Mitoxin polymer as well as preparation method and application thereof

Abstract

The invention provides a melittin polymer, which is formed by at least connecting disulfide bonds between two melittin variant molecules after a melittin variant is formed by mutating alanine at the fourth site of an amino acid sequence of a melittin monomer into cysteine and mutating isoleucine at the twentieth site into cysteine in the amino acid sequence of the melittin monomer. The preparation method of the melittin polymer comprises the following steps: obtaining a melittin variant through escherichia coli recombinant expression, and purifying and oxidizing the obtained melittin variant through a nickel column to obtain the melittin polymer, and carrying out gel filtration chromatography on the obtained melittin polymer to obtain the purified melittin polymer. The invention also provides an application of the melittin polymer, and the melittin polymer is used for preparing antibacterial drugs or antibacterial feed additives. The prepared melittin polymer can effectively reduce the toxicity to red blood cells, has relatively strong environmental responsiveness and stability, and can effectively prolong the antibacterial aging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bee venom polymer, its preparation method, and its application. Background Technology

[0002] Melittin is the main active peptide in bee venom, possessing broad-spectrum antibacterial activity and exerting its effects by disrupting bacterial cell membranes. However, it exhibits strong toxicity to the membranes of normal cells (such as erythrocytes and epithelial cells) and is easily degraded by gastrointestinal proteases, greatly limiting its application.

[0003] In existing technologies, attempts to reduce bee venom toxicity through chemical modification or sequence alteration often result in reduced antibacterial activity or excessively high preparation costs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a bee venom polymer, its preparation method and application, which can effectively reduce erythrocyte toxicity, has strong environmental responsiveness and stability, and can effectively prolong the antibacterial effect.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a bee venom polymer, wherein the bee venom polymer is formed by at least two bee venom variants linked by intermolecular disulfide bonds; The bee venom variant is formed by mutating alanine at position 4 of the bee venom monomer amino acid sequence to cysteine ​​and isoleucine at position 20 to cysteine. The amino acid sequence of the bee venom monomer is shown in SEQ ID No. 1; the amino acid sequence of the bee venom variant is shown in SEQ ID No. 2.

[0006] Preferably, the intermolecular disulfide bond is formed by the oxidative coupling of thiol groups of cysteine ​​in the bee venom variant.

[0007] Preferably, the bee venom polymer is a bee venom dimer, bee venom trimer, or a higher-order bee venom polymer.

[0008] The method for preparing the bee venom polymer in this invention is as follows: S1. Bee venom variants were obtained through recombinant expression in Escherichia coli; S2. The bee venom variant obtained in S1 is purified by nickel column chromatography and then oxidized to obtain bee venom polymers. S3. The bee venom polymer obtained in S2 is subjected to gel filtration chromatography to obtain purified bee venom polymer.

[0009] Preferably, the oxidation conditions in S2 are: pH value of 7.0 to 8.0, temperature of 20°C to 37°C, and time of 4 hours.

[0010] Preferably, the purity of the bee venom variant purified by the nickel column in S2 is >98%; and the purity of the purified bee venom polymer in S3 is >95%.

[0011] The present invention also provides the application of the bee venom polymer prepared by the above preparation method, wherein the bee venom polymer is used to prepare antibacterial drugs or antibacterial feed additives.

[0012] Preferably, the bee venom polymer is used to inhibit the activity of Staphylococcus aureus and / or Escherichia coli.

[0013] Compared with the prior art, the present invention has the following advantages: 1. The bee venom polymer of the present invention reduces cell membrane damage through steric hindrance, and in vitro experiments show that its hemolysis rate on erythrocytes is reduced by 77.9% compared with that of bee venom monomer.

[0014] 2. The bee venom polymer of the present invention has strong environmental responsiveness. It releases bee venom variants in a high-reducing environment of bacteria (e.g., on the surface of bacterial cell membranes) and retains antibacterial activity against Gram-positive bacteria (e.g., Staphylococcus aureus) and Gram-negative bacteria (e.g., Escherichia coli). The minimum inhibitory concentration (MIC) of the bee venom variant is comparable to that of the bee venom monomer.

