Fusion antibacterial peptide for inhibiting mycobacterium tuberculosis as well as preparation method and application of fusion antibacterial peptide

Abstract

The invention belongs to the technical field of fusion antibacterial peptides, and particularly relates to a fusion antibacterial peptide for inhibiting mycobacterium tuberculosis as well as a preparation method and application thereof. The fusion antibacterial peptide is composed of PK34 and LL-37, and the amino acid sequence of the fusion antibacterial peptide is as shown in SEQ ID NO: 1. The preparation method comprises the following steps: synthesizing exogenous antibacterial peptides PK34 and LL-37 and a fused antibacterial peptide PK34 / LL-37 by using a solid-phase synthesis method; then, the inhibition effect of the antibacterial peptide on infection of the two pathogens is explored through extracellular and intracellular experiments. The antibacterial peptide LL-37 has the advantages of two polypeptides, can specifically bind to the surface of mycobacterium tuberculosis to achieve the effect of targeted killing of latent mycobacterium tuberculosis in macrophages, and has the advantages of resisting bacteria, especially mycobacterium tuberculosis, resisting inflammation and promoting tissue repair of the antibacterial peptide LL-37. And the PK34 can specifically recognize glycolipid on the surface of the mycobacterium tuberculosis and efficiently kill the mycobacterium tuberculosis, and can be specifically combined with intracellular mycobacterium to achieve the purpose of efficiently killing the intracellular mycobacterium tuberculosis in a targeted manner.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion antimicrobial peptides, and in particular relates to a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, a preparation method and an application thereof. Background Art

[0002] In recent years, antimicrobial peptides with high antibacterial activity and low resistance to drug resistance and serious adverse reactions have gradually come into the sight of researchers. Antimicrobial peptides (AMPs) are cationic polypeptides produced by organisms. They are widely present in various organisms from protozoa to humans. They have killing effects on Gram-positive cocci, Gram-negative bacilli and even some fungi and protozoa. They may become a new way to treat tuberculosis. For example, antimicrobial peptides such as human α-defensin (hAD), human β-defensin 1-2 (hBD-1, hBD-2), human neutrophil defensin 1-3 (hNP1, 2, 3), cathelicidin LL-37 (LL37), NK-lysin and porcine antimicrobial peptides PG-1, PR-39 have good effects against Mycobacterium tuberculosis, and do not have a series of adverse reactions and side effects such as liver damage, gastrointestinal symptoms, and allergic reactions of tuberculosis chemotherapy drugs.

[0003] As a bacterial virus, bacteriophages can effectively interact with host bacteria. Since bacteriophages can specifically lyse host bacteria, many bacteriophages such as Staphylococcus aureus phage and Pseudomonas aeruginosa phage have been used in clinical trials. Mycobacterium phage D29 has become a research hotspot because it can specifically lyse Mycobacterium tuberculosis. In vitro experiments show that D29 has a good sterilization effect. However, if it is to enter the body to fight against Mycobacterium tuberculosis, especially against intracellular infection, the sterilization efficiency is not high due to the small number of D29 entering macrophages. Therefore, the researchers found a small molecule peptide PK34 from mycobacterium phage D29, which can specifically bind to the most abundant sugar ester on the surface of Mycobacterium tuberculosis (trehalose-6,6'-dimycolate, TDM). TDM is called the core factor of Mycobacterium tuberculosis. Injecting TDM into the tail vein of mice can fully simulate a series of immunopathological reactions that occur in the lungs after Mycobacterium tuberculosis infection. Therefore, TDM is an important target for the development of anti-tuberculosis drugs. In addition, PK34 also has the ability to kill tuberculosis bacteria and fight inflammation. In vivo anti-tuberculosis animal model experiments have shown that PK34 has the ability to eliminate tuberculosis bacteria in vivo comparable to rifampicin. PK34 inhibits the massive secretion of inflammatory factors by inhibiting the activation of mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), but at the same time maintains a certain level of inflammatory cytokines to maintain the normal immune capacity of experimental animals. This specificity, efficient sterilization and anti-inflammatory function make PK34 a promising adjuvant drug for targeted treatment of tuberculosis infection, or as a template for the development of anti-tuberculosis drugs. However, it is difficult for PK34 to exert its effect on intracellular tuberculosis bacteria through macrophages, which hinders the progress of PK34 in anti-tuberculosis treatment. It has also become a key issue in the application of new and highly efficient antimicrobial peptides such as PK34 in the treatment of intracellular infections.

[0004] Cathelicidin LL-37 (LL37) is a linear cationic α-helical peptide bioactive antimicrobial peptide composed of 37 amino acids. It exists in the innate immune cells of humans or mammals as an inactive precursor hCAP-18. It is continuously expressed in human neutrophils, natural killer cells, mast cells and other cells, and stored in the cytoplasm in the form of granules. For macrophages, LL-37 expression can be induced by environmental factors, such as when bacteria or bacterial cell wall components come into contact, tissues are infected or damaged, and vitamin D3 increases, macrophages will be induced to express hCAP-18. In addition, studies have shown that exogenous increase or endogenous overexpression of LL-37 or hCAP18 in macrophages can improve the efficiency of intracellular killing of mycobacteria. Its antibacterial mechanism is: the cationic helical secondary structure of LL-37 makes it positively charged, and it can bind to the surface of the negatively charged bacterial cell membrane through charge attraction. Then LL-37 passes through the outer cell membrane to the inner bacterial membrane, causing the destruction of the bacterial cell membrane and leading to bacterial death. Since the surface of eukaryotic cell membranes is neutral and contains cholesterol that can reduce the interaction of LL-37 with the membrane, these characteristics inhibit the interaction of LL-37 with eukaryotic cells (including protozoa, fungi, plant and animal cells, etc.).

[0005] It is also difficult to control the efficacy of antimicrobial peptides in macrophages in vivo to exert their anti-intracellular infection effects. For example, LL-37 enters cells through the active endocytosis of macrophages or the penetration of antimicrobial peptides to exert its effects. If it is an intracellular infection, due to the active uptake of macrophages or the penetration of antimicrobial peptides, the concentration of antimicrobial peptides at the site of infection, and the function of self-expression of antimicrobial peptides, will be affected by the state of the cells themselves. In many cases, antimicrobial peptides cannot reach the concentration of inhibition or sterilization in the cell. Other antimicrobial peptides need to enter the bacteria and act on bacterial DNA or other target organelles in the cytoplasm to kill bacteria, such as PR39. Then reaching the center of inflammation or entering macrophages is not the last step. Antimicrobial peptides also need to pass through the cell wall and three-layer biomembrane of Mycobacterium tuberculosis to enter the bacteria and interact with specific target organelles to cause bacterial death. How we can maximize our functions in macrophages in a targeted manner has become a key issue we need to solve. Summary of the invention

[0006] In view of the existing technology, LL-37 not only has a broad-spectrum antibacterial activity and rapid bactericidal activity, but also can affect the expression of inflammatory factors and play an important role in the body's immune regulation. PK34, as a small molecule polypeptide separated from the bacteriophage of Mycobacterium tuberculosis, can specifically bind to the most abundant sugar ester on the surface of Mycobacterium tuberculosis, and its killing effect on Mtb is comparable to that of rifampicin. However, when the two act alone, the bactericidal specificity of LL-37 is not as good as PK34, and the immunoregulatory effect of PK34 is not as good as LL-37. The present invention provides a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, a preparation method and an application, which not only overcomes the disadvantage that antimicrobial drugs are not easy to reach the target, but also avoids the instability of a single antimicrobial peptide, and reduces costs and toxic side effects.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, characterized in that the fusion antimicrobial peptide is composed of PK34 and LL-37, and its amino acid sequence is shown in SEQ ID NO:1.

