A HAL-2@MoS2, its preparation method and application

Abstract

The present invention discloses a HAL-2@MoS2, its preparation method and applications, belonging to the field of synthesis technology. The HAL-2@MoS2 in the present invention is prepared by binding the antibacterial peptide HAL-2 to MoS2, wherein the amino acid sequence of the antibacterial peptide HAL-2 is shown as SEQ ID NO.1. The binding of the nanomaterial molybdenum disulfide increases the antibacterial activity of the HAL-2 antibacterial peptide by 45%. HAL-2@MoS2 has the potential to become a novel antibacterial drug.

Description

Technical Field

[0001] The present invention belongs to the field of synthesis technology, and particularly relates to HAL-2@MoS2, a preparation method thereof, and an application thereof. Background Art

[0002] The structure of molybdenum disulfide (MoS2) includes: a transition metal atom (i.e., molybdenum atom) and a pair of atoms from the 16th column of the periodic table including sulfur and selenium elements (this element family is known as chalcogen elements), and it is a transition metal sulfide with a two-dimensional sheet structure. At the same time, the antibacterial effect of molybdenum disulfide itself is not strong, which limits its application in the field of biological antibacterial. Therefore, molybdenum disulfide is mostly combined with other antibacterial agents to form a composite to achieve better effects.

[0003] The antibacterial peptide HAL-2 is isolated from the venom of the bee Hallictus sexcinctus, also known as canavanine, and it is the shortest linear cationic α-helical antibacterial peptide in nature. Summary of the Invention

[0004] One object of the present invention is to provide HAL-2@MoS2, which is prepared by binding the antibacterial peptide HAL-2 to MoS2; the amino acid sequence of the antibacterial peptide HAL-2 is shown in SEQ ID NO.1.

[0005] Preferably, the preparation process of the MoS2 is as follows:

[0006] (1) Weigh 0.25 g of sodium molybdate dihydrate, place it in a container, add 25 mL of deionized water, and dissolve it completely. Adjust the pH to 6.5.

[0007] (2) Weigh 0.5 g of cysteine, place it in a container, add deionized water, and after completely dissolving it, add it to the solution in step (1).

[0008] (3) React the solution at 200 °C for 24 h.

[0009] (4) After the reaction is completed, take the precipitate, wash it alternately with absolute ethanol and distilled water several times, and then dry it to obtain MoS2 powder.

[0010] Preferably, the preparation process of the antibacterial peptide HAL-2 is as follows:

[0011] (1) Activation of Wang resin: Weigh 2 g of Wang resin into a reaction tube, soak it in DCM for a few minutes to swell the resin, and then drain the DCM.

[0012] (2) Modified resin: Design the SD of Wang resin to be 0.5. According to the calculation formula: amount of amino acid = 2g (amount of resin) × 0.5 (SD) × Mw (relative molecular mass of amino acid) × 1.33 (multiple is 3 starting from the second amino acid) ÷ 1000, the dosage of the first amino acid glycine is: 0.395g. Use DMF (dimethylformamide) as the solvent for the resin. According to the calculation formula: HOBt = 2g (amount of resin) × 0.5 (SD) × 138 × 3 ÷ 1000, the mass of HOBt is 0.414g. According to the calculation formula: DMAP = 2g (amount of resin) × 0.5 (SD) × 380 × 3 ÷ 1000, the mass of DMAP is 0.183g. Add 1ml of DIC and react for 4 - 6h;

[0013] (3) Capping: Drain the solution, add DMF, add 1ml of acetic anhydride and DIEA (diisopropylethylamine), react for 30 - 60min, and wash with DMF several times. The added mass of acetic anhydride and DIEA is both 1 / 2 of the resin;

[0014] (4) Deprotection: Add a piperidine solution prepared by piperidine:DMF solution = 1:4. After reacting for 10min, drain the solution, then add the piperidine solution and react for 5min, then drain the solution again, and then wash with DMF several times and wash with methanol several times, and wash with DMF again several times;

