High-temperature-resistant antibacterial protein Cystatin B as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses a high-temperature-resistant antibacterial protein Cystatin B sourced from marine fish as well as a preparation method and application of the high-temperature-resistant antibacterial protein Cystatin B. The high-temperature-resistant antibacterial protein Cystatin B is derived from pomfret, and the amino acid sequence of the high-temperature-resistant antibacterial protein Cystatin B is as shown in SEQ ID NO. 1; the nucleotide sequence of the gene for coding the high-temperature-resistant antibacterial protein Cystatin B is as shown in SEQ ID NO.2, and the sequences of primers adopted for cloning the gene are as shown in SEQ ID NO.3-4. The high-temperature-resistant antibacterial protein Cystatin B has a remarkable antibacterial effect on gram-negative bacteria such as escherichia coli, vibrio parahaemolyticus and edwardsiella tarda as well as gram-positive bacteria such as staphylococcus aureus; and after being treated at the temperature of 100 DEG C, the antibacterial agent still can keep relatively high antibacterial activity. The antibacterial protein is simple in preparation method, can replace traditional antibiotics, and can quickly and efficiently prevent and treat various common bacterial diseases in aquaculture.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a heat-resistant antibacterial protein Cystatin B derived from marine fish (silver pomfret), and a preparation method and application thereof. Background Art

[0002] In recent years, the development of China's marine aquaculture industry has been rapid, and the output of marine fish farming reached 1.9256 million tons in 2022. However, problems such as slow growth and frequent diseases have severely restricted the development of the marine fish farming industry. Currently, antibiotics are usually used to improve these problems, but the overuse of antibiotics has led to the generation of drug-resistant strains, causing food safety and water environmental pollution problems, and seriously threatening human health and public health safety. Therefore, it is extremely 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 destroying 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] Cysteine protease inhibitors are a class of naturally occurring regulatory proteins that specifically inhibit the activities of cysteine proteases such as papain, cathepsin B, H, and L, and participate in key physiological processes such as cellular protein metabolism, immune response, inflammation regulation, and apoptosis. Cystatin B is a typical member of the cysteine protease superfamily type I, with a molecular weight of about 11 kDa, characterized by the absence of disulfide bonds and glycosylation modifications. It is a protease inhibitor widely present in organisms, mainly participating in various physiological processes such as protein degradation, immune response, and inflammation regulation by regulating the activity of cysteine protease. It is mainly distributed in the cytoplasm and nucleus, and regulates the stability of lysosomes and the protein degradation pathway by binding to lysosomal proteases. There is currently no report on establishing a connection between cystatin B and antibacterial effects or developing cystatin B that can be used as an antibacterial protein. Summary of the Invention

[0005] One object of the present invention is to provide a heat-resistant antibacterial protein Cystatin B, which is derived from silver pomfret, and its amino acid sequence is as shown in SEQ ID NO.1. The heat-resistant antibacterial protein Cystatin B has heat resistance and antibacterial properties, can replace traditional antibiotics, and can quickly and efficiently prevent and treat various common bacterial diseases in aquaculture.

[0006] Furthermore, the high-temperature resistant antibacterial protein Cystatin B still maintains more than 90% antibacterial activity after being treated at 100°C for 5 minutes; after heating for 45 minutes, the antibacterial efficiency drops to about 60% - 65%.

[0007] The second object of the present invention is to provide a gene encoding the high-temperature resistant antibacterial protein Cystatin B, and its amino acid sequence is shown as SEQ ID NO.2.

[0008] The third object of the present invention is to provide a preparation method of the high-temperature resistant antibacterial protein Cystatin B, including the following steps: cloning the Cystatin B gene (SEQ ID NO.2) onto the pET-28a vector through EcoRⅠ and HindⅢ restriction enzyme sites to construct a recombinant expression vector pET-28a-Cystatin B; transforming the recombinant expression vector into DH5α competent cells, inducing expression by IPTG, extracting, and purifying to obtain the high-temperature resistant antibacterial protein Cystatin B.

[0009] Furthermore, the primer sequences used for cloning the gene encoding the Cystatin B protein are shown as SEQ ID NO.3 - 4.

[0010] Furthermore, the IPTG induction expression method is as follows: DH5α competent cells carrying the pET-28a-Cystatin B plasmid are cultured in LB medium containing 50 μg / mL kanamycin at 37°C until the OD600 reaches 0.4 - 0.6; 0.5 mM IPTG is added to induce protein expression, and the culture is incubated in a shaker at 26°C at 180 rpm / min for 12 hours.