[0008] A method for preparing a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, wherein the fusion antimicrobial peptide is synthesized by a solid phase synthesis method, and the synthesis steps include:

[0009] (1) 2-Chlorotrityl Chloride Resin was placed in a reaction tube, DMF was added, and the reaction was shaken for 60 min to allow the resin to swell. The solvent was filtered out through a sand core, and an excess of Fmoc was added to protect the first amino acid at the C-terminus. Then, an excess of DIEA was added, and finally, DMF was added to dissolve the mixture, and the mixture was shaken for 30 min. The mixture was capped with methanol for 30 min, and the first amino acid was connected.

[0010] (2) Remove DMF, add 20% piperidine DMF solution, remove after 5 minutes, and then add 20% piperidine DMF solution for 15 minutes to achieve deprotection; wash with DMF, methanol, and DMF twice each; add Fmoc-protected amino acid, then add HBTU, DIEA and DMF, and shake for 45 minutes; wash with DMF once, methanol twice, and DMF twice;

[0011] (3) Repeat step (2) to connect the amino acids in SEQ ID NO: 1 from right to left until the Fmoc protecting group of the last amino acid is removed. Wash the resin with DMF twice, DCM three times, and methanol four times, and drain for 10 min.

[0012] (4) Use TFA, water, EDT, and TIS to prepare a cutting solution, cut for 180 min, blow the lysate dry with nitrogen as much as possible, precipitate ether, centrifuge to remove the supernatant, wash the precipitate with ether six times, and then evaporate to dryness at room temperature;

[0013] (5) Take a small amount of the crude product, dissolve it in H2O / ACN, analyze it on an HPLC analyzer to determine the peak time corresponding to the target peak, collect the target peak solution, and freeze-dry the qualified target peak solution to obtain the fusion antimicrobial peptide.

[0014] Furthermore, the specific steps of (1) include:

[0015] Put 2-Chlorotrityl Chloride Resin into a reaction tube, add 15 ml / g of MF, shake for 60 min to make the resin swell, filter out the solvent through a sand core, add 3 times molar excess of Fmoc to protect the first amino acid at the C-terminus, then add 10 times molar excess of DIEA, finally add DMF to dissolve, shake for 30 min, use methanol to cap the tube for 30 min, and connect the first amino acid.

[0016] Furthermore, the specific steps of (2) include:

[0017] Remove DMF, add 20% piperidine DMF solution at a concentration of 15 ml / g, remove after 5 minutes, and then add 20% piperidine DMF solution at a concentration of 15 ml / g for 15 minutes to achieve deprotection; wash twice with 10 ml / g of DMF, methanol, and DMF respectively; add 3-fold molar excess of Fmoc-protected amino acid, then add 3-fold molar excess of HBTU and 10-fold molar excess of DIEA, then add DMF to dissolve, and shake for 45 minutes; wash once with 10 ml / g of DMF, wash twice with 10 ml / g of methanol, and wash twice with 10 ml / g of DMF.

[0018] Furthermore, the specific steps of (3) include:

[0019] Repeat (2) to connect the amino acids in SEQ ID NO: 1 from right to left in sequence until the Fmoc protecting group of the last amino acid is removed. Wash the resin with 10 ml / g DMF twice, 10 ml / g DCM three times, and 10 ml / g methanol four times, and drain for 10 min.

[0020] Furthermore, the specific steps of (4) include:

[0021] The cutting solution was prepared by volume percentage of TFA 95%, water 2%, EDT 2%, and TIS 1%. The cutting was performed for 180 min. The lysate was blown dry as much as possible with nitrogen gas, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed with ether six times, and then evaporated to dryness at room temperature.

[0022] Furthermore, the specific steps of (5) include:

[0023] Take a small amount of crude product, dissolve it in H2O / ACN, analyze it on an HPLC analyzer to determine the peak time corresponding to the target peak, and use a C18 reverse phase chromatography preparation system: Wavelength: 220nm; Flow Rate: 15ml / min; Inj.Vol: 20mL Column Temp: 25℃; Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution, and lyophilize the qualified target peak solution to obtain the fusion antimicrobial peptide.

[0024] The application of a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis is prepared by the above method, and the fusion antimicrobial peptide is used for non-therapeutic intracellular or extracellular killing of Mycobacterium tuberculosis.

[0025] The application of a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, wherein the fusion antimicrobial peptide is prepared by the above method, and the fusion antimicrobial peptide is used for non-therapeutic extracellular promotion of the proliferation of macrophages or A549 cells, or promoting the expression of IL-2 and IL-10 in macrophages, and inhibiting the expression of inflammatory factors TNF-α and IP-10.

[0026] The application of a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis is prepared by the above method, and the fusion antimicrobial peptide is used for non-therapeutic intracellular inhibition of TNF-αmRNA and IL-6mRNA expression.

[0027] The benefits of the present invention are:

[0028] The present invention has the advantages of two polypeptides, which can specifically bind to the surface of Mycobacterium tuberculosis to achieve the effect of targeted killing of latent Mycobacterium tuberculosis in macrophages. Through the antibacterial, especially Mycobacterium tuberculosis, anti-inflammatory and tissue repair advantages of the antimicrobial peptide LL-37, and the ability of PK34 to specifically identify the surface glycolipids of Mycobacterium tuberculosis and effectively kill Mycobacterium tuberculosis, these two characteristics enable the fusion antimicrobial peptide LL-37 / PK34 to specifically bind to intracellular mycobacteria, thereby achieving the purpose of effectively targeting and killing intracellular Mycobacterium tuberculosis. This not only overcomes the disadvantage that antimicrobial drugs are difficult to reach the target, but also avoids the instability of a single antimicrobial peptide, thereby reducing costs and toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HPLC and MS diagrams of the antimicrobial peptides of the present invention;

[0030] Figure 2 The Western blot results of the antimicrobial peptides PK34, LL-37, and PK34 / LL-37 of the present invention are shown in FIG.

