaker ferritin heavy chain antibacterial and insecticidal fragment LcFet-H51, including but not limited to the preparation of antibacterial agents, insecticides, or feed additives.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The applicant constructed a prokaryotic expression vector containing the gene encoding the large yellow croaker ferritin heavy chain antibacterial and insecticidal fragment LcFet-H51, induced expression, sonicatedly disrupted, and purified the fragment to obtain LcFet-H51. The corresponding nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. LcFet-H51 can be obtained using any protein preparation method, including but not limited to prokaryotic and eukaryotic expression and synthesis.

[0009] The nucleotide sequence SEQ ID NO.1 and amino acid sequence SEQ ID NO.2 of LcFet-H51 are both within the scope of protection of this invention.

[0010] Applications of LcFet-H51, an antibacterial and insecticidal fragment of ferritin heavy chain from large yellow croaker, include the preparation of antibacterial drugs or insecticides, or feed additives, using the antimicrobial peptides provided by this invention.

[0011] In the applications described above, the bacteria and parasites that the antibacterial / insecticides can inhibit or kill include, but are not limited to: Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio vulnificus, and scutiformis.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] The antibacterial and antiparasitic ferritin heavy chain fragment LcFet-H51 screened in this invention effectively inhibits the growth of both Gram-positive and Gram-negative bacteria, particularly exhibiting strong bactericidal activity against Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus, which are difficult to control with chemical agents. Simultaneously, LcFet-H51 demonstrates a strong insecticidal effect against scutellaria barbata within a short period. Hemolysis experiments have shown that LcFet-H51 is non-toxic to fish hemocytic cells. LcFet-H51 holds promise for future research in the biological control of aquatic animal diseases, and may be developed and utilized as a drug or feed additive. Attached Figure Description

[0014] Figure 1 Results of expression and purification of LcFet-H51 recombinant protein.

[0015] Figure 2 The inhibitory / bacterial effects of LcFet-H51 and the control against Staphylococcus aureus (A), Vibrio parahaemolyticus (B), and Vibrio vulnificus (C) are shown. D represents the diameter of the inhibition zone.

[0016] Figure 3 The minimum inhibitory concentration of LcFet-H51 against Staphylococcus aureus, Vibrio vulnificus, and Vibrio parahaemolyticus is given.

[0017] Figure 4 Scanning electron microscopy (SEM) results of the inhibitory / bactericidal effects of LcFet-H51 and the control group on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus. A, Control group: Staphylococcus aureus; B, LcFet-H51 group: Staphylococcus aureus; C, Control group: Vibrio parahaemolyticus; D, LcFet-H51 group: Vibrio parahaemolyticus; E, Control group: Vibrio vulnificus; F, LcFet-H51 group: Vibrio vulnificus.

[0018] Figure 5 The killing effect of LcFet-H51 and the control on scutellaria was shown. A, control group of scutellaria; B, LcFet-H51 treated with scutellaria for 15 min; C, LcFet-H51 treated with scutellaria for 30 min; D, LcFet-H51 treated with scutellaria for 1 h; E, LcFet-H51 treated with scutellaria for 2 h.

[0019] Figure 6 The figure shows the stability test results of LcFet-H51. Detailed Implementation

[0020] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional technologies in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0022] Example 1: Obtaining the C-terminal fragment LcFet-H51 of the H chain of ferritin from large yellow croaker

[0023] Through computer simulation, genome alignment, charge and structure analysis of large yellow croaker, the C-terminal fragment LcFet-H51 of the H chain (heavy chain) of ferritin in large yellow croaker was obtained. The nucleotide sequence corresponding to its encoding gene is as follows:

[0024] TGTGATTTCATCGAGACACACTATCTGGACGAGCAGGTGAAGTCCATCAAGGAACTGGC

[0025] AGACTGGGTGACCAACTTGCGTCGCATGGGAGCTCCTCAGAACGGCATGGCCGAATACC

[0026] TGTTTGACAAACACACCCTGGGCAAAGAAAGCAGC

[0027] The amino acid sequence corresponding to the C-terminal fragment LcFet-H51 of the ferritin H chain in large yellow croaker is as follows:

[0028] CDFIETHYLDEQVKSIKELADWVTNLRRMGAPQNGMAEYLFDKHTLGKESS

[0029] Preparation of the C-terminal fragment LcFet-H51 of the H chain of ferritin from large yellow croaker:

[0030] (1) Construction of expression vector pET-32a-LcFet-H51: The gene fragment encoding the C-terminal fragment of the H chain of ferritin from large yellow croaker containing EcoRI and XhoI restriction sites was recombined and ligated with the pET-32a vector to obtain the fusion expression vector pET-32a-LcFet-H51 containing the target gene.

[0031] (2) The correctly sequenced fusion expression vector pET-32a-LcFet-H51 plasmid was transformed into Escherichia coli DH5α highly competent cells by heat shock method for amplification.

[0032] (3) Extract the plasmid and then transform it into Escherichia coli BL21 cells to obtain the pET-32a-LcFet-H51 fusion expression strain.

[0033] (4) At 20℃, expression was induced for 16 h with 0.1 mol / L inducer.

[0034] (5) The induction solution was centrifuged at 7000 r / min at 4℃ and washed three times with 1 mol / L PBS buffer before harvesting the cells. The precipitate was resuspended in pre-cooled buffer A (1 mol / L PBS, 20 mmol / L imidazole, pH 7.4) and the mixture was sonicated using a cell disruptor.

[0035] (6) Centrifuge at 4℃ and 12000r / min for 20 minutes to separate the supernatant and precipitate, and analyze them separately by SDS-PAGE.

[0036] (7) The supernatant was filtered through a 0.22 μm filter membrane and then purified using a His-tag column. Non-target proteins were first washed with buffer B (1 mol / L PBS, 35 mmol / L imidazole, pH 7.4), and then the target proteins were collected with elution buffer C (1 mol / L PBS buffer containing 500 mmol / L imidazole, pH 7.4).

[0037] (8) Remove high concentrations of imidazole using a dialysis bag, and finally freeze-dry the eluent.

[0038] Figure 1 The results of expression and purification of the C-terminal fragment LcFet-H51 of the H chain of ferritin from large yellow croaker were obtained, and finally, soluble purified protein was obtained.

[0039] Example 2: Determination of the antibacterial / bacterial effect and minimum inhibitory concentration (MIC) of LcFet-H51. Indicator bacteria preparation: Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus were selected as indicator bacteria. The indicator bacteria were inoculated into LB liquid medium at a 1%-2% v / v inoculum and cultured at 37℃ (Staphylococcus aureus), 28℃ (Vibrio parahaemolyticus and Vibrio vulnificus), and 120-150 rpm for 8-10 h with shaking. The bacterial OD... 600 Adjusted to version 1.0.

[0040] Double-layer plate preparation: First, pour solid LB medium as the lower layer plate and let it stand for 3-5 minutes; then take 300 μL of indicator bacteria and mix gently with 3 mL of semi-solid medium, quickly pour it onto the lower layer plate, gently shake it to mix evenly, and after the semi-solid medium solidifies, divide the petri dish into several areas, and place a sterile Oxford cup in each area.

[0041] Antibacterial activity assay: The concentration of purified LcFet-H51 protein was adjusted to 500 μg / mL, and 50 μL was slowly added to an Oxford cup. The cup was incubated at 37℃ or 28℃ for 18 h. The size of the inhibition zone was measured and photographed. The pET-32a empty vector protein was used as a control.

[0042] Figure 2 The inhibitory / bacterial effects of LcFet-H51 protein and empty carrier protein on Staphylococcus aureus (A), Vibrio vulnificus (B) and Vibrio parahaemolyticus (C) were investigated.

[0043] The purified LcFet-H51 protein was diluted with PBS to 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.63 μg / mL. The antibacterial activity was determined using the same method as described above to determine the minimum inhibitory concentration.