[0015] (5) Detection: Take a small amount of resin and place it in a test tube, add two drops of ninhydrin, one drop of phenol, one drop of pyridine, and react at 100°C for 1 - 3min. Observe the color of the solution. If the solution is blue or brown, it indicates that the deprotection is successful;

[0016] (6) Coupling of amino acids: Add the next amino acid, HOBt, and DIC, use DMF as the solvent, react for 50min, then wash alternately with DMF and methanol several times. Take a small amount of resin for ninhydrin detection, it is colorless; if it is colored, then re - add amino acid + HOBt + DIC;

[0017] Repeat steps (2) and (3) to connect the amino acids until all amino acids are coupled;

[0018] (7) Drain: Add methanol solution to blow up the coupled resin so that the resin is fully mixed in the methanol solution. After sufficient mixing, draw off most of the methanol, leaving a small amount of methanol. Do not drain the resin completely, and then add methanol solution to blow up the resin again. After mixing, drain the methanol solution until the resin is in granular form;

[0019] (8) Cutting: Transfer the drained resin into a centrifuge tube, add 20 ml of cutting reagent, which is prepared from trifluoroacetic acid (TFA): ethanedithiol (EDT): triisopropylsilane (Tis): water = 95:2:2:1, and react on a shaker for 2 h;

[0020] (9) Filtration: After the cutting reaction is completed, transfer the resin polypeptide solution into a new reaction tube, and filter the polypeptide solution into another clean centrifuge tube with an ear pipette;

[0021] (10) Centrifugation: Add ether to sediment in the centrifuge tube containing the polypeptide solution, centrifuge, and the lower layer precipitate is HAL-2.

[0022] The second object of the present invention is to provide a preparation method of HAL-2@MoS2, and the preparation method of HAL-2@MoS2 is as follows:

[0023] (1) Weigh 0.10 g of HAL-2 antimicrobial peptide and place it in a container, add 40 mL of PBS buffer solution with a pH of 7.4, and ultrasonicate until completely dissolved;

[0024] (2) Weigh 0.05 g of MoS2 and place it in a container, add 10 mL of PBS buffer solution with a pH of 7.4, ultrasonicate until uniformly dispersed, pour the MoS2 dispersion into the HAL-2 antimicrobial peptide solution and stir, then incubate on a shaker for 10 - 14 h, and then perform lyophilization to obtain the final product of HAL-2@MoS2.

[0025] Preferably, the incubation conditions are 37 °C and 185 rpm.

[0026] The third object of the present invention is to provide the application of HAL-2@MoS2 in the preparation of antibacterial reagents.

[0027] The fourth object of the present invention is to provide the application of the preparation method of HAL-2@MoS2 in the preparation of antibacterial reagents.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] In the present invention, the combination of the nanomaterial molybdenum disulfide improves the antibacterial activity of HAL-2 antimicrobial peptide by 45%, and HAL-2@MoS2 has the potential to become a new antibacterial drug. Description of the Drawings

[0030] Figure 1 It is the mass spectrum of HAL-2 in Example 1.

[0031] Figure 2 It is the ultraviolet spectrum in Example 2.

[0032] Figure 3It is the infrared spectrogram in Example 2.

[0033] Figure 4 It is the scanning electron microscope images of MoS2 and HAL-2@MoS2 in Example 2.

[0034] Figure 5 It is the photo-thermal analysis effect diagram of three materials in Example 2.

[0035] Figure 6 It is the photo-thermal analysis diagram of three materials in Example 2.

[0036] Figure 7 It is the bacteriostatic circle effect diagram of three materials against Escherichia coli in Example 3.

[0037] Figure 8 It is the bacteriostatic circle effect diagram of three materials against Staphylococcus aureus in Example 3.

[0038] Figure 9 It is the CFU effect diagram of HAL-2 antimicrobial peptide, MoS2 and HAL-2@MoS2 against Escherichia coli in Example 3.

[0039] Figure 10 It is the CFU survival rate diagram of three materials against Escherichia coli in Example 3.