[0031] Figure 3 CCK-8 assay for cell viability after co-culturing antimicrobial peptides and macrophages in the present invention

[0032] 1: 0μg / mL; 2: 0.625μg / mL; 3: 1.25μg / mL; 4: 2.5μg / mL; 5: 5μg / mL; 6: 10μg / mL; 7: 20μg / mL; 8: 40μg / mL; 9: 80μg / mL. a, d: antimicrobial peptide PK34 co-culture group; b, e: antimicrobial peptide LL-37 co-culture group; c, f: fusion antimicrobial peptide PK34 / LL-37 co-culture group; *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 4 The cell viability of A549 cells after the antimicrobial peptide was detected by CCK8 in the present invention

[0034] *P<0.05, **P<0.01,***, #P<0.001;

[0035] Figure 5 The results of the antimicrobial peptide and rifampicin anti-tuberculosis experiment of the present invention are shown in FIG.

[0036] a: control group; b: antimicrobial peptide PK34 group; c: antimicrobial peptide LL-37 group; d: antimicrobial peptide PK34 / LL-37 group; e: rifampicin group; 0: zero adjustment group; 1: 160 μg / mL; 2: 80 μg / mL; 3: 40 μg / mL; 4: 20 μg / mL; 5: 10 μg / mL; 6: 5 μg / mL; 7: 2.5 μg / mL;

[0037] Figure 6 ELISA detection of antimicrobial peptides PK34, LL-37, and PK34 / LL-37 regulating changes in LPS-induced macrophage inflammatory factors in the present invention

[0038] *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 7 Acid-fast staining of Mtb in the present invention

[0040] a: Mtb acid-fast staining; b: Mtb-infected RAW264.7 macrophage acid-fast staining;

[0041] Figure 8 Acid-fast staining of RAW264.7 macrophages infected with Mtb treated with antimicrobial peptides according to the present invention

[0042] a: normal group; b: infection group; c: antimicrobial peptide PK34; d: antimicrobial peptide LL-37; e: antimicrobial peptide PK34 / LL-37; f: rifampicin; *P<0.05, **P<0.01, ***P<0.001;

[0043] Fig. 9 Colony count of RAW264.7 macrophages infected with Mtb treated with antimicrobial peptides according to the present invention

[0044] a: normal group; b: infection group; c: antimicrobial peptide PK34; d: antimicrobial peptide LL-37; e: antimicrobial peptide PK34 / LL-37; f: rifampicin; *P<0.05, **P<0.01, ***P<0.001;

[0045] Fig.10 RT-PCR detection of cytokines in RAW264.7 macrophages infected with Mtb treated with antimicrobial peptides in the present invention a: normal group; b: infection group; c: antimicrobial peptide PK34; d: antimicrobial peptide LL-37; e: antimicrobial peptide PK34 / LL-37; f: rifampicin; *P<0.05, **P<0.01, ***P<0.001;

[0046] Fig.11 ELISA detection of cytokines in RAW264.7 macrophages infected with Mtb treated with antimicrobial peptides according to the present invention

[0047] a: normal group; b: infection group; c: antimicrobial peptide PK34; d: antimicrobial peptide LL-37; e: antimicrobial peptide PK34 / LL-37; f: rifampicin; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0048] The specific implementation manner will be further described below in conjunction with the accompanying drawings.

[0049] The reagents and instruments in the following examples are all conventional experimental reagents and instruments.

[0050] Embodiment 1:

[0051] A fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, characterized in that: the fusion antimicrobial peptide is composed of PK34 and LL-37, and its amino acid sequence is shown in SEQ ID NO: 1:

[0052] PRVIETKVHGREVTGLARNVSEENVDRLAKRWIKLLGDFFRKSKEKIGKEFKRIVQRIKDFL

[0053] RNLVPRTES

[0054] A method for preparing a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, wherein the fusion antimicrobial peptide is synthesized by a solid phase synthesis method, and the synthesis steps include:

[0055] (1) 2-Chlorotrityl Chloride Resin was placed in a reaction tube, 15 ml / g of MF was added, and the mixture was shaken for 60 min to allow the resin to swell. The solvent was filtered off through a sand core, and a 3-fold molar excess of Fmoc was added to protect the first amino acid at the C-terminus. Then, a 10-fold molar excess of DIEA was added. Finally, DMF was added to dissolve the mixture, and the mixture was shaken for 30 min. The mixture was capped with methanol for 30 min, and the first amino acid was connected.

[0056] (2) Remove DMF, add 20% piperidine DMF solution at a concentration of 15 ml / g, remove it after 5 minutes, and then add 20% piperidine DMF solution at a concentration of 15 ml / g for 15 minutes to achieve deprotection; Detection: Draw off the piperidine solution, take a dozen resins, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105°C-110°C for 5 minutes, and turn dark blue for a positive reaction.

[0057] Use 10ml / g DMF, methanol, and DMF to wash twice respectively; add 3 times molar excess of Fmoc protected amino acid, then add 3 times molar excess of HBTU and 10 times molar excess of DIEA, then add DMF to dissolve, and shake for 45 minutes; detection: take a dozen resins, wash three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol solution, heat at 105℃-110℃ for 5 minutes, colorless is a negative reaction.

[0058] The product was washed once with 10 ml / g of DMF, twice with 10 ml / g of methanol, and twice with 10 ml / g of DMF.

[0059] (3) Repeat step (2) to connect the amino acids in SEQ ID NO: 1 from right to left until the Fmoc protecting group of the last amino acid is removed. Wash the resin with 10 ml / g DMF twice, 10 ml / g DCM three times, and 10 ml / g methanol four times, and drain for 10 min.

[0060] (4) Prepare a cutting solution with the following volume percentages: TFA 95%, water 2%, EDT 2%, and TIS 1%. Cut for 180 min, blow the lysate dry with nitrogen as much as possible, precipitate ether, remove the supernatant by centrifugation, wash the precipitate with ether six times, and then evaporate to dryness at room temperature.

[0061] (5) Take a small amount of crude product, dissolve it in H2O / ACN, analyze it on an HPLC analyzer to determine the peak time corresponding to the target peak, and use a C18 reverse phase chromatography preparation system: Wavelength: 220nm; Flow Rate: 15ml / min; Inj.Vol: 20mL Column Temp: 25℃; Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution, and lyophilize the qualified target peak solution to obtain the fusion antimicrobial peptide.

[0062] Identification: Take a small amount of the finished peptide for molecular weight identification by MS and purity identification by HPLC. The powdered peptide is sealed and stored at -20 degrees.

[0063] At the same time, the antimicrobial peptides PK34 and LL-37 were synthesized by solid phase synthesis.