[0044] like Figure 3 As shown, the minimum inhibitory concentrations (MICs) of LcFet-H51 protein against Staphylococcus aureus, Vibrio vulnificus, and Vibrio parahaemolyticus are 31.25-62.5 μg / mL, 15.625-31.25 μg / mL, and 15.625-31.25 μg / mL, respectively.

[0045] Example 3: Sterilization mechanism of LcFet-H51

[0046] Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus were selected as indicator bacteria, and the bacterial concentration was adjusted to 1×10⁻⁶ using working medium. 8 CUF / mL (OD) 600 =1.0). The purified LcFet-H51 protein was added to the bacterial solution at a final concentration of 500 μg / mL, and the mixture was incubated in 24-well plates at 28°C for 2 h. After fixing the samples with 2.5% glutaraldehyde at 4°C for 3 h, they were dehydrated in serial fractions of ethanol. Finally, the samples were thoroughly dehydrated with 100% ethanol and air-dried in a clean bench. The bacterial cells were observed under a scanning electron microscope. The pET-32a empty vector protein was used as a control.

[0047] Figure 4 The results of scanning electron microscopy observations of the inhibitory / bactericidal effects of LcFet-H51 protein and empty carrier protein on Staphylococcus aureus, Vibrio parahaemolyticus and Vibrio vulnificus. Figure 4 The empty vector protein control group of Staphylococcus aureus (Staphylococcus aureus) showed that... Figure 4 A) Clustered in a grape-like pattern, the bacteria are plump and have smooth, flat cell surfaces; Vibrio parahaemolyticus ( Figure 4 C) and Vibrio vulnificus ( Figure 4E) is short rod-shaped, with its surface covered with pili and flagella. After co-culturing LcFet-H51 protein with indicator bacteria for 2 hours, Staphylococcus aureus (… Figure 4 B) Distorted and deformed shape, wrinkled and collapsed cell membranes, cell shrinkage, and leakage of contents; Vibrio parahaemolyticus ( Figure 4 D) and Vibrio vulnificus ( Figure 4 F) The disappearance of pili and the smooth bacterial surface indicate a significant reduction or loss of bacterial adhesion ability; some Vibrio species, like Staphylococcus aureus, exhibit cell membrane wrinkling and collapse, cell shrinkage, and leakage of contents; in addition, some Vibrio species also show perforation on their surface. This unique bactericidal mechanism of LcFet-H51 protein acting on the bacterial cell membrane is unlikely to induce microbial resistance.

[0048] Example 4: Insecticide Effect of LcFet-H51

[0049] The collected large yellow croaker shield ciliates were cultured in a 16℃ incubator, with sterilized filtered seawater replaced 1-2 times daily. On days 3-4, when the shield ciliates had adapted to the culture environment and were able to stably proliferate, they were ready for experiments. Approximately 300 shield ciliate larvae were added to a 48-well plate containing 500 μg / mL LcFet-H51 protein solution, for a total volume of 1.5 mL. The mixture was fixed for 3 hours at 4℃ with 200 μL of 5.0% glutaraldehyde. Sterile seawater was used as a control instead of LcFet-H51 protein. The larvae were observed under an optical microscope.

[0050] Figure 5 The killing effect of LcFet-H51 protein and control on scutellaria barbata was evaluated. Figure 5 A shows the control group of scutellaria, which are plump, swim freely, have neat and intact cilia, and their internal storage granules are light black. After 15 minutes of treatment with LcFet-H51 protein, the scutellaria swim slowly, the color of their internal storage granules darkens, and their cilia become disordered. Figure 5 B); After 30 minutes, the insect body begins to shrink, internal stored particles accumulate, the color darkens, the cilia become disordered, and the insect body ruptures. Figure 5 C); 1 hour later, the worm body ruptured severely, a large amount of contents leaked out, and most of the cilia disappeared. Figure 5 D); 2 hours later, the entire worm disintegrated. Figure 5 E).

[0051] Example 5: Stability determination of LcFet-H51

[0052] Adjusting the LcFet-H51 protein concentration: Take the purified LcFet-H51 protein and pET-32a empty vector protein into 2mL centrifuge tubes respectively, and adjust the concentration of LcFet-H51 protein and empty vector protein to 500μg / mL with PBS buffer for later use.