[0011] Furthermore, the extraction method is as follows: collecting the bacterial liquid induced by IPTG, centrifuging at 12,000×g for 5 minutes at 4°C, collecting the precipitate, and then resuspending it with lysis buffer; subjecting the resuspended solution to ultrasonic fragmentation on ice and centrifuging at 12,000×g for 30 minutes at 4°C to clarify the lysate to collect the soluble supernatant.

[0012] Furthermore, the purification method is as follows: using Ni-NTA affinity chromatography for protein purification, the supernatant is applied to a pre-equilibrated Ni-NTA column and washed with washing buffer to remove non-specifically bound proteins; the target protein is eluted with elution buffer, the elution fraction containing Cystatin B is collected, and dialyzed overnight at 4°C with PBS to remove imidazole to obtain the purified Cystatin B protein.

[0013] A fourth object of the present invention is to provide the application of the heat-resistant antibacterial protein Cystatin B in the field of preparing feeds for preventing and treating aquatic bacterial diseases. The application method is to add Cystatin B as an antibacterial component to the feed.

[0014] Furthermore, the bacteria include Gram-positive bacteria and Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Escherichia coli, Vibrio parahaemolyticus, and Edwardsiella tarda.

[0015] The present invention has the following beneficial effects:

[0016] The present invention first reveals that the Cystatin B protein derived from marine fish has broad-spectrum antibacterial activity, can effectively inhibit the growth of a variety of pathogenic bacteria, and can be used to prepare broad-spectrum antibacterial agents. Applying it to the field of aquaculture can effectively protect farmed fish from bacterial infections, reduce the use of antibiotics, reduce the risk of drug resistance in aquatic animals, effectively avoid the risks of food safety and environmental pollution caused by the abuse of antibiotics, and promote the green and sustainable development of the aquaculture industry. Moreover, the Cystatin B protein has excellent heat-resistant activity, can still maintain high antibacterial activity after high-temperature treatment, has good stability, and has a wide range of applicable scenarios and scopes. Description of the Drawings

[0017] Figure 1 It is the SDS-PSGE diagram of the verification expression of the heat-resistant antibacterial protein Cystatin B in Example 1. Lane M: Molecular weight marker. Lane 1: Uninduced pET-28a. Lane 2: pET-28a induced with 0.5 mM IPTG. Lane 3: Uninduced Cystatin B. Lane 4: Supernatant of Cystatin B induced with 0.5 mM IPTG. Lane 5: Purified Cystatin B (sample eluted with elution buffer containing 250 mM imidazole).

[0018] Figure 2 It is the antibacterial effect test of the antibacterial protein Cystatin B on Escherichia coli (1), Vibrio parahaemolyticus (2), Edwardsiella tarda (3), and Staphylococcus aureus (4) in Example 2. a and b are negative controls of PBS and 0.5 mM-pET-28a, and c is the antibacterial group added with Cystatin B.

[0019] Figure 3 It is the effect of incubating the heat-resistant protein Cystatin B at 55 °C, 65 °C, 75 °C, 85 °C, and 100 °C for 5 minutes on the activities of Cystatin B against papain and cathepsin K. (A) is the result of papain, and (B) is the result of cathepsin K

[0020] Figure 4 To show the effect of the heat-resistant protein Cystatin B in Example 3 on the antibacterial activity of Cystatin B at different heating times at 100°C. The "0 min" group represents untreated Cystatin B as the negative control group. The significance between the control group and the experimental group is indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Detailed implementation manners

[0021] 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.

[0022] Example 1: Preparation of the heat-resistant antibacterial protein Cystatin B

[0023] The heat-resistant antibacterial protein Cystatin B provided in this example is derived from the heat-resistant antibacterial protein PaCystatin B of silver pomfret. The following provides the relevant sequence information of this protein:

[0024] (1) Amino acid sequence of the heat-resistant antibacterial protein PaCystatin B of silver pomfret (SEQ ID NO.1):

[0025] MISGAPGPEEDASEEIQDMCESVKHYAEQRAGKSYNVFIARCFRTQIVCG TNYFIKVHVGGADHVHIRVHKKLPCHGEDLELTDMQESKSHDDPIEYF

[0026] (2) Nucleotide sequence of the coding gene PaCystatin B of the heat-resistant antibacterial protein PaCystatin B of silver pomfret (SEQ ID NO.2):