[0064] Experimental results:

[0065] 1. Purity and molecular weight detection of antimicrobial peptides

[0066] The antimicrobial peptides PK34, LL-37, and PK34 / LL-37 were synthesized by solid phase synthesis. The peaks of the synthetic antimicrobial peptides were symmetrical by HPLC, and the purity was as high as 98.14% ( Figure 1 a) 98.53%( Figure 1 b) 98.81% ( Figure 1 c). In addition, the molecular weight of the synthetic PK34 was determined by MS analysis to be 660.71 [M+6H]6+ (theoretical molecular weight is 3957.56) ( Figure 1 d), LL-37 molecular weight is 750.3 [M + 6H] 6 + (theoretical molecular weight is 4493.33) ( Figure 1 e), PK34 / LL-37 molecular weight is 894.6 [M+10H]10+ (theoretical molecular weight is 8935.37) ( Figure 1 f), which is consistent with the theoretical molecular weight, indicating that the amino acid sequence of the synthetic antimicrobial peptide is accurate.

[0067] 2. Western blot identification of antimicrobial peptides

[0068] The synthesized antimicrobial peptides were subjected to Western blot with the corresponding polyclonal antibodies of PK34 and LL-37. Figure 2 Both expressed proteins could bind to their corresponding polyclonal antibodies and were successfully identified by Western blot.

[0069] Embodiment 2:

[0070] The invention discloses an application of a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis. The fusion antimicrobial peptide is used for non-therapeutic extracellular killing of Mycobacterium tuberculosis, extracellular promotion of proliferation of macrophages or A549 cells, or promotion of expression of IL-2 and IL-10 in macrophages, and inhibition of expression of inflammatory factors TNF-α and IP-10.

[0071] 1. Cell Culture

[0072] (1) RAW264.7 cell recovery

[0073] ①Take out the cells from the liquid nitrogen tank.

[0074] ②Incubate in a 37℃ water bath.

[0075] ③After rewarming, aspirate the cell suspension into a 15 mL centrifuge tube.

[0076] ④Centrifuge at 800rpm for 3 minutes.

[0077] ⑤Discard the supernatant and add fresh culture medium to resuspend.

[0078] ⑥ Transfer to a culture bottle and culture in a cell culture incubator.

[0079] (2) A549 cell recovery

[0080] ① Add 8 mL of 1640 complete medium to a 15 mL centrifuge tube.

[0081] ②Take out the cells from -80℃ and immediately freeze and thaw them in a 37℃ water bath with shaking.

[0082] ③Add the cells to the culture medium prepared in advance.

[0083] ④800rpm, 3min, discard the supernatant as much as possible.

[0084] ⑤ Add 3 mL of fresh 1640 complete medium and mix by gently pipetting.

[0085] ⑥ Move to a clean culture bottle and culture in a cell culture incubator.

[0086] ⑦ After two days of culture, if the cells have not adhered to the wall or are in poor condition, they need to be revived.

[0087] 2. CCK-8 assay to detect the effect of antimicrobial peptides on host cell viability

[0088] (1) Cell inoculation: Prepare a cell suspension and inoculate RAW264.7 cells at a concentration of 1×10 4 / mL was inoculated into 96-well plates, 100 μL per well, and drugs were added the next day. The cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0089] (2) Experimental groups: blank group (culture medium + CCK-8), control group (cells + culture medium + water + CCK-8), experimental group (cells + culture medium + antimicrobial peptide + CCK-8).

[0090] (3) Antimicrobial peptide co-incubation: Antimicrobial peptides PK-34, LL-37, and PK34 / LL-37 were added the next day after plating. Antimicrobial peptide concentrations were 0 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL. Where n=3. This time was recorded as co-incubation time 0. OD values ​​were detected after 24 h, 48 h, and 72 h of co-incubation with antimicrobial peptides.

[0091] (4) Add CCK-8 to detect host cell viability: After the cell incubation is completed, the cell medium is replaced, and 10 μL of CCK-8 reagent is added to each well under light-proof conditions, and the reaction is carried out in a cell culture incubator for 4 h.

[0092] (5) Detection: The absorbance value of each group of samples at 450 nm was detected by an ELISA instrument, and the analysis data was recorded and analyzed to calculate the macrophage survival rate. Cell survival rate (%) = [OD (experimental) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0093] Experimental results:

[0094] The effects of antimicrobial peptides PK34, LL-37, and PK34 / LL-37 on macrophage viability were determined using the classic CCK-8 assay. The cell viability after 24h, 48h, and 72h of action of the three antimicrobial peptides at different concentrations is shown in the figure ( Figure 3 a, b, c). At 24h of incubation, there was no significant difference in the effect of different concentrations of antimicrobial peptide PK34 on cell proliferation, and there was no statistical significance between the concentrations. However, at 48h of incubation, with the increase in the concentration of antimicrobial peptide PK34, the macrophage viability showed a downward trend, and the higher the concentration, the more obvious the decline. When the concentration of antimicrobial peptide PK34 reached 80μg / mL (90.32±2.13)%, the macrophage viability decreased significantly compared with the control group (100.00±6.49)% (P<0.001). At 72h of incubation, the macrophage viability of the experimental group was lower than that of the control group (100.00±1.98)%, especially when the antimicrobial peptide concentration was 80μg / mL (72.60±11.49)%. The cell viability was the lowest. Pairwise comparisons between the groups were statistically significant (P<0.001) ( Figure 3 a). According to the entire time change curve, it can be seen that the antimicrobial peptide PK34 reduces the activity of macrophages in a concentration-dependent manner. The higher the concentration of antimicrobial peptide PK34, the more obvious the decrease in macrophage activity ( Figure 3d). However, the antimicrobial peptide LL-37 showed a completely different effect. With the addition of the antimicrobial peptide LL-37, the macrophage viability was significantly increased compared with the control group without the addition of the antimicrobial peptide. Unlike PK34, at 24h of incubation, with the increase of LL-37 concentration, the macrophage viability showed an upward trend, but the trend was not very significant. At 48h of incubation, the macrophage viability of the experimental group increased significantly. Compared with the control group (100.00±4.11)%, the macrophage viability increased with the increase of LL-37 concentration, but when the concentration of LL-37 reached 20μg / mL (218.34±21.12)%, the cell viability reached the maximum. When the concentration of LL-37 was 80μg / mL (190.31±31.53)%, the cell viability decreased instead (P<0.001). LL-37 was incubated for 72h, and the cell viability decreased significantly compared with that at 48h, which may be related to the large consumption of nutrients in the culture medium. However, at 72 h, the overall trend of cell viability was similar to that at 48 h. The cell viability was the highest at a concentration of 20 μg / mL (167.67±10.39)%, and then the cell viability showed a downward trend with increasing concentration (P<0.001)( Figure 3 b). In addition, the absorbance ratio curve showed that the addition of LL-37 could increase the viability of macrophages. When the concentration of LL-37 was 20 μg / mL, the increase in cell viability was most obvious ( Figure 3 e). The antimicrobial peptide PK34 / LL-37 showed the same effect as LL-37 in affecting cell viability. After 48 hours of incubation, the macrophage viability of the PK34 / LL-37 concentration of 20μg / mL (209.34±32.93)% was still on the rise compared with the control group (100.00±4.82)%, and then gradually decreased with the increase in concentration (P<0.001). Regarding the phenomenon of overall decrease in cell viability after 72 hours, we considered that it was due to the lack of nutrients. However, when the antimicrobial peptide concentration was 20μg / mL (164.22±21.15)%, the cell viability was still higher than that of other concentration groups (P<0.001) ( Figure 3 c). The results of the cell absorbance change curve are roughly the same as this result ( Figure 3 f). The experiment proved that the antimicrobial peptide PK34 affected cell proliferation in a concentration-dependent manner, while the antimicrobial peptide LL-37 and the fusion antimicrobial peptide PK34 / LL-37 promoted cell proliferation.