[0053] Antimicrobial peptides were treated at different temperatures: LcFet-H51 protein solutions were heat-treated at 25℃, 50℃, 75℃, and 100℃ for 30 min and then set aside.

[0054] Different pH treatments for LcFet-H51: Adjust the pH of the LcFet-H51 protein solution to 3, 5, 7, 11, and 12 for later use.

[0055] Ultraviolet radiation treatment of antimicrobial peptides: LcFet-H51 protein solution was irradiated under ultraviolet light for 15, 30, 45 and 60 min.

[0056] Antibacterial activity was determined by the agarose diffusion method, the same as in Example 3.

[0057] Figure 6 Figure A shows the results of the thermostability experiment of LcFet-H51 protein. When the temperature is above 50℃, the antibacterial activity of LcFet-H51 protein against Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus is reduced, but the effect is not significant; at 100℃, the antibacterial activity of LcFet-H51 protein against the three indicator bacteria is 91.74%, 91.94%, and 94.31% of that at 25℃, respectively. Figure 6 Figures B and 6C show the results of pH and UV tolerance experiments on LcFet-H51 protein. The optimal pH for LcFet-H51 protein to function is 7, but its antibacterial activity did not change significantly at acidity levels of 3 and alkalinity levels of 12. UV radiation also had almost no effect on the antibacterial activity of LcFet-H51 protein.

[0058] Example 6: Cytotoxicity of LcFet-H51:

[0059] Fresh large yellow croaker blood was collected and centrifuged at 5000 rpm for 10 min at 4℃. The supernatant was discarded, and the precipitate was resuspended three times with 0.2 M PBS buffer (pH = 7.2). The resuspended blood cells were then centrifuged at 3000 rpm for 5 min at 4℃ to obtain blood cells. The blood cells were diluted to 1% with TBS buffer of the same concentration as the PBS buffer, and the purified LcFet-H51 protein was adjusted to different concentrations. 50 μl of the blood cell suspension and 50 μl of LcFet-H51 protein at different concentrations were incubated in 96-well cell culture plates at 37℃ for 1 h. The positive control was sterile ultrapure water (completely hemolyzed), and the negative control was TBS buffer (non-hemolyzed). After 1 h of incubation, the cells were centrifuged at 4000 rpm for 10 min to remove cell debris. 100 μl of the supernatant was transferred to a new 96-well cell culture plate, and the absorbance at 520 nm was measured. Calculation formula:

[0060] Hemolysis rate (%) = (Experimental group A520 – Negative control A520) / (Positive control A520 – Negative control A520) × 100%

[0061] The results (Table 1) show that LcFet-H51 protein is almost non-toxic to fish blood cells and can be used as an alternative to antibiotics (the LcFet-H51 protein concentration in Table 1 refers to the protein concentration before mixing with the blood cell suspension).

[0062] Table 1. Hemolytic activity of LcFet-H51 on large yellow croaker hemocytes

[0063]

[0064] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A large yellow croaker ferritin heavy chain antibacterial and antiparasitic fragment LcFet-H51, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the antibacterial and antiparasitic fragment LcFet-H51 of the ferritin heavy chain of large yellow croaker as described in claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

3. The application of the LcFet-H51 antibacterial and anti-insect fragment of the large yellow croaker ferritin heavy chain as described in claim 1 or the encoding gene as described in claim 2 in the preparation of antibacterial agents, characterized in that: The antibacterial agent is used to inhibit Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus.

4. The application of the LcFet-H51 antibacterial and insecticidal fragment of the large yellow croaker ferritin heavy chain as described in claim 1 or the encoding gene as described in claim 2 in the preparation of insecticides, characterized in that: The insecticide is used to kill shield ciliates.

5. The application of the LcFet-H51 antibacterial and antiparasitic fragment of the large yellow croaker ferritin heavy chain as described in claim 1 or the encoding gene as described in claim 2 in the preparation of feed additives, characterized in that: The feed additive is used to inhibit Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus and / or kill large yellow croaker ciliates.

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