[0027] ATGATAAGCGGAGCACCTGGACCTGAAGAAGATGCTAGCGAAGAAATTCAGGATATGTGTGAAAGTGTGAAGCATTATGCGGAGCAAAGAGCAGGAAAATCCTATAATGTCTTCATTGCCAGGTGTTTCAGGACACAGATTGTATGTGGGACCAACTACTTCATAAAGGTCCACGTGGGAGGCGCCGATCATGTTCACATCCGTGTTCACAAAAAACTCCCATGTCATGGAGAAGACCTGGAGCTGACTGATATGCAGGAATCCAAGTCTCATGATGACCCTATTGAATACTTTTAG

[0028] This embodiment also provides a preparation method of the above-mentioned high-temperature resistant antibacterial protein Cystatin B, including cloning, induced expression, extraction and purification steps, which are specifically as follows:

[0029] (1) Cloning and identification of Cystatin B gene

[0030] Primers were designed for the Cystatin B gene by Primer Premier 5.0 software, and the designed and synthesized primers are shown as follows:

[0031] F: 5’-ccggaattcATGATAAGCGGAGCACCTGGACCTG (SEQ ID NO:3, the lowercase letters are EcoRⅠ restriction enzyme site and protection base);

[0032] R: cccaagcttCTAAAAGTATTCAATAGGGTCATCATGAGACTT (SEQ ID NO:4, the lowercase letters are HindⅢ restriction enzyme site and protection base).

[0033] PCR amplification was carried out using the designed primers to obtain the Cystatin B gene fragment. The PCR reaction system was: 2×TaqMaster Mix 5μL, 0.2μL of each upstream and downstream primer, 1μL of cDNA, 3.6μL of ddH2O. The PCR reaction program was 92℃ for 5 min, (92℃ for 1 min, 60℃ for 30 s)×35 cycles, 72℃ for 1 min, and 72℃ for 5 min.

[0034] Purify the PCR amplification product using a DNA purification kit (such as QIAquick Gel Extraction Kit). Digest the purified PCR product and pET-28a with EcoRⅠ and HindⅢ restriction enzymes respectively, and ligate them overnight at 16°C using T4 DNA ligase, thereby cloning the Cystatin B gene (SEQ ID NO.2) into the pET-28a vector to construct the recombinant expression vector pET-28a-Cystatin B.

[0035] The recombinant plasmid (pET-28a-cystatin B) was transformed into competent DH5α Escherichia coli cells, and colonies were selected on an LB agar plate containing 50 μg / mL kanamycin. The colonies were subjected to PCR amplification, and the PCR products were electrophoresed on an agarose gel. The colonies with electrophoretic band sizes consistent with the target gene fragment size were sent for DNA sequencing identification. After the sequence was verified to be correct by sequencing, they were determined to be positive clones.

[0036] (2) IPTG-induced expression

[0037] The competent DH5α cells carrying the pET-28a-Cystatin B plasmid (i.e., the selected positive clones) were cultured in LB medium containing 50 μg / mL kanamycin at 37°C until the optical density at 600 nm (OD600) reached 0.4–0.6; 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the culture was incubated in a shaker at 26°C at 180 rpm / min for 12 hours.

[0038] (3) Protein extraction

[0039] Harvest the cells by centrifugation at 12,000×g for 5 minutes at 4°C, and then resuspend them in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0). Lyse Escherichia coli by sonication on ice, and clarify the lysate by centrifugation at 12,000×g for 30 minutes at 4°C to collect the soluble supernatant.

[0040] (4) Protein purification

[0041] Protein purification was carried out using Ni-NTA affinity chromatography. The supernatant was applied to a pre-equilibrated Ni-NTA column and washed with a washing buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 8.0) to remove non-specifically bound proteins. The target protein was eluted with an elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0). The eluted fraction containing Cystatin B was collected and dialyzed overnight at 4 °C against PBS (pH 7.4) to remove imidazole, yielding purified Cystatin B protein.

[0042] Figure 1 An SDS-PAGE gel (M: molecular weight marker. Lane 1: uninduced pET-28a; Lane 2: pET-28a induced with 0.5 mM IPTG; Lane 3: uninduced Cystatin B; Lane 4: supernatant of Cystatin B induced with 0.5 mM IPTG; Lane 5: purified Cystatin B) was used to verify the purification of Cystatin B protein. It can be seen that Cystatin B protein was successfully separated and purified by column chromatography. The eluted and dialyzed Cystatin B protein sample corresponding to Lane 5 was collected for subsequent antibacterial and high-temperature resistance performance tests.