[0040] Figure 11 It is the effect diagram of three materials on CFU of Staphylococcus aureus in Example 3. Detailed implementation method

[0041] Example 1

[0042] 1. Synthesis of HAL-2 antimicrobial peptide:

[0043] (1) Solution preparation

[0044] Prepare phenol solution: Weigh 40 g of phenol into a 50 ml beaker, and then add 10 ml of absolute ethanol to it for dissolution and standby.

[0045] Prepare ninhydrin solution: Weigh 5 g of ninhydrin into a 100 ml conical flask, and then add absolute ethanol to the conical flask to 100 ml for dissolution and standby.

[0046] Piperidine preparation: Mix piperidine and DMF in a ratio of 1:4. Measure 100 ml of piperidine into a wash bottle, and then measure 400 ml of DMF and add it to the wash bottle for mixing and standby.

[0047] (2) Cleaning of reaction tubes

[0048] Clean by ultrasonic cleaning, then clean with pure water, and finally wash with DCM (dichloromethane). Place it on the experimental bench and drain the excess water in the reaction tube.

[0049] (3) Activation of Wang resin

[0050] Weigh 2 g of Wang resin into a reaction tube, soak it in DCM for a few minutes to swell the resin, and then drain the DCM.

[0051] (4) Modify the resin

[0052] Design the SD of Wang resin to be 0.5. According to the calculation formula: amount of amino acid = 2 g (resin amount) × 0.5 (SD) × Mw (relative molecular mass of amino acid) × 1.33 (multiplier is 3 starting from the second amino acid) ÷ 1000, the dosage of the first amino acid glycine is: 0.395 g. Use DMF (dimethylformamide) as the solvent for the resin. According to the calculation formula: HOBt = 2 g (resin amount) × 0.5 (SD) × 138 × 3 ÷ 1000, the mass of HOBt (1-hydroxybenzotriazole) is 0.414 g. According to the calculation formula: DMAP = 2 g (resin amount) × 0.5 (SD) × 380 × 3 ÷ 1000, the mass of DMAP (dimethylaminopyridine) is 0.183 g. Add 1 ml of DIC (N,N'-diisopropylcarbodiimide) and react for 4 - 6 h.

[0053] (5) Capping

[0054] Drain, add DMF, add 1 ml of acetic anhydride and DIEA (diisopropylethylamine), react for 30 - 60 min, and wash with DMF three times. The addition amounts of acetic anhydride and DIEA are both 1 / 2 of the resin.

[0055] (6) Deprotection

[0056] Add piperidine solution (prepared by mixing piperidine:DMF solution = 1:4), after reacting for ten minutes, drain the solution, then add piperidine solution and react for five minutes, then drain the solution again, and then wash with DMF twice and methanol twice, and wash with DMF twice again.

[0057] (7) Detection

[0058] Take a small amount of resin and place it in a test tube, add two drops of ninhydrin, one drop of phenol, one drop of pyridine, and react at 100 °C for 1 - 3 min, and observe the color of the solution. If the solution is blue or brown, it indicates that deprotection is successful.

[0059] (8) Conjugate amino acid

[0060] Add the next amino acid, HOBt, and DIC, use DMF as the solvent, react for 50 min, and then wash (alternately wash with DMF and methanol four times). Take a small amount of resin for ninhydrin detection, and it is colorless. (If it is colored, re-add amino acid + HOBt + DIC)

[0061] Repeat steps (5) and (6) to connect the amino acids until all amino acids are completely coupled. (Note: If not completed on the same day, after the reaction, wash, drain, place, and turn off the suction filtration. Remember, do not deprotect.)

[0062] (9) Drain

[0063] Add methanol solution to blow up the coupled resin, so that the resin is fully mixed in the methanol solution. After sufficient mixing, draw off most of the methanol, leaving a small amount of methanol. Do not drain the resin completely. Then add methanol solution to blow up the resin again. After mixing, drain the methanol solution until the resin becomes granular.

[0064] (10) Cleavage

[0065] Transfer the drained resin to a 50 ml centrifuge tube, add 20 ml of cleavage reagent (TFA (trifluoroacetic acid): EDT (ethanedithiol): Tis (triisopropylsilane): water = 95:2:2:1), and react on a shaker for 2 h.