[0015] 3. The multimer structure of bee venom in this invention reduces the degradation of bee venom by gastric and intestinal proteases and enhances its stability in the gastrointestinal tract; it controls the release rate and prolongs the antibacterial effect, extending the 5-minute duration of bee venom monomer to more than 30 minutes.

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is an α-helical structure diagram of the bee venom variant and bee venom monomer in Embodiment 1 of the present invention.

[0018] Figure 2 This is a predicted spatial structure diagram of the bee venom polymer in Embodiment 1 of the present invention.

[0019] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of bee venom polymers in Example 1 of the present invention.

[0020] Figure 4 This is an SDS-PAGE gel electrophoresis image of bee venom polymers, bee venom variants, and bee venom monomers in Example 1 of the present invention.

[0021] Figure 5This is a graph showing the hemolysis rate of bee venom polymers and bee venom monomers in Example 3 of the present invention. Detailed Implementation

[0022] Example 1 The bee venom polymer of this embodiment is formed by at least two bee venom variants linked by intermolecular disulfide bonds. These bee venom variants are obtained by mutating the 4th alanine to cysteine ​​and the 20th isoleucine to cysteine ​​in the amino acid sequence of the bee venom monomer (natural bee venom). The amino acid sequence of the bee venom monomer is shown in SEQ ID No. 1; the amino acid sequence of the bee venom variant is shown in SEQ ID No. 2; and so on. Figure 1 As shown, bee venom variants and bee venom monomers have similar α-helical structures; the spatial structure of bee venom polymers is predicted, such as... Figure 2 As shown, the intermolecular disulfide bonds are formed by the oxidative coupling of cysteine ​​thiol groups.

[0023] The preparation method of the bee venom polymer in this embodiment is as follows: S1. The base sequence of the bee venom variant is synthesized and constructed into the pET-28a vector to obtain a recombinant plasmid. The recombinant plasmid is transformed into Escherichia coli BL121 and expressed by isopropyl-β-D-thiogalactoside (IPTG) to obtain the bee venom variant. S2. The bee venom variant obtained in S1 was purified by nickel column chromatography to a purity of 98.14%, and dissolved in a 10 mM phosphate buffer solution with a pH of 7.4. After standing at 37°C for 4 hours, bee venom polymers were obtained. The formation of bee venom polymers was detected by high-performance liquid chromatography (HPLC), and the results are as follows. Figure 3 As shown, the chromatogram contains multiple main chromatographic peaks that are regularly distributed, corresponding to polymers with different degrees of polymerization; S3. The bee venom polymer obtained in S2 was subjected to gel filtration chromatography to obtain a bee venom polymer with a purity of 96.52%. The molecular weight of the bee venom polymer was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows. Figure 4 As shown, the molecular weight of the bee venom polymer is twice or more than that of the bee venom variant and the bee venom monomer.

[0024] Experimental parameters in this embodiment: In step S2, the purity of the bee venom variant purified by nickel column should be >98%. In step S2, the pH value can also be 7.0 to 8.0; In step S2, the temperature can also be 20℃~37℃; In step S3, the purity of the bee venom polymer should be >95%; At least two bee venom variants can be linked by intermolecular disulfide bonds to form bee venom dimers, bee venom trimers, or higher-order bee venom polymers.

[0025] Example 2 This example is an antibacterial test of the bee venom polymer prepared in Example 1.

[0026] (a) Antibacterial activity test of bee venom polymers: 1. Experimental materials: Gram-positive bacteria: Staphylococcus aureus (ATCC 25923); Gram-negative bacteria: Escherichia coli (ATCC 25922).

