Antibacterial protein SmH2A.1 as well as preparation method and application thereof
By preparing the antibacterial protein SmH2A.1 from turbot, the problem of insufficient specificity of the existing antibacterial protein is solved, and the broad-spectrum antibacterial effect is achieved. It is applied in aquaculture, food and cosmetics fields, reducing the risk of drug resistance.
Patent Information
- Application Number
- CN202510467403.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-11
AI Technical Summary
Existing antibacterial proteins can usually only target a certain pathogen specifically, and it is difficult to replace antibiotics for large-scale application. The abuse of antibiotics leads to the generation of drug-resistant strains, threatening food safety and public health safety.
A kind of antibacterial protein SmH2A.1 derived from turbot was developed, which acted through multiple mechanisms and had broad-spectrum antibacterial properties. It was cloned on the pET-32a vector through BamHI and SalI restriction sites, and was recombinantly expressed and purified, and prepared into an antibacterial agent for use in aquaculture and other fields.
SmH2A.1 protein has broad-spectrum antibacterial activity, can effectively inhibit a variety of pathogens, reduce the use of antibiotics, and reduce the risk of drug resistance. It is suitable for applications in aquaculture, food and cosmetics and other fields.
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Figure CN120289609A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to an antibacterial protein SmH2A.1 derived from turbot, and a preparation method and application thereof. Background Art
[0002] Aquaculture is one of the fastest-growing food industries in the global economy. However, its rapid development is accompanied by the overuse of antibiotics, especially in low- and middle-income countries in Asia and Africa. The abuse of antibiotics has led to the emergence of drug-resistant strains, causing food safety and water environmental pollution problems, and seriously threatening human health and public health security. The World Health Organization (WHO) pointed out that drug-resistant bacteria caused approximately 1.3 million deaths in 2019, and it is expected that this figure will rise to 10 million by 2050, far exceeding the number of deaths caused by cancer, and resulting in direct economic losses of more than $2.0 trillion per year. WHO has listed antibiotic resistance as one of the top ten public health threats faced by humanity and has specifically identified 15 drug-resistant bacteria that cause serious problems. Therefore, it is urgent to develop green, safe, and effective alternatives to antibiotics.
[0003] Antibacterial proteins / peptides are natural defensive substances produced by organisms to resist the invasion of pathogenic microorganisms. Different from traditional antibiotics, antibacterial proteins / peptides act through multiple mechanisms, such as disrupting bacterial cell membranes, inhibiting cell wall synthesis, and interfering with key intracellular metabolic processes, making it difficult for bacteria to develop drug resistance. In addition, antibacterial proteins / peptides have the advantages of low toxicity, low allergenicity, and thermal stability, and have broad application prospects in the fields of medicine, food preservation, and agriculture.
[0004] However, existing antibacterial proteins usually can only specifically target a certain pathogen and are difficult to replace antibiotics for large-scale application. Therefore, developing new antibacterial proteins with broad-spectrum antibacterial properties has become the key to solving the problem of antibiotic resistance. Summary of the Invention
[0005] To solve the above problems, one of the objectives of the present invention is to provide an antibacterial protein SmH2A.1, which is derived from turbot and has an amino acid sequence as shown in SEQ ID NO.1. As a new antibacterial protein, the turbot antibacterial protein SmH2A.1 not only has broad-spectrum antibacterial properties, but also can effectively kill pathogenic bacteria through multiple mechanisms, and at the same time has low toxicity and high stability, making it suitable for popularization and application in aquaculture.
[0006] Another objective of the present invention is to provide a gene encoding the antibacterial protein SmH2A.1, and its nucleotide sequence is as shown in SEQ ID NO.2.
[0007] A third object of the present invention is to provide a preparation method of the antibacterial protein SmH2A.1, comprising the following steps: cloning the gene encoding SmH2A.1 onto a pET-32a vector through BamHⅠ and SalⅠ restriction enzyme cleavage sites to construct a recombinant expression vector pET-32a-SmH2A.1; transforming the recombinant expression vector pET-32a-SmH2A.1 into BL21(DE3) competent cells, and inducing expression, extracting, and purifying through IPTG to obtain the turbot antibacterial protein SmH2A.1.
[0008] Further, the IPTG induction expression method is as follows:
[0009] Transform the recombinant expression vector pET-32a-SmH2A.1 into BL21(DE3) competent cells, and culture overnight at 37°C and 180 rpm / min in a shaker;
[0010] After overnight culture, pipette 10 mL of the bacterial solution into 1 L of LB liquid medium with Amp resistance, and culture at 37°C and 180 rpm / min in a shaker for 4 h;
[0011] Then, detect the OD value of the bacterial solution every half hour. When the OD value reaches 0.4 - 0.6, add 0.5 mM inducer IPTG;
[0012] Place it in a shaker and culture overnight at 16°C and 180 rpm / min to complete the induction culture.