[0095] 3. CCK8 assay to detect the effect of antimicrobial peptides on A549 cell viability

[0096] (1) Experimental groups: blank group (culture medium + CCK-8), control group (cells + culture medium + water + CCK-8), experimental group (cells + culture medium + antimicrobial peptide + CCK-8).

[0097] (2) Cell inoculation: A549 cells were plated at 1×10 4 / mL, 100 μL per well was evenly inoculated into a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator.

[0098] (3) Co-incubation with antimicrobial peptides: PK-34, LL-37, and PK34 / LL-37 were added to the wells at concentrations of 0 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, respectively. Each well was tested in triplicate and incubated for 24 h, 48 h, and 72 h before detection.

[0099] (4) CCK-8 detection of cell viability: After the co-incubation, the culture medium in the wells was discarded, and 100 μL of culture medium containing 10% CCK8 was added to each well in a dark place and placed in a cell culture incubator for 4 h.

[0100] (5) Detection: Detect the OD value of each well at 450 nm, and analyze and compare the effects of different types, concentrations of antimicrobial peptides and different incubation times on the viability of A549 cells. Cell viability (%) = [OD (experimental) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0101] Experimental results:

[0102] like Figure 4As shown in Figures A, B, and C, different statistical graphs and different angles were used to analyze and compare the cell survival rates of antimicrobial peptides LL-37, PK34, and PK34 / LL-37 at different concentrations after acting on A549 cells for 24h, 48h, and 72h, respectively. As shown in Figure A, when acting for 24h, the three antimicrobial peptides with concentrations of 0.625μg / mL, 1.25μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, and 40μg / mL did not inhibit the viability of A549 cells, and when the concentration was 20μg / mL, PK34 could increase the cell survival rate compared with the control group, which was statistically significant (P<0.01); at a high concentration of 80μg / mL, antimicrobial peptides LL-37 and PK34 / LL-37 could reduce cell viability, which was statistically significant (P<0.001). However, when the antimicrobial peptides acted for 48 hours, all concentrations of LL-37 inhibited cell viability, while the antimicrobial peptides PK34 and PK34 / LL-37 did not affect cell viability. Similarly, when the antimicrobial peptides acted for 72 hours, LL-37 affected cell viability from 0.125 μg / mL, and the effect of PK34 on cells was the same as that at 48 hours, and it also did not affect cell viability. At 72 hours, the effect of the fusion antimicrobial peptide PK34 / LL-37 on cell viability did not decrease but increased. Starting from 10 μg / mL, the effect of PK34 / LL-37 on cell viability gradually increased. Figure B shows that when the action time was 24 hours and 48 hours, the cell viability of the PK34 group was the highest, followed by PK34 / LL-37, and the lowest was LL-37. When the concentration was 2.5μg / mL, 5μg / mL, and 10μg / mL, the cell viability of each group was still the same as that of 24h and 48h: the cell viability of the PK34 group was still the highest, followed by PK34 / LL-37, and the lowest was LL-37. At the lowest concentrations of 0.625μg / mL and 1.25μg / mL, LL-37 had little effect on cell viability, while at 20μg / mL, 40μg / mL, and 80μg / mL, the cell viability of the PK34 / LL-37 group was significantly higher than that of LL-37 and PK34. In Group C, the cell growth curves of the three antimicrobial peptides at different concentrations were compared with the cell growth curves of the control group, and the effects of the three antimicrobial peptides on cell proliferation at different concentrations were clearly shown.As shown in Figure C, after most concentrations of antimicrobial peptide LL-37 acted on A549 cells, their cell growth curves were all below the control curve, indicating that LL-37 had an inhibitory effect on the proliferation of A549 cells. The cell growth curves of antimicrobial peptide PK34 at various concentrations were close to the cell growth curve of the control group, and there was not much difference, indicating that PK34 had basically no effect on the growth of A549 cells. On the contrary, the fusion antimicrobial peptide PK34 / LL-37 showed that the cell growth curves of 10μg / mL, 20μg / mL, 40μg / mL, and 80μg / mL PK34 / LL-37 were higher than those of the control group, indicating that the fusion antimicrobial peptide had the effect of promoting cell proliferation. In addition, Figure C also shows that the cell survival rate at 72h was lower than that at 48h, which is speculated to be caused by the exhaustion of nutrients in the cell culture medium and the lack of nutrition in the cells. In summary, from the perspective of long-term efficacy, the antimicrobial peptide LL-37 has an inhibitory effect on the proliferation of A549 cells, the antimicrobial peptide PK34 has basically no effect on the growth of A549 cells, and the fusion antimicrobial peptide PK34 / LL-37 at 10μg / mL, 20μg / mL, 40μg / mL, and 80μg / mL has a promoting effect on the proliferation of A549 cells.

[0103] 4. Extracellular antibacterial assay

[0104] (1) Preparation of bacterial suspension

[0105] Aseptically take 2-3 single colonies of Mtb strains into a 1.5mL EP tube, add 1mL of 7H9 medium containing 0.05% Tween-80 and 10% OADC, and repeatedly aspirate with a 1mL sterile syringe until there are no obvious bacterial clumps to the naked eye, and collect the supernatant after settling at 4℃ overnight. Measure the OD600 value of the bacterial solution, the number of bacteria / mL = OD600×108×dilution factor.

[0106] (2) Experimental groups: zero adjustment group (7H9 culture medium), control group (Mtb + culture medium), PK34 group (Mtb + PK34 + culture medium), LL-37 group (Mtb + LL-37 + culture medium), PK34 / LL-37 group (Mtb + PK34 / LL-37 + culture medium). The experiment was repeated three times.

[0107] (3) Inoculation: Mtb at 5×10 5 CFU / mL were inoculated in a 96-well plate, and 100 μL of bacterial suspension was added to each well.

[0108] (4) Antimicrobial peptide infection: Add the drug directly after plating. Adjust the concentration of antimicrobial peptide to between 0.625-160 μg / mL, and perform duplicate wells for each concentration.

[0109] (5) Incubate at 37°C for 7 days, add 30 μL of 0.02% resazurin solution to the control group, and incubate at 37°C for another 24 h. If the zeroing group turns pink, add 30 μL of 0.02% resazurin solution to all experimental wells and incubate for another 24 h.