[0027] Bee venom samples: Bee venom monomer (Shanghai Aladdin Biochemical Technology Co., Ltd.) natural bee venom with a purity ≥98%, prepared as a stock solution with a mass concentration of 1.0 mg / mL using sterile ultrapure water; Bee venom polymer prepared in Example 1 (mass concentration of 1.0 mg / mL); Reduced bee venom monomer (mass concentration of 1.0 mg / mL): 1.0 mg of the bee venom polymer prepared in Example 1 was added to 1 mL of reduction reaction buffer (100 mmol / L of tris(hydroxymethyl)aminomethane hydrochloride, pH 8.5, containing 5 mmol / L of ethylenediaminetetraacetic acid), and incubated at 37°C for 30 min to obtain a reduced bee venom monomer with a mass concentration of 1.0 mg / mL.

[0028] 2. Experimental methods: The minimum inhibitory concentration (MIC) was determined using the microbroth dilution method. Different bee venom samples were diluted to 10 concentration gradients: 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 100 μg / mL, and 200 μg / mL. These gradients were then mixed with bacterial suspension (1×10⁻⁶). 6 The CFU / mL concentrations were mixed at a 1:1 volume ratio and incubated at 37°C for 24 hours. The bacterial survival rate at each concentration was then determined, and each experiment was repeated three times.

[0029] 3. Experimental Results: The results are shown in Table 1. The MICs of the melitoxin polymer against Staphylococcus aureus and Escherichia coli were 3.82±0.32 μg / mL and 14.55±1.65 μg / mL, respectively, which were slightly higher than those of the melitoxin monomer. The MICs of the reduced melitoxin monomer against Staphylococcus aureus and Escherichia coli were comparable to those of the melitoxin monomer.

[0030] Table 1. Detection of Minimum Inhibitory Concentration of Bee Venom Polymer (II) Antibacterial activity test of bee venom polymers in simulated acidic and alkaline environments: 1. Experimental materials: Bee venom polymers and bee venom monomers: Same as Experiment (I); Pepsin solution: Take 2.0g sodium chloride and 3.2g pepsin (Shanghai Aladdin Biochemical Technology Co., Ltd.) with a purity ≥98% and an activity ≥2500U / mg, add 800mL of ultrapure water to dissolve, adjust the pH to 2.0 with concentrated hydrochloric acid to simulate the acidic environment in the stomach, and make up to 1000mL; Intestinal protease solution: Take 6.8g potassium dihydrogen phosphate and 10.0g trypsin (Shanghai Aladdin Biochemical Technology Co., Ltd.) with a purity ≥98% and an activity ≥250U / mg, add 800mL of ultrapure water to dissolve, adjust the pH to 7.4 with a 0.1mol / L sodium hydroxide aqueous solution, and make up to 1000mL.

[0031] 2. Experimental methods: Take 1 mL of melitoxin polymer stock solution (1.0 mg / mL) and 1 mL of melitoxin monomer stock solution (1.0 mg / mL), then add 9 mL of pepsin solution and intestinal protease solution to each, and mix gently. Incubate at 37°C with constant temperature shaking, and take samples to terminate the reaction at 5 min, 30 min, and 60 min. The residual antibacterial rate of the short peptide samples after treatment with gastric and intestinal proteases is detected by the micro-broth dilution method. Each experiment is repeated 3 times.

[0032] 3. Experimental Results: The results are shown in Table 2. After 5 min of pepsin treatment, the residual antibacterial rate of the meliofemoral venom polymer was 6.38 times higher than that of the meliofemoral venom monomer; after 30 min of treatment, it still retained an antibacterial rate of 31.6 ± 2.9%. After 5 min of trypsin treatment, the residual antibacterial rate of the meliofemoral venom polymer was 4.17 times higher than that of the meliofemoral venom monomer; after 30 min of treatment, it still retained an antibacterial rate of 24.2 ± 1.8%.