[0013] Further, the extraction method is as follows:
[0014] Collect the bacterial solution induced by IPTG, centrifuge to discard the supernatant, and collect the bacterial cells;
[0015] Resuspend the bacterial cells with PBS solution and gently pipette to mix evenly; use an ultrasonic disruptor to disrupt the bacterial cells, and the program is set as: power 400 W, ultrasonic for 2 s, stop for 4 s, and ultrasonic for a total of 30 min. The whole process of ultrasonic disruption is carried out on ice; centrifuge to collect the supernatant.
[0016] Further, the purification method is as follows:
[0017] Add the extracted protein supernatant to a chromatography column (Ni-NTA column, hereinafter referred to as nickel column), block the lower outlet, and allow the protein supernatant to fully bind to the nickel column for 30 min, and then repeatedly load the effluent onto the column three times;
[0018] Elute the protein in the nickel column with imidazole solutions of different concentrations, pass through the nickel column in ascending order, and collect the eluted solutions respectively;
[0019] The collected eluted liquid was loaded into a dialysis bag, and imidazole in the eluate was removed by dialysis to obtain the purified antibacterial protein SmH2A.1.
[0020] A fourth object of the present invention is to provide the application of the antibacterial protein SmH2A.1 in the preparation or as an antibacterial agent, and the antibacterial agent uses the antibacterial protein SmH2A.1 as the antibacterial component.
[0021] Furthermore, the antibacterial agent can be specifically applied to fields such as food, aquatic products, cosmetics, etc., such as being used as a cosmetic additive, an aquatic feed additive, a food preservative, etc.
[0022] Furthermore, the antibacterial protein SmH2A.1 can inhibit the activities of Gram-positive bacteria and Gram-negative bacteria. The Gram-negative bacteria include Escherichia coli (E. coli), and the Gram-positive bacteria include Staphylococcus aureus (S. aureus).
[0023] Furthermore, in the presence of Ca 2+ the antibacterial protein SmH2A.1 can cause agglutination reactions in Escherichia coli and Staphylococcus aureus.
[0024] The present invention has the following beneficial effects:
[0025] The present invention first reveals that the SmH2A.1 protein derived from turbot has broad-spectrum antibacterial activity and can effectively inhibit the growth of various pathogenic bacteria. Based on this discovery, the SmH2A.1 protein can be used to prepare a broad-spectrum antibacterial agent, which can be applied to the aquaculture field to protect farmed fish from bacterial infections, thereby reducing the use of antibiotics, reducing the risk of drug resistance, promoting the green and sustainable development of the aquaculture industry, and can also be extended to antibacterial applications in fields such as food and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is the SDS-PAGE diagram for verifying the induced expression of the antibacterial protein SmH2A.1 in Example 1 (wherein, lane M: protein marker; lane 1: protein extract of the expression strain before induction; lanes 2-5: protein extracts of the expression strain after induction with 0.5 mM inducer IPTG; lane 6: protein in the supernatant after induction and ultrasonic disruption; lane 7: protein in the precipitate after induction and ultrasonic disruption);
[0027] Figure 2SDS-PAGE gel for the purification verification of the antibacterial protein SmH2A.1 in Example 1 (where lane M: protein marker; lane 1: protein before column purification in the supernatant; lane 2: protein in the solution after passing through the column in the supernatant; lanes 3 - 8: target protein eluted with imidazole at different concentrations (30, 90, 120, 150, 200, and 250 mM));
[0028] Figure 3 Antibacterial efficiency of the antibacterial protein SmH2A.1 and the tag protein rTRX (control) at different concentrations against Escherichia coli (A) and Staphylococcus aureus (B) in Example 2.
[0029] Figure 4 Agglutination results of the antibacterial protein SmH2A.1 and the tag protein rTRX against Escherichia coli and Staphylococcus aureus in Example 3. Detailed implementation manners
[0030] To better understand the content of the present invention, the technical solutions will be further described below in conjunction with the detailed implementation manners. It should be noted that the present invention is not limited to these implementation manners. Unless otherwise specified, the technologies involved in the present invention are all conventional technologies in the art, and the reagents or materials used are all obtained through commercial channels.