[0066] (11) Filtration

[0067] After the cleavage reaction is completed, transfer the resin polypeptide solution to a new reaction tube, and filter the polypeptide solution into another clean 50 ml centrifuge tube using an ear bulb.

[0068] (12) Centrifugation

[0069] Add ether to precipitate in the centrifuge tube containing the polypeptide solution, centrifuge at 3000 r for 5 min, repeat three times, and the precipitate at the lower layer of the centrifuge tube is HAL-2.

[0070] (13) Characterization

[0071] The mass spectrum of HAL-2 is Figure 1 , and the mass-to-charge ratio is shown in Table 1.

[0072] Table 1

[0073]

[0074] Result analysis shows that the main peaks in the mass spectrum are the same as the mass-to-charge ratio of HAL-2.

[0075] 2. The synthesis process of MoS2 is as follows:

[0076] (1) Weigh 0.25 g of sodium molybdate dihydrate, place it in a 100 mL beaker, add 25 mL of deionized water, ultrasonicate for 5 min to completely dissolve it, and adjust the pH to 6.5 using 0.1 mol HCl.

[0077] (2) Weigh 0.5 g of cysteine, place it in a 100 mL beaker, add 50 mL of deionized water, sonicate for 10 min, and after complete dissolution, add it to the above solution.

[0078] (3) Dispense the solution into a reaction kettle, place it in an electrothermal constant temperature drying oven, and react at 200 °C for 24 h.

[0079] (4) After the reaction is completed, take the precipitate, centrifuge at 12000 r / min for 10 min, wash it twice alternately with absolute ethanol and distilled water, and dry the product at 80 °C for 4 h to obtain MoS2 powder.

[0080] Example 2

[0081] 1. The synthesis process of HAL-2@MoS2 is as follows:

[0082] Since Halictine antimicrobial peptide is isolated from the venom of the bee Hallictus sexcinctus, also known as canaryine, which is the shortest linear cationic α-helical antimicrobial peptide in nature. That is, HAL-2 antimicrobial peptide is positively charged, and the sequence of HAL-2 antimicrobial peptide (SEQ ID NO.1) is: GKWMSLLKHILK, while MoS2 is negatively charged. Therefore, HAL-2 antimicrobial peptide is loaded on MoS2 using the principle of electrostatic adsorption. The specific preparation process is as follows:

[0083] (1) Prepare phosphate buffered saline (PBS): 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, add 800 mL of distilled water, and adjust the pH of the solution to 7.4 with HCl.

[0084] (2) Weigh 0.10 g of HAL-2 antimicrobial peptide and place it in a 100 mL beaker, add 40 mL of buffer solution, sonicate until completely dissolved, and set aside for later use.

[0085] (3) Weigh 0.05 g of the prepared MoS2 and place it in a beaker, add 10 mL of buffer solution, sonicate (150 W, 30 min) until evenly dispersed. Pour the MoS2 dispersion into a beaker containing 40 mL of HAL-2 antimicrobial peptide solution, stir magnetically at 37 °C for 3 h, transfer the mixture to a conical flask, incubate it in a shaker (conditions: 37 °C, 185 rpm) for 12 h, transfer it to a 50 mL centrifuge tube, place it in a -18 °C refrigerator, and then perform freeze-drying to obtain HAL-2@MoS2.

[0086] 2. Morphology and structure characterization

[0087] (1) Characterization by ultraviolet spectrophotometer: Take a small amount of the prepared HAL-2 antimicrobial peptide, MoS2, and HAL-2@MoS2 products respectively and place them in 2 mL centrifuge tubes. Add ultrapure water and dissolve them by ultrasonic treatment. After running the baseline with ultrapure water, dilute the sample solutions respectively and start scanning. The results are as Figure 2 shown, Figure 2 The HAL-2 antimicrobial peptide has an absorption peak at 280 nm, while molybdenum disulfide has no absorption peak, indicating that this is a unique feature of the HAL-2 antimicrobial peptide. And HAL-2@MoS2 also has an absorption peak at 280 nm, indicating that the HAL-2 antimicrobial peptide has been successfully loaded onto the nanomaterial molybdenum disulfide.