[0043] Example 2: In vitro antibacterial activity detection of the high-temperature resistant and antibacterial protein Cystatin B

[0044] The antibacterial activity was evaluated against Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-negative bacteria (Escherichia coli E. coli, Vibrio parahaemolyticus V. parahaemolyticus, and Edwardsiella tarda E. tarda) by the Oxford cup method. The specific steps are as follows:

[0045] 1. Bacterial activation: The cryopreserved S. aureus, E. coli, V. parahaemolyticus, and E. tarda were separately plated on solid media. After overnight incubation, single colonies were picked and transferred to EP tubes containing 1 mL of LB liquid medium, and activated at 37 °C for 5 h in a shaker.

[0046] 2. Cultivation: After activation, 100 μL of the bacterial solution was pipetted into an EP tube containing 1 mL of LB liquid medium and cultured at 37 °C for 5 h in a shaker. The number of bacteria in the bacterial solution was counted using a hemocytometer.

[0047] 3. Dilution: The bacterial solution was centrifuged at 6000 rpm / min for 10 min, the supernatant was aspirated, and the bacterial cells were resuspended in PBS. This operation was repeated 3 times. The bacterial cells were diluted to 1×10^5 CFU / mL with PBS.

[0048] 4. Cultivation: First, use a disposable plastic spreader to evenly spread 200 μL of the bacterial solution on the surface of the culture medium. Then, use sterile forceps to place 3 Oxford cups on the plate. Slowly add 200 μL of Cystatin B, PBS (negative control), and empty pET-28a vector (0.5 mM) into the Oxford cups using a pipette. Incubate at 37 °C (Vibrio parahaemolyticus at 28 °C) for 12 - 16 hours.

[0049] 5. Observation: Observe the colony growth in the control group and the antibacterial group.

[0050] The results are as Figure 2 shown. In the antibacterial effect tests of antibacterial protein Cystatin B against Escherichia coli (1), Vibrio parahaemolyticus (2), Edwardsiella tarda (3), and Staphylococcus aureus (4), obvious inhibition zones were produced. Compared with the PBS and empty plasmid controls, Cystatin B showed a significant inhibitory effect on these four bacteria.

[0051] Example 3: Detection of the thermal stability of the heat-resistant antibacterial protein Cystatin B

[0052] The thermal stability of Cystatin B was evaluated by measuring the enzyme inhibition activity and antibacterial efficiency of Cystatin B protein at different high temperatures, compared with the untreated control group. Specifically, the Cystatin B protein was incubated at 55 °C, 65 °C, 75 °C, 85 °C, and 100 °C for 5 minutes, and then the inhibition activities against papain and cathepsin K were determined using the azocasein hydrolysis method. The specific steps are as follows:

[0053] 1. Heat treatment of cystatin B: Incubate the cystatin B protein at 55 °C, 65 °C, 75 °C, 85 °C, and 100 °C for 5 minutes.

[0054] 2. Preparation of the protease solution to be tested: Take a 1.5 ml centrifuge tube, mix 2 μL of papain solution (1 mg / mL, Meryer, China) or cathepsin K solution (1 mg / mL, Meryer, China) with the heat-treated cystatin B. The final enzyme / inhibitor ratio is 512. Make up the reaction system to 20 μL with buffer, and incubate the reaction mixture at 25 °C for 15 minutes.

[0055] 3. Enzymatic hydrolysis reaction: Add 120 μL of 0.5% azocasein solution (Meryer, China), and further incubate the mixture at 37 °C for 30 minutes.

[0056] 4. Termination of the reaction: Terminate the reaction by adding 150 μL of 10% trichloroacetic acid, then centrifuge and collect the supernatant.

[0057] 5. Absorbance measurement: Measure the absorbance at 440 nm using a UV spectrophotometer. Calculate the relative inhibitory activity of rPaCystatin B against each enzyme using the following formula: Relative activity (%) = 100% × (1 - A440 of cystatin B / A440 of the control group).

[0058] The results are as Figure 3 shown, and it can be seen that the enzyme inhibitory activities of Cystatin B protein against papain (A) and cathepsin K (B) are not affected by temperature.