[0031] Example 1: Preparation of the turbot antibacterial protein SmH2A.1
[0032] This example first provides the relevant sequences of the turbot antibacterial protein SmH2A.1, including:
[0033] (1) Amino acid sequence of the turbot antibacterial protein SmH2A.1 (SEQ ID NO.1):
[0034] MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAVRNDEELNKLLGGVTIAQGGVLPNIQAVLLPKKTEKPAKSK
[0035] (2) Nucleotide sequence of the coding gene SmH2A.1 of the turbot antibacterial protein SmH2A.1 (SEQ ID NO.2):
[0036] ATGTCTGGCAGAGGGAAAACCGGAGGCAAAGCCCGGGCCAAGGCCAAGTCTCGCTCCTCTCGCGCCGGACTCCAGTTCCCGGTGGGCCGGGTCCACAGGCTACTGCGCAAGGGCAACTATGCGGAGCGCGTCGGCGCCGGGGCTCCGGTGTACTTGGCCGCCGTGCTGGAGTATCTGACGGCCGAGATCCTGGAGCTGGCAGGCAACGCGGCCCGGGACAACAAGAAGACCCGGATCATCCCGCGGCACCTCCAGCTGGCCGTGCGCAACGACGAGGAGCTCAACAAGCTGCTGGGAGGTGTGACCATCGCTCAGGGCGGCGTGCTGCCCAACATCCAGGCTGTCCTCCTGCCCAAGAAGACCGAGAAGCCGGCCAAGAGCAAGTAG
[0037] This example also provides a preparation method for the turbot antibacterial protein SmH2A.1, including cloning, induced expression, extraction and purification steps, which are specifically as follows:
[0038] (1) Clone the coding gene SmH2A.1 (SEQ ID NO.2) of the turbot antibacterial protein SmH2A.1 into the pET-32a vector through the BamHⅠ / SalⅠ restriction enzyme digestion site to construct the recombinant expression vector pET-32a-SmH2A.1. The specific process is as follows:
[0039] a. Design primers for the SmH2A.1 gene using Primer Premier 5.0 software. The designed and synthesized primers are as follows:
[0040] Forward primer: CTGACTCGTGTGACTAC (SEQ ID NO:3);
[0041] Reverse primer: CAGAGCAGTCTGTGAATA (SEQ ID NO:4).
[0042] b. Perform PCR amplification using the designed primers to obtain the SmH2A.1 gene fragment. The PCR reaction system is 10ul, and the PCR reaction program is: denaturation (95°C, 30 seconds); annealing (56°C, 30 seconds) and extension (72°C, 1 minute), cycling 30 times; then, perform final extension (72°C, 5 minutes); finally, store the PCR product at 4°C.
[0043] c. Purify the PCR amplification product using a DNA purification kit (such as QIAquick Gel Extraction Kit). Digest the purified PCR product and pET-32a with BamHⅠ and SalⅠ restriction enzymes respectively, and ligate them overnight at 16°C using T4 DNA ligase, thereby cloning the SmH2A.1 gene (SEQ ID NO.2) into the pET-32a vector to construct the recombinant expression vector pET-32a-SmH2A.1.
[0044] (2) Transform the recombinant expression vector pET-32a-SmH2A.1 into BL21(DE3) competent cells and culture them overnight in a shaker at 37°C and 180 rpm / min.
[0045] (3) After overnight culture, pipette 10 mL of the bacterial solution into 1 L of LB liquid medium with Amp resistance and culture it in a shaker at 37°C and 180 rpm / min for 4 h.
[0046] (4) Detect the OD value of the bacterial solution every half hour. When the OD value reaches 0.4 - 0.6, add 0.5 mM inducer IPTG.
[0047] (5) Place it in a shaker and culture it overnight at 16°C and 180 rpm / min.
[0048] (6) After overnight culture, pipette 300 μL of the bacterial solution as the sample after protein induction. The remaining bacterial solution is aliquoted into 50 mL centrifuge tubes and centrifuged at 6000 rpm / min at 4°C for 20 min. Discard the supernatant and collect the bacterial cells.
[0049] (7) Resuspend the bacterial cells with 30 mL of PBS solution and gently pipette to mix evenly. Use an ultrasonic cell disruptor to lyse the bacterial cells. The program is set as follows: power 400 W, ultrasound for 2 s, stop for 4 s, and ultrasound for a total of 30 min. The whole process of ultrasonic disruption is carried out on ice.
[0050] (8) Add the filtered protein supernatant to the chromatography column, block the lower outlet, and allow the protein supernatant to fully bind to the nickel column for 30 min. Then, repeat the process of loading the effluent onto the column three times.