[0088] (2) Characterization by infrared spectroscopy: Take a small amount of the HAL-2 antimicrobial peptide, MoS2, and HAL-2@MoS2 products respectively and place them in 2 mL centrifuge tubes. Then add an appropriate amount of dried potassium bromide, and dry them in an oven. Place the dried products in a mortar and crush them into powder. Pour the powder into a tablet press, press the pressure to exceed 20, time the tablet pressing for 1 min, release the pressure, put the pressed tablet into an infrared spectrometer, and start scanning. The results are as Figure 3 shown, Figure 3 The HAL-2 antimicrobial peptide has an obvious vibration peak at 1200 nm while molybdenum disulfide has no peak at this position, and the material combined with the HAL-2 antimicrobial peptide and molybdenum disulfide also has a vibration peak at this position, indicating that the HAL-2 antimicrobial peptide has been successfully loaded onto the nanomaterial molybdenum disulfide.

[0089] (3) Characterization by scanning electron microscope: Add silicon wafers to ethanol and ultrasonically clean them for 1 - 2 min, then dry them. Then take a small amount of MoS2 and HAL-2@MoS2 respectively, dissolve them in water, drop a drop of the sample on the silicon wafer, dry it, place it on a scanning electron microscope, and start scanning. The results are as Figure 4 shown, Figure 4 In a, it is the scanning electron microscope image of MoS2, Figure 4 In b, it is the scanning electron microscope image of HAL-2@MoS2. The electron microscope image of molybdenum disulfide is nanospheres (about 500 nm in size), while the electron microscope image of the HAL-2 antimicrobial peptide loaded on molybdenum disulfide has changed from spherical to flaky (about 200 nm in size), indicating that the HAL-2 antimicrobial peptide has been successfully loaded onto molybdenum disulfide.

[0090] (4) Characterization by photothermal method: Take 200 μg of the sample respectively and place them in 2 mL centrifuge tubes. Add 1 mL of deionized water and dissolve them by ultrasonic treatment to prepare a 200 μg / mL sample solution, with deionized water as the control. Install the instrument, take a photo every minute, and perform photothermal treatment for 15 min. The results are as Figure 5 and Figure 6 shown, where Figure 5 is the photothermal analysis effect of the three materials, Figure 6It is a photothermal analysis diagram. From this, it can be seen that the HAL-2 antimicrobial peptide has almost no photothermal effect, while the photothermal effect of molybdenum disulfide is very obvious. At the same time, the combined material has a photothermal effect but not as strong as that of molybdenum disulfide, indicating that the HAL-2 antimicrobial peptide reduces the photothermal effect of molybdenum disulfide. This shows that the HAL-2 antimicrobial peptide has been successfully loaded onto the nanomaterial molybdenum disulfide.

[0091] Example 3

[0092] In this example, the antibacterial activity of the synthetic material in Example 2 was analyzed, and the process is as follows:

[0093] (1) Determination of the inhibition zone

[0094] Beef extract peptone medium: The component content per 1000 mL of deionized water: 5 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 20 g of agar. Sterilize at 121 °C in a high-pressure steam sterilizer for 20 min.

[0095] LB liquid medium: The component content per 1000 mL of deionized water: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride. Sterilize at 121 °C in a high-pressure steam sterilizer for 20 min.

[0096] Activation of Escherichia coli: Take out the preserved Escherichia coli strain from the refrigerator, operate in a laminar flow hood, pick up a small amount of bacteria with an inoculation loop and transfer it to LB liquid medium, and culture it in a shaker for 12 h under the conditions of 37 °C and 200 r / min.

[0097] Activation of Staphylococcus aureus: Take out the preserved Staphylococcus aureus strain from the refrigerator, operate in a laminar flow hood, pick up a small amount of bacteria with an inoculation loop and transfer it to LB liquid medium, and culture it in a shaker for 12 h under the conditions of 37 °C and 200 r / min.