[0110] (6) The concentration of antimicrobial peptide at which the culture medium does not turn pink is the minimum inhibitory concentration (MIC).

[0111] Experimental results:

[0112] The results of the determination of the minimum inhibitory concentration of antimicrobial peptides PK34, LL-37, PK34 / LL-37 and rifampicin against tuberculosis are shown in the figure ( Figure 5 ): After adding resazurin to the zeroing group, the culture medium did not turn pink, proving that there was no contamination and the experimental results were credible. After adding resazurin solution to the control group, the culture medium turned red, indicating that Mycobacterium tuberculosis grew actively. When the concentration of antimicrobial peptide PK34 was 10μg / mL, the culture medium did not turn red, proving that it had an antibacterial effect. When the concentration of antimicrobial peptide LL-37 was lower than 40μg / mL, the culture medium turned red and had no antibacterial effect. When the concentration was 20μg / mL, the culture medium containing resazurin in the antimicrobial peptide PK34 / LL-37 group did not turn red, and it was able to inhibit bacteria, and the antibacterial effect increased with the increase of the concentration of antimicrobial peptides. From the in vitro antibacterial experiment, it can be seen that the minimum inhibitory concentration of rifampicin is 1μg / mL, the minimum inhibitory concentration of antimicrobial peptide PK34 is 10μg / mL, and the MIC concentrations of antimicrobial peptide LL-37 and antimicrobial peptide PK34 / LL-37 are 40μg / mL and 20μg / mL, respectively.

[0113] 5. Effect of antimicrobial peptides on regulating LPS-induced inflammatory factors in RAW264.7 macrophages

[0114] (1) Cell inoculation: RAW264.7 macrophages were seeded at 20×10 4 / mL was inoculated into a 24-well plate, 500 μL of culture medium was added to each well, and cultured overnight in a cell culture incubator. Infection was carried out the next day.

[0115] (2) Cell treatment: On the day after plating, antimicrobial peptides PK34, LL-37, and PK34 / LL-37 were added to the corresponding culture medium at a concentration of 20 μg / mL. LPS was added 2 hours later to stimulate macrophage differentiation. This time was recorded as time 0, and the cell supernatant was collected 8 hours, 16 hours, and 24 hours after infection.

[0116] (3) Experimental groups: blank + solvent, blank + LPS, PK34 + solvent, PK34 + LPS, LL-37 + solvent, LL-37 + LPS, PK34 / LL-37 + solvent, PK34 / LL-37 + LPS. The solvent group was added with the same amount of sterile water as the LPS group. n = 3.

[0117] (4) After 8 h, 16 h, and 24 h of treatment, the cell supernatant was collected and centrifuged for detection of cytokines using ELISA.

[0118] Experimental results:

[0119] Two hours after the addition of antimicrobial peptides, LPS was added to induce macrophages, and the supernatants were collected at different times. The cytokine levels were detected by ELISA ( Figure 6 ). Data analysis found that after LPS treatment of macrophages for 16 hours, antimicrobial peptides LL-37 and PK34 / LL-37 could inhibit the expression of inflammatory factors TNF-α and IP-10 ( Figure 6 A, B), exerted a good anti-inflammatory effect, and the effect of the fusion antimicrobial peptide PK34 / LL-37 was better than that of LL-37 (P < 0.05), while PK34 could not inhibit the expression of these two factors, but promoted the expression of TNF-α and IP-10 under the synergistic stimulation of LPS. Antimicrobial peptides LL-37 and PK34 / LL-37 can stimulate the expression and release of IL-2 in macrophages RAW264.7 before LPS treatment ( Figure 6 C), and thus can resist the inhibitory effect of LPS on IL-2 after stimulating macrophages RAW264.7, thereby improving the immune capacity of cells. Antimicrobial peptides PK34, LL-37, and PK34 / LL-37 can all increase the expression level of IL-10 in macrophages RAW264.7 ( Figure 6 D), especially the antimicrobial peptides LL-37 and PK34-LL37, can also promote the increase of IL-10 levels before LPS stimulation. After 16 hours of LPS treatment, the three antimicrobial peptides will further enhance the release of anti-inflammatory factor IL-10, which has a significant anti-inflammatory effect.

[0120] Example 3

[0121] The invention discloses an application of a fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis. The fusion antimicrobial peptide is used for non-therapeutic intracellular killing of Mycobacterium tuberculosis and intracellular inhibition of the expression of TNF-αmRNA and IL-6mRNA.

[0122] 1. Preparation of Mtb bacterial suspension and establishment of Mtb infection RAW264.7 macrophage model

[0123] (1) Preparation of bacterial suspension.

[0124] (2) Acid-fast staining to determine the uniformity of bacterial suspension

[0125] Take a drop of Mtb bacterial suspension and drop it on a clean glass slide. After flame fixation, stain it with acid fast. After staining, observe it under a microscope and take pictures.

[0126] (3) Preparation of infected macrophage model

[0127] Cell inoculation: RAW264.7 macrophages were cultured at 20 × 10 4 / mL was inoculated into 12-well plates and infected the next day. The cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0128] Cell infection: Inoculated cells were incubated with Mtb bacterial suspension at an infection multiplicity of 10, and the medium was changed after 6 hours of infection. The cells were washed with PBS three times to remove the unphagocytosed Mtb bacterial suspension, which was recorded as infection time 0.

[0129] 2. Preparation of parasite suspension and establishment of RAW264.7 macrophage model of parasite infection

[0130] (1) Preparation of insect suspension

[0131] Use an inverted microscope to observe the state of the worms. When the worms are in the active promastigote growth phase, perform the infection experiment. Take an appropriate amount of worm culture, centrifuge at room temperature at 2000×g for 5 minutes, and discard all the supernatant. Add an appropriate amount of DMEM medium to resuspend the worms and count them until the worm density reaches 10 8 .

[0132] (2) Preparation of infected macrophage model

[0133] Cell seeding.

[0134] Cell infection: According to the results of the preliminary experiment, the inoculated cells were co-incubated with the prepared parasite suspension at an infection multiplicity of MOI = 10. After 6 hours of infection, the cells were washed three times with serum-free DMEM medium to remove the unphagocytosed parasites. Fresh serum-containing DEME medium was added for further infection, which was recorded as infection time 0. After 24 hours of infection, the three antimicrobial peptides were added to the corresponding culture medium at a concentration of 20 μg / mL.

[0135] Experimental results:

[0136] Before cell infection, the supernatant of the precipitated Mtb bacterial suspension was taken, and acid-fast staining and microscopic examination were performed, and Mtb was uniformly distributed ( Figure 7 a); After the cells were infected, they were again examined by acid-fast staining, and the number and distribution of cells phagocytosed bacteria met the requirements ( Figure 7 b).