[0051] (9) Elute the protein in the nickel column with imidazole solutions of different concentrations. The concentrations are set as: 30 mM, 90 mM, 120 mM, 150 mM, 200 mM, and 250 mM. Use 30 mL of each concentration solution and pass them through the nickel column in ascending order, and collect the eluted solutions respectively.
[0052] (10) Load the collected elution liquid into a dialysis bag. Prepare 1 L of PBS buffer and add it to a 1 L beaker as the dialysis solution. Place the dialysis bag containing the elution liquid into it and stir using a magnetic stirrer. Replace the fresh dialysis solution every 8 h and repeat the operation 5 times to remove imidazole from the elution liquid, obtaining the purified antibacterial protein SmH2A.1.
[0053] Figure 1 SDS-PAGE diagram for verifying the induced expression of antibacterial protein SmH2A.1 (where lane M: protein marker; lane 1: protein extract of the expression strain before induction; lanes 2-5: protein extracts of the expression strain after induction with 0.5 mM inducer IPTG; lane 6: protein in the supernatant after induction and ultrasonic disruption; lane 7: protein in the precipitate after induction and ultrasonic disruption). It can be seen that IPTG successfully induced the expression of SmH2A.1 protein, and the separation effect in the supernatant after ultrasonic disruption is better and the purity is higher. Therefore, this supernatant was selected for further purification by chromatography column.
[0054] Figure 2 SDS-PAGE diagram for verifying the purification of antibacterial protein SmH2A.1 (where lane M: protein marker; lane 1: protein in the supernatant before column purification; lane 2: protein in the solution after the supernatant passes through the column; lanes 3-8: target proteins eluted with imidazole at different concentrations (30, 90, 120, 150, 200, and 250 mM)). It can be seen that imidazole at each concentration except 30 mM successfully eluted the antibacterial protein SmH2A.1 and had a relatively high purity. The protein size is 22 kDa. Among them, the elution volume corresponding to lane 6 at a concentration of 150 mM is the largest and the elution effect is the best. Collect the elution liquid at a concentration of 150 mM, and obtain the antibacterial protein SmH2A.1 after removing imidazole by dialysis for subsequent antibacterial ability detection.
[0055] Example 2: In vitro antibacterial activity detection of turbot antibacterial protein SmH2A.1
[0056] 1. Activation: Spread the cryopreserved Staphylococcus aureus and Escherichia coli on solid media respectively. After overnight culture, pick single colonies into EP tubes containing 1 mL of LB liquid medium and activate them at 37 °C in a shaker for 5 h.
[0057] 2. Culture: After activation, pipette 100 μL of the bacterial solution into an EP tube containing 1 mL of LB liquid medium and culture it at 37 °C in a shaker for 5 h. Use a hemocytometer to count the number of bacteria in the bacterial solution.
[0058] 3. Dilution: Centrifuge the bacterial solution at 6000 rpm / min for 10 min, discard the supernatant, and resuspend the bacterial cells with PBS. Repeat this operation 3 times. Dilute the bacterial cells with PBS to 1×10 5 CFU / mL.
[0059] 4. Incubation: Dilute SmH2A.1 and the tagged protein rTRX to prepare concentration gradients of 100 μL each (0, 25, 50, 75, 100 μg / mL). Add 20 μL of the diluted bacterial solution to each concentration and incubate at room temperature for 1 h.
[0060] 5. Plating: Pipette 20 μL of each of the above recombinant protein - bacterial solution mixtures at each concentration and spread them evenly on the LB solid medium. Set up three parallel replicates for each concentration and place the solid medium in a 37°C constant temperature incubator overnight.
[0061] 6. Counting: Observe the growth status of colonies on the solid medium of the control group (0 μg / mL) and the experimental groups, and count the number of colonies.
[0062] 7. Calculation: Bacteriostatic rate = (control group - experimental group) / control group × 100%.
[0063] Figure 3 The bacteriostatic efficiency of the antibacterial protein SmH2A.1 and the tagged protein rTRX (control) against Escherichia coli (A) and Staphylococcus aureus (B) at different concentrations. According to Figure 3 the detection results, compared with the tagged protein rTRX, the SmH2A.1 protein has good bactericidal activity against both Gram - positive and Gram - negative bacteria, that is, it has good broad - spectrum antibacterial activity and can be used to prepare antibacterial agents.