[0098] Preparation of the material liquid medicine: Weigh 5 mg of the purified HAL-2 antimicrobial peptide, add 1 ml of deionized water to dissolve it, and prepare a 5 mg / ml liquid medicine for storage in the refrigerator. Weigh 200 μg of the dried molybdenum disulfide, add 1 mL of deionized water and ultrasonicate until dissolved to prepare a 200 μg / mL molybdenum disulfide solution for storage at room temperature. Weigh 200 μg of HAL-2@MoS2 whose morphology and structure have been characterized, add 1 mL of deionized water and ultrasonicate until dissolved to prepare a 200 μg / mL solution for storage at room temperature.

[0099] Preparation of the inhibition zone: Turn on the ultraviolet lamp for sterilization 30 minutes in advance when using the laminar flow hood. Take 4 test tubes containing 4.5 ml of sterile water, numbered 1 - 4. Take 0.5 mL from the test tube containing the activated bacterial solution and put it into test tube No. 1. After mixing, take 0.5 ml from test tube No. 1 and transfer it to test tube No. 2, and so on. By the time it reaches test tube No. 4, the bacterial solution has been diluted 10,000 times. Soak the sterilized filter paper in the medicinal liquid for 10 minutes in advance. Take 100 μL of the Escherichia coli bacterial solution and Staphylococcus aureus bacterial solution after dilution to a concentration of 10,000 times respectively, and spread them evenly on the plate. Use forceps to pick up the filter paper, wipe off the excess medicinal liquid, and place it in the middle of the plate. Use the filter paper soaked in sterile water as a control, and do three parallel groups for each. After the operation, place the plate in an incubator at 37 °C. Incubate Escherichia coli for 12 h, while incubate the positive bacterium Staphylococcus aureus for 24 h. Figure 7 Figure showing the inhibition zones of three materials against Escherichia coli, where a) is the blank, b) is HAL-2, c) is MoS2, and d) is HAL-2@MoS2; Figure 8 Figure showing the inhibition zones of three materials against Staphylococcus aureus, where a) is the blank, b) is HAL-2, c) is MoS2, and d) is HAL-2@MoS2. Table 2 shows the inhibition zone results of HAL-2, MoS2 and HAL-2@MoS2.

[0100] Table 2

[0101] Inhibition zone (mm) Escherichia coli Staphylococcus aureus Blank control 6 6 HAL-2 8 9 <![CDATA[MoS2]]> 6 6 <![CDATA[HAL-2@MoS2]]> 12 13

[0102] It can be seen from this that the antibacterial peptide HAL-2 and HAL-2@MoS2 have obvious antibacterial effects on Gram-negative bacteria and Gram-positive bacteria. The diameters of the inhibition zones of HAL-2 against Escherichia coli and Staphylococcus aureus are 8 mm and 9 mm respectively; the diameters of the inhibition zones of HAL-2@MoS2 against Escherichia coli and Staphylococcus aureus are 12 mm and 13 mm respectively. This shows that the antibacterial activity of HAL-2@MoS2 is stronger than that of the single antibacterial peptide HAL-2. The antibacterial mechanism of MoS2 is mainly through photothermal generation. Under the condition without photothermal, its antibacterial activity is almost non-existent, and the effect is the same as that of the control group.

[0103] (2) Determination of colony-forming units (CFU)

[0104] The preparation of the culture medium, the activation of the bacterial strain, the concentration of the material medicinal liquid preparation and the dilution of the bacterial solution are all the same as the above steps and methods.

[0105] Steps for making CFU: Respectively take 100 μL of Escherichia coli bacterial liquid and Staphylococcus aureus bacterial liquid and place them in 2-mL sterilized centrifuge tubes. Then, add 100 μL of HAL-2 antibacterial peptide liquid medicine, MoS2 solution, and HAL-2@MoS2 solution to each of them respectively, mix evenly. Subject the mixed MoS2 liquid and HAL-2@MoS2 liquid to photothermal treatment for 5 min. Then, respectively take 100 μL of the solution and evenly coat it on the plate. Also, respectively take 100 μL of the non-photothermal MoS2 liquid and HAL-2@MoS2 liquid and evenly coat them on the plate. Use 100 μL of bacterial liquid and 100 μL of sterile water as controls, and perform three parallel groups each. Place them in an incubator at 37 °C and culture for 12 h.