[0137] 3. Effect of exogenous antimicrobial peptides on the survival of Mtb in RAW264.7 macrophages

[0138] (1) Acid-fast staining

[0139] Cell seeding: RAW264.7 macrophages at 20×10 4 / mL was inoculated into a 24-well plate, 500μL of culture medium was added to each well, and infection was carried out the next day. The 24-well plate should be built with a slide for acid-fast staining. Cultured overnight in a cell culture incubator.

[0140] Cell infection: The day after plating, inoculated cells were co-incubated at a multiplicity of infection of MOI = 10. After 6 hours of infection, the medium was changed and washed three times with PBS to remove unphagocytosed Mtb. This time was recorded as infection time 0. Experimental detection was performed 24 hours after infection.

[0141] Dosing: Dosing began at infection time 0. Referring to the experimental results of CCK-8, three antimicrobial peptides, PK34, LL-37, and PK34 / LL-37, were added to the corresponding culture medium at a concentration of 20 μg / mL, and rifampicin was added to the corresponding culture medium at a concentration of 1 μg / mL.

[0142] Experimental groups: a normal group, b infection group, c antimicrobial peptide PK34 group, d antimicrobial peptide LL-37 group, e antimicrobial peptide PK34 / LL-37 group, f rifampicin group, n=3.

[0143] The slides in the 24-well plate were taken out, dried, flame-fixed, and acid-fast stained. They were observed under a microscope and photographed.

[0144] (2) Colony CFU count

[0145] After 24 hours of infection, the supernatant was discarded and the cells were washed with PBS three times for 3 minutes each time. 200 μL of 0.1% Triton X-100 was added to each well to lyse the cells. After the cells were completely lysed, the cells were diluted in a gradient manner and 100 μL was inoculated in Roche culture medium. The cells were cultured in a 37°C constant temperature incubator for 21 days and then the colonies were counted.

[0146] Experimental results:

[0147] The change in the survival rate of Mtb in infected cells is an important indicator of the effect of antimicrobial peptides on host cells. Three antimicrobial peptides and the drug rifampicin were added to RAW264.7 macrophages infected with Mtb, and the effect of exogenous antimicrobial peptides on the survival of Mtb in infected macrophages was investigated by acid-fast staining and colony counting experiments. The experimental results showed that compared with the infection group (176.33±12.50), the number of intracellular Mtb in the three antimicrobial peptides decreased to varying degrees. The number of intracellular Mtb in the antimicrobial peptide PK34 group (80.67±7.51) was slightly higher than that in the antimicrobial peptide LL-37 group (57.33±13.58), which was statistically significant (P<0.05); the results of the antimicrobial peptide PK34 / LL-37 group (64.67±4.73) and the rifampicin group (66.33±14.01) were similar, and both groups could cause a significant decrease in the number of Mtb in infected cells compared with the infection group (P<0.001), which was statistically significant ( Figure 8 ).

[0148] The experimental results of colony counting were slightly different from those of acid-fast staining. The colony numbers of the antimicrobial peptide PK34 group (4.48±0.80) and the antimicrobial peptide LL-37 group (5.53±0.94) were similar to those of the rifampicin group (5.15±0.23) (P>0.05), with no statistical significance. However, the colony number of the antimicrobial peptide PK34-LL37 group (3.90±0.20) was significantly lower than that of the antimicrobial peptide LL-37 group (P<0.01), and the antibacterial effect was consistent with the extracellular antibacterial results, showing stronger antibacterial ability ( Fig. 9 ).

[0149] 4. RT-PCR detection of changes in cytokine transcription levels in RAW264.7 macrophages infected with Mtb

[0150] (1) Extraction of total cell RNA

[0151] Cell seeding: RAW264.7 macrophages at 20×10 4 / mL was inoculated into a 12-well plate, 1mL per well, infected the next day, and cultured in a cell culture incubator overnight.

[0152] Cell infection: The day after plating, inoculated cells were co-incubated with an infection multiplicity of MOI = 10. The medium was changed 6 hours after infection, and the cells were washed 3 times with PBS to remove the unphagocytosed Mtb bacterial solution. This time was recorded as infection time 0. Experimental detection was performed 24 hours after infection.

[0153] Add medicine.

[0154] Experimental groups.

[0155] Extraction of cellular RNA: The experimental steps for RNA extraction refer to the instructions of Solebao "RNA Extraction Solution". The concentration of RNA and A260 / A280 were detected by spectrophotometer. The samples were packaged and stored in a -80℃ refrigerator.

[0156] cDNA synthesis.

[0157] (2) Real-time PCR detection

[0158] Fluorescence quantitative primer synthesis:

[0159] The nucleotide sequences of mouse cytokines TNF-α, IL-6, IL-10, and IL-12 were retrieved using GenBank, and primers were designed and synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.

[0160] Fluorescence quantitative PCR reaction:

[0161] 5. ELISA to detect changes in cytokine translation levels in RAW264.7 macrophages infected with Mtb

[0162] The cell supernatants of different experimental groups were collected respectively, and the expression levels of cytokines TNF-α, IL-6, IL-10, and IL-12 were detected according to the instructions of the ELISA kit. The OD450 was detected by a microplate reader, and the results were analyzed.

[0163] Experimental results:

[0164] To explore the effect of antimicrobial peptides on inflammatory factors in macrophages infected with Mtb, we used RT-PCR and ELISA experiments to detect the regulation of antimicrobial peptides on inflammatory factors in the RAW264.7 macrophage infection model. Compared with the normal group, the mRNA expression of TNF-α in the Mtb infection group increased by about 3 times (P < 0.001); after rifampicin was added to the infected cells, the expression of TNF-α mRNA decreased (P < 0.01); compared with the rifampicin group, the expression of TNF-α mRNA of antimicrobial peptide PK34 was slightly reduced (P < 0.01), while the results of antimicrobial peptide LL-37 and antimicrobial peptide PK34 / LL-37 were similar, and the effect of regulating the reduction of TNF-α mRNA was more significant (P < 0.001) ( Fig.10 A). The experimental results of ELISA were similar, and both showed that the expression of TNF-α increased after infection with Mtb, but the increase of TNF-α in the experimental group containing antimicrobial peptides was not obvious. Compared with rifampicin, antimicrobial peptides, antimicrobial peptide LL-37, could more significantly inhibit the increase of TNF-α mRNA expression (P < 0.01), and the difference was statistically significant ( Fig.11 A).

[0165] Compared with the normal group, the expression of IL-6 mRNA in the Mtb infection group increased significantly (P < 0.001). After adding the antimicrobial peptide PK34, the change of IL-6 mRNA was not obvious, and the difference was not statistically significant. However, the expression of IL-6 mRNA in the rifampicin group decreased (P < 0.05), and the decrease of IL-6 mRNA in the LL-37 group and the PK34 / LL-37 group was more obvious, which more significantly inhibited the increase of IL-6 mRNA expression caused by infection (P < 0.01), which was statistically significant ( Fig.10 B). In the ELISA experimental results, the addition of antimicrobial peptides and rifampicin can inhibit the increase in IL-6 expression caused by infection. Among them, LL-37 has the most significant effect in inhibiting IL-6 expression (P < 0.001), and the difference is statistically significant ( Fig.11 B).