[0064] Example 3: Examination of the Bacterial Agglutination Ability of the Turbot Antibacterial Protein SmH2A.1
[0065] 1. Dilution: Centrifuge the Staphylococcus aureus and Escherichia coli bacterial solutions at 6000 rpm / min for 10 min, discard the supernatant, and resuspend the bacterial cells with PBS. Repeat this operation 3 times. Dilute the bacterial cells to 1×10 8 CFU / ml with PBS, and at the same time dilute SmH2A.1 and the tagged protein rTRX to 100 μg / mL.
[0066] 2. Incubation: Mix 25 μL of each of the two bacterial solutions with 25 μL of SmH2A.1 and the tagged protein rTRX respectively. At the same time, set up a group with added CaCl2 and a group without added CaCl2, with a CaCl2 concentration of 10 mM, and place them in a 37°C constant temperature incubator for 1 h.
[0067] 3. Observation: Observe the agglutination of the two bacteria under an optical microscope.
[0068] Figure 4 The agglutination results of the antibacterial protein SmH2A.1 and the tagged protein rTRX against Escherichia coli (A) and Staphylococcus aureus (B). According to Figure 4In the detection results, compared with the tagged protein rTRX, the SmH2A.1 protein showed significant bacteriagglutination activity against Escherichia coli and Staphylococcus aureus, and this activity was typically Ca 2+ -dependent, which could effectively promote bacterial agglutination and thus better inhibit the growth of bacteria.
[0069] This specific implementation manner is only an interpretation of the present invention and not a limitation thereof. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.
Claims
1. An antibacterial protein SmH2A.1, characterized in that, It is derived from turbot, and its amino acid sequence is shown in SEQ ID NO.
1.
2. A gene encoding the antibacterial protein SmH2A.1 according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.
2.
3. The preparation method of the antibacterial protein SmH2A.1 according to claim 1, characterized in that, It includes the following steps: Clone the gene described in Claim 2 onto the pET-32a vector through the BamHⅠ and SalⅠ restriction enzyme cleavage sites to construct the recombinant expression vector pET-32a-SmH2A.1; transform the recombinant expression vector pET-32a-SmH2A.1 into BL21(DE3) competent cells, induce expression by IPTG, extract and purify to obtain the antibacterial protein SmH2A.
1.
4. The preparation method according to claim 3, characterized in that, The method for IPTG-induced expression is as follows: Transform the recombinant expression vector pET-32a-SmH2A.1 into BL21(DE3) competent cells, and culture overnight at 37°C and 180 rpm / min in a shaker; After overnight culture, pipette 10 mL of the bacterial solution into 1 L of LB liquid medium with Amp resistance, and culture at 37°C and 180 rpm / min in a shaker for 4 h; Then, detect the OD value of the bacterial solution every half hour. When the OD value reaches 0.4 - 0.6, add 0.5 mM inducer IPTG; Put it into a shaker and culture overnight at 16°C and 180 rpm / min to complete the induction culture.
5. The preparation method according to claim 4, characterized in that, The extraction method is as follows: Collect the bacterial solution induced by IPTG, centrifuge to discard the supernatant, and collect the bacterial cells; Resuspend the bacterial cells with PBS solution and gently pipette to mix evenly; use an ultrasonic cell disruptor to disrupt the bacterial cells, and the program is set as: power 400 W, ultrasound for 2 s, stop for 4 s, and ultrasound for a total of 30 min. The whole process of ultrasonic disruption is carried out on ice; centrifuge to collect the supernatant.
6. The preparation method according to claim 5, characterized in that, The purification method is as follows: Add the extracted protein supernatant to the chromatography column, block the lower outlet, and allow the protein supernatant to fully bind to the nickel column for 30 min, and then load the effluent onto the column three times repeatedly; Elute the protein in the nickel column with imidazole solutions of different concentrations, pass through the nickel column in ascending order, and collect the eluted solutions respectively; Load the collected eluted liquid into a dialysis bag, and remove the imidazole in the eluate by dialysis to obtain the purified antibacterial protein SmH2A.
1.
7. Use of the antibacterial protein SmH2A.1 according to claim 1 in the preparation or as an antibacterial agent, characterized in that, The antibacterial agent uses the antibacterial protein SmH2A.1 as the antibacterial component.
8. The application according to claim 7, characterized in that, It can be applied to the fields of food, aquatic products, and cosmetics.
9. The application according to claim 7, wherein The antibacterial protein SmH2A.1 can inhibit the activities of Gram-positive bacteria and Gram-negative bacteria. The Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.
10. The application according to claim 9, wherein In the presence of Ca 2+ The antibacterial protein SmH2A.1 can cause agglutination reactions in Escherichia coli and Staphylococcus aureus.