[0106] The CFU effects of HAL-2 antibacterial peptide, MoS2, and HAL-2@MoS2 on Escherichia coli are as Figure 9 shown, where a) is the blank, b) is HAL-2, c) is MoS2, d) is MoS2 (photothermal), e) is HAL-2@MoS2, and f) is HAL-2@MoS2 (photothermal); Figure 10 Figure of the CFU survival rate of three materials against Escherichia coli, where a) is the blank; b) is HAL-2; c) is MoS2; d) is MoS2 (photothermal); e) is HAL-2@MoS2; f) is HAL-2@MoS2 (photothermal); The CFU effects of Staphylococcus aureus are as Figure 11 shown, where a) is the blank, b) is HAL-2, c) is MoS2, d) is MoS2 (photothermal), e) is HAL-2@MoS2, and f) is HAL-2@MoS2 (photothermal).

[0107] It can be seen from this that HAL-2 antibacterial peptide and HAL-2@MoS2 without photothermal treatment still have obvious antibacterial activities, while for MoS2 without photothermal treatment, its antibacterial activity is still no different from the control. When the MoS2 solution is mixed evenly with the bacterial liquid and subjected to photothermal treatment for 5 min, it already has antibacterial effects, and when the HAL-2@MoS2 solution is mixed with the bacterial liquid and subjected to photothermal treatment for 5 min, the antibacterial effects produced are stronger. And through comparison, it can be obtained that these liquid medicines have better antibacterial activities against Gram-positive bacterium Staphylococcus aureus.

[0108] (3) Determination of the minimum inhibitory concentration (MIC)

[0109] Prepare the liquid medium as above. Prepare the concentration of HAL-2 antibacterial peptide to be 5 mg / mL, and the concentrations of both the MoS2 solution and the HAL-2@MoS2 solution to be 200 μg / mL.

[0110] The minimum inhibitory concentration was analyzed by the micro-dilution method. 100 μL of LB medium was added to each well of a sterile 96-well plate. Then, 100 μL of the prepared medicinal solution was added to the first well. After mixing, it was serially diluted until 100 μL of the mixed solution was discarded from the last well after mixing. 100 μL of the prepared bacterial dilution was added to each well. At the same time, a positive control group (adding bacteria without adding medicine) and a negative control group (not adding bacteria and not adding medicine) were set up. In addition, an antibiotic control group needs to be set up before the formal experiment, that is, a control experiment of antibiotics is carried out for each strain of bacteria. The medicinal solution is replaced with an antibiotic to determine the maximum solvent content that can ensure the survival of all test strains, so that the solvent content used to prepare the medicinal solution is much lower than this value. It was placed in a constant temperature incubator at 37°C. Escherichia coli was cultured for 12 h, and Staphylococcus aureus was cultured for 24 h and then taken out. The absorbance value at OD600 was measured in an enzyme-linked immunosorbent assay instrument. There were three parallels in each group.

[0111] The analysis results of the minimum inhibitory concentration (MIC) of HAL-2 antimicrobial peptide, MoS2, and HAL-2@MoS2 against Escherichia coli and Staphylococcus aureus determined by the micro-dilution method are shown in Table 3 below. It can be seen from this that the minimum inhibitory concentration of HAL-2 antimicrobial peptide against Escherichia coli is 78.00 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is 39.00 μg / mL. Since the antibacterial mechanism of molybdenum disulfide itself is generated by photothermal, in the case where the MoS2 medicinal solution does not undergo photothermal, it has almost no antibacterial activity. After photothermal treatment, its minimum inhibitory concentration against Escherichia coli is 50.00 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is 25.00 μg / mL. The minimum inhibitory concentration of the HAL-2@MoS2 medicinal solution against Escherichia coli is 12.50 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is 6.25 μg / mL without photothermal treatment; after photothermal treatment for 5 min, the antibacterial effect is stronger, and the minimum inhibitory concentration against Escherichia coli is 3.125 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is also 3.125 μg / mL.