[0059] To further evaluate the stability of Cystatin B protein at 100 °C, incubate the Cystatin B protein at 100 °C for 5, 15, 25, 35, and 45 minutes. After incubation, evaluate its antibacterial activities against Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, and Edwardsiella tarda using the OD600 method. In addition, each experiment is repeated three times, and one-way ANOVA is performed using GraphPad Prism 9.0 software. The specific steps are as follows:

[0060] 1. Heat treatment of cystatin B: Incubate the cystatin B protein at 100 °C for 0 (control), 5, 15, 25, 35, and 45 minutes.

[0061] 2. Bacterial activation: Spread the cryopreserved Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, and Edwardsiella tarda on solid media respectively. After overnight incubation, 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.

[0062] 3. Culturing the bacterial solution: Pick single colonies and inoculate them into LB liquid medium, and culture them at 37 °C with shaking (200 rpm) until the logarithmic growth phase (OD600 ≈ 0.5 - 0.6). Dilute the bacterial solution to OD600 = 0.1.

[0063] 4. Antibacterial experiment: Add 90 μL of bacterial suspension (OD600 = 0.1) and 10 μL of the treated cystatin B solution to each well of a 96-well plate, and set up a negative control (add only 10 μL of medium and 90 μL of bacterial solution) and a positive control (add only 10 μL of antibiotic and 90 μL of bacterial solution), and incubate them statically at 37 °C for 12–24 h. Monitor OD600 (bacterial growth) every 2–3 h.

[0064] 5. Calculation of the inhibition rate: The formula for calculating the antibacterial efficiency: Inhibition rate (%) = [1 - (OD value of the treatment group - initial OD value) / (OD value of the control group - initial OD value)] × 100%.

[0065] The results are asFigure 4 As shown, it can be seen that the Cystatin B protein still retains more than 90% of its antibacterial activity after being treated at 100°C for 5 minutes; after heating for 45 minutes, the antibacterial efficiency drops to about 60% - 65%.

[0066] This specific embodiment 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. A heat-resistant antibacterial protein Cystatin B, characterized in that, It is derived from silver pomfret, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The thermotolerant antibacterial protein Cystatin B according to claim 1, characterized in that, When treated at 100 °C for 5 minutes, Cystatin B still retains more than 90% of its antibacterial activity; after heating for 45 minutes, the antibacterial efficiency drops to 60% - 65%.

3. Encoding the gene of the heat-resistant antibacterial protein Cystatin B described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

4. The preparation method of the high-temperature resistant antibacterial protein Cystatin B according to claim 1, characterized in that, It includes the following steps: Clone the gene described in claim 3 onto the pET-28a vector through the EcoRⅠ and HindⅢ restriction enzyme sites to construct the recombinant expression vector pET-28a-Cystatin B; transform the recombinant expression vector into DH5α competent cells, induce expression, extract, and purify by IPTG to obtain the high-temperature resistant antibacterial protein Cystatin B.

5. The preparation method according to claim 4, characterized in that The primer sequences used for cloning the gene encoding the Cystatin B protein are shown in SEQ ID NO.3 - 4.

6. The preparation method according to claim 4, characterized in that, The method for IPTG-induced expression is as follows: DH5α competent cells carrying the pET-28a-Cystatin B plasmid are cultured in LB medium containing 50 μg / mL kanamycin at 37 °C until OD600 reaches 0.4 - 0.6; add 0.5 mM IPTG to induce protein expression, and incubate the culture in a shaker at 26 °C at 180 rpm / min for 12 hours.

7. The preparation method according to claim 6, characterized in that, The extraction method is as follows: Collect the bacterial liquid induced by IPTG, centrifuge at 12,000×g at 4 °C for 5 minutes, collect the precipitate, and then resuspend it with lysis buffer; ultrasonically disrupt the resuspended solution on ice and centrifuge the lysate at 12,000×g at 4 °C for 30 minutes to clarify it, in order to collect the soluble supernatant.

8. The preparation method according to claim 7, characterized in that, The purification method is as follows: Use Ni-NTA affinity chromatography for protein purification. The supernatant is applied to a pre-equilibrated Ni-NTA column and washed with washing buffer to remove non-specifically bound proteins; the target protein is eluted with elution buffer, and the elution fraction containing Cystatin B is collected and dialyzed overnight at 4 °C with PBS to remove imidazole, obtaining the purified Cystatin B protein.

9. Use of the high-temperature resistant antibacterial protein Cystatin B described in claim 1 or 2 in the field of preparing feeds for preventing and treating aquatic bacterial diseases.

10. The application according to claim 9, characterized in that, The bacteria include Gram-positive bacteria and Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Escherichia coli, Vibrio parahaemolyticus, and Edwardsiella tarda.

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