[0033] Table 2. Antibacterial activity tests of bee venom polymers in simulated acidic and alkaline environments. Note: Different lowercase letters in the same line indicate significant differences (P<0.05).

[0034] In summary, compared with bee venom monomers, the bee venom polymers prepared by this invention have similar antibacterial activity and stronger stability, and can be used in the preparation of antibacterial drugs or antibacterial feed additives.

[0035] Example 3 This example is a hemolysis test of the bee venom polymer prepared in Example 1.

[0036] 1. Experimental materials: Bee venom polymers and bee venom monomers: Same as Experiment (I) in Example 2, different bee venom samples were diluted to mass concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL and 64 μg / mL respectively.

[0037] 2. Experimental methods: Fresh rabbit blood was collected, plasma was removed by centrifugation, and a 2% (w / w) red blood cell suspension was prepared using phosphate-buffered saline (PBS). 100 μL of the red blood cell suspension and 100 μL of different concentrations of bee venom samples were added to each well of a 96-well plate and incubated at 37°C for 10 min. PBS was used as a negative control (0% hemolysis), and 0.1% (w / w) Triton X-100 was used as a positive control (100% hemolysis). The plates were centrifuged at 4°C and 3000 r / min for 10 min, and the absorbance of the supernatant was measured at 540 nm. The toxicity was evaluated by comparing the supernatant with that of bee venom monomers.

[0038] 3. Experimental Results: The results are as follows Figure 5 As shown, "**" indicates a highly significant difference (P<0.01); at a mass concentration of 4 μg / mL (approximately equal to 1×MIC of meliofemoral polymer against Staphylococcus aureus), the hemolysis rate of meliofemoral polymer was 3.5%; at a mass concentration of 16 μg / mL (approximately equal to 1×MIC of meliofemoral polymer against Escherichia coli), the hemolysis rate of meliofemoral polymer was 21.3%, which was 77.9% lower than that of meliofemoral polymer.

[0039] In summary, compared with bee venom monomers, the bee venom polymers prepared in this invention have lower hemolysis rates and cytotoxicity, as well as higher gastrointestinal stability and biosafety.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A melittin multimer, characterized in that, The melittin multimer is formed by at least two melittin variants connected by intermolecular disulfide bonds; The melittin variant is formed by mutating alanine at position 4 and isoleucine at position 20 in the amino acid sequence of a melittin monomer to cysteine; The amino acid sequence of the melittin monomer is shown in SEQ ID No. 1, and the amino acid sequence of the melittin variant is shown in SEQ ID No.

2.

2. A melittin multimer according to claim 1, characterized in that The intermolecular disulfide bond is formed by oxidation coupling of the sulfhydryl groups of the cysteines in the melittin variants.

3. A melittin multimer according to claim 1, wherein The melittin multimer is a melittin dimer, a melittin trimer, or a higher-order melittin multimer.

4. A method of preparing a melittin multimer according to any one of claims 1 to 3, characterized in that, The method comprises: S1, obtaining a melittin variant by recombinant expression in E. coli; S2, purifying the melittin variant obtained in S1 by a nickel column, and then oxidizing to obtain a melittin multimer; S3, purifying the melittin multimer obtained in S2 by gel filtration chromatography to obtain a purified melittin multimer.

5. The method of claim 4, wherein, The oxidation conditions in S2 are: pH 7.0-8.0, temperature 20-37℃, and time 4h.

6. The method of claim 4, wherein, The purity of the melittin variant purified by the nickel column in S2 is >98%, and the purity of the purified melittin multimer in S3 is >95%.

7. Use of a melittin multimer as claimed in any one of claims 1-3, characterized in that, The melittin multimer is used for preparing an antibacterial drug or an antibacterial feed additive.

8. The use of a mast cell degranulation inhibitor according to claim 7, wherein the mast cell degranulation inhibitor is a mast cell degranulation inhibitor according to claim 1. The melittin multimer is used for inhibiting the activity of Staphylococcus aureus and / or E. coli.

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