[0112] Table 3

[0113] MIC (μg / mL) Escherichia coli Staphylococcus aureus HAL-2 78.125 39.062 MoS2 (not photothermal) 100.00 100.00 MoS2 (photothermal) 50.00 25.00 HAL-2@MoS2 (not photothermal) 12.50 6.25 HAL-2@MoS2 (photothermal) 3.125 3.125

[0114] In this invention, the synthesized materials MoS2 and HAL-2@MoS2 were characterized by means of ultraviolet spectroscopy, infrared spectroscopy, scanning electron microscopy, and photothermal analysis, which proved that the HAL-2 antimicrobial peptide was successfully loaded onto the nanomaterial molybdenum disulfide. The antibacterial activity of the synthesized materials was analyzed, and the results showed that the diameters of the inhibition zones of HAL-2 antimicrobial peptide, MoS2, and HAL-2@MoS2 against Escherichia coli were 8 mm, 6 mm, and 12 mm respectively, and the diameters of the inhibition zones against Staphylococcus aureus were 9 mm, 6 mm, and 13 mm respectively; the CFU antibacterial effects of HAL-2 antimicrobial peptide and non-photothermal HAL-2@MoS2 against Escherichia coli and Staphylococcus aureus were both obvious, while the CFU effect of non-photothermal MoS2 was not obvious, the CFU determination effect of photothermal MoS2 was slightly stronger, and the CFU effect of photothermal HAL-2@MoS2 was the most obvious. The MICs of HAL-2, MoS2, and HAL-2@MoS2 against Escherichia coli were 78 μg / mL, 50 μg / mL, and 3.125 μg / mL respectively, and the MICs against Staphylococcus aureus were 39 μg / mL, 25 μg / mL, and 3.125 μg / mL respectively. The experimental results proved that the combination of the nanomaterial molybdenum disulfide increased the antibacterial activity of the HAL-2 antimicrobial peptide by 45%, showing the potential to become a new type of antibacterial drug.

[0115] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kind of HAL-2@MoS2, characterized in that, The HAL-2@MoS2 is prepared by binding the antimicrobial peptide HAL-2 to MoS2; the amino acid sequence of the antimicrobial peptide HAL-2 is shown in SEQ ID NO.

1.

2. The HAL-2@MoS2 according to claim 1, characterized in that, The preparation process of the MoS2 is as follows: (1) Weigh 0.25 g of sodium molybdate dihydrate, place it in a container, add 25 mL of deionized water, and dissolve it completely. Adjust the pH to 6.

5. (2) Weigh 0.5 g of cysteine, place it in a container, add deionized water, and after complete dissolution, add it to the solution in step (1). (3) React the solution at 200 °C for 24 h. (4) After the reaction, take the precipitate, wash it alternately with anhydrous ethanol and distilled water several times, and then dry it to obtain MoS2 powder.

3. The preparation method of a kind of HAL-2@MoS2 according to claim 1, characterized in that, The preparation method of the HAL-2@MoS2 is as follows: (1) Weigh 0.10 g of HAL-2 antimicrobial peptide and place it in a container. Add 40 mL of PBS buffer solution with a pH of 7.4 and ultrasonicate until completely dissolved. (2) Weigh 0.05 g of MoS2 and place it in a container. Add 10 mL of PBS buffer solution with a pH of 7.4 and ultrasonicate until evenly dispersed. Pour the MoS2 dispersion into the HAL-2 antimicrobial peptide solution and stir. Then incubate it in a shaker at 37 °C and 185 rpm for 10 - 14 h, and then perform freeze-drying to obtain the final product of HAL-2@MoS2.

4. The preparation method of HAL-2@MoS2 according to claim 3, characterized in that, The conditions for the incubation are 37 °C and 185 rpm.

5. The application of the HAL-2@MoS2 according to claim 1 or 2 in the preparation of antibacterial reagents.

6. The application of the preparation method of HAL-2@MoS2 according to claim 3 or 4 in the preparation of antibacterial reagents.

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