[0166] After infection with Mtb, the expression of IL-10mRNA increased by nearly 20 times. The expression of IL-10mRNA in the antimicrobial peptide PK34 group was similar to that in the infection group, and could not change the increase in IL-10mRNA expression caused by infection, and the difference was not statistically significant. Compared with the infection group, the addition of the antimicrobial peptide LL-37 could stimulate the expression of IL-10mRNA (P < 0.001). Although the antimicrobial peptide PK34 / LL-37 and rifampicin group could also cause an increase in IL-10mRNA expression, there was no significant difference between them and the infection group ( Fig.10 C). At the translation level, the IL-10 expression level in the antimicrobial peptide LL-37 group was still significantly higher than that in the infection group (P < 0.001), and the IL-10 expression level was similar to that in the antimicrobial peptide PK34 / LL-37 group, with no statistically significant difference ( Fig.11 C).

[0167] The results of RT-PCR and ELISA experiments showed that after infection with Mtb, the expression level of IL-12 in cells increased. However, the expression level of IL-12 mRNA in the experimental group with the addition of antimicrobial peptides and rifampicin was significantly reduced ( Fig.10 D). However, in the ELISA test, the IL-12 concentrations in each experimental group were similar, only 1 / 2 of that in the infection group. The differences between the experimental groups were not obvious and had no statistical significance ( Fig.11 D).

[0168] The a-helical structure of LL-37 in the fusion polypeptide of the present invention can lead PK34 to enter macrophages through the cell membrane. Intracellularly, PK34 leads LL-37 to specifically recognize and bind to TDM on the surface of tuberculosis bacteria and destroy bacterial activity. LL-37 in the fusion polypeptide can be inserted into the lipid bilayer of the bacterial cell membrane to destroy the integrity of the bacterial cell membrane. At the same time, LL-37 in the fusion polypeptide also has an immunomodulatory effect, limiting inflammation to the lesion site.

[0169] The fusion antimicrobial peptide PK34 / LL-37 of the present invention has significant advantages in targeted bactericidal killing. It has the advantages of both LL-37 and PK34 polypeptides, can specifically bind to the surface of Mycobacterium tuberculosis, and achieve the effect of targeted killing of latent Mycobacterium tuberculosis in macrophages outside cells and inside macrophages. It has powerful effects in eliminating pathogens and inhibiting inflammation, and has a stronger effect in promoting cell proliferation.

[0170] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis, characterized by: The fusion antimicrobial peptide consists of PK34 and LL-37, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The fusion antimicrobial peptide is synthesized by solid phase synthesis, and the synthesis steps include: (1) 2-Chlorotrityl Chloride Resin was placed in a reaction tube, DMF was added, and the reaction was shaken for 60 min to allow the resin to swell. The solvent was filtered out through a sand core, and an excess of Fmoc was added to protect the first amino acid at the C-terminus. Then, an excess of DIEA was added, and finally, DMF was added to dissolve the mixture, and the mixture was shaken for 30 min. The mixture was capped with methanol for 30 min, and the first amino acid was connected. (2) Remove DMF, add 20% piperidine DMF solution, remove after 5 minutes, and then add 20% piperidine DMF solution for 15 minutes to achieve deprotection; wash with DMF, methanol, and DMF twice each in sequence; Add Fmoc protected amino acid, then add HBTU, DIEA and DMF, shake for 45 min; wash once with DMF, twice with methanol, and twice with DMF; (3) Repeat step (2) to connect the amino acids in SEQ ID NO: 1 from right to left until the Fmoc protecting group of the last amino acid is removed. Wash the resin with DMF twice, DCM three times, and methanol four times, and drain for 10 min. (4) Using TFA; water; EDT; TIS prepared cutting solution, cut for 180 min, blow the lysate dry with nitrogen as much as possible, precipitate ether, centrifuge to remove supernatant, wash the precipitate with ether six times, and then evaporate to dryness at room temperature; (5) Take a small amount of the crude product, dissolve it in H2O / ACN, analyze it on an HPLC analyzer to determine the peak time corresponding to the target peak, collect the target peak solution, and freeze-dry the qualified target peak solution to obtain the fusion antimicrobial peptide.

3. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The specific steps of (1) include: Put 2-Chlorotrityl Chloride Resin into a reaction tube, add 15 ml / g of MF, shake for 60 min to make the resin swell, filter out the solvent through a sand core, add 3 times molar excess of Fmoc to protect the first amino acid at the C-terminus, then add 10 times molar excess of DIEA, finally add DMF to dissolve, shake for 30 min, use methanol to cap the tube for 30 min, and connect the first amino acid.

4. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The specific steps of (2) include: Remove DMF, add 20% piperidine DMF solution at a concentration of 15 ml / g, remove after 5 minutes, and then add 20% piperidine DMF solution at a concentration of 15 ml / g for 15 minutes to achieve deprotection; wash twice with 10 ml / g of DMF, methanol, and DMF respectively; add 3-fold molar excess of Fmoc-protected amino acid, then add 3-fold molar excess of HBTU and 10-fold molar excess of DIEA, then add DMF to dissolve, and shake for 45 minutes; wash once with 10 ml / g of DMF, wash twice with 10 ml / g of methanol, and wash twice with 10 ml / g of DMF.

5. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The specific steps of (3) include: Repeat (2) to connect the amino acids in SEQ ID NO: 1 from right to left in sequence until the Fmoc protecting group of the last amino acid is removed. Wash the resin with 10 ml / g DMF twice, 10 ml / g DCM three times, and 10 ml / g methanol four times, and drain for 10 min.

6. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The specific steps of (4) include: The volume percentage is TFA 95%; Water 2%; EDT 2%; The cutting solution was prepared with 1% TIS, and the cutting was carried out for 180 min. The lysate was blown dry as much as possible with nitrogen, ether was precipitated, and the supernatant was removed by centrifugation. The precipitate was washed with ether for six times and then evaporated to dryness at room temperature.

7. The method for preparing the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, characterized in that: The specific steps of (5) include: Take a small amount of crude product, dissolve it in H2O / ACN, analyze it on HPLC analyzer to determine the peak time corresponding to the target peak, and use C18 reverse phase chromatography preparation system: Wavelength: 220nm; Flow Rate: 15ml / min; Inj.Vol: 20mL ColumnTemp: 25℃; Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; The target peak solution is collected, and the qualified target peak solution is freeze-dried to obtain the fusion antimicrobial peptide.

8. The use of the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, wherein the fusion antimicrobial peptide is prepared by the method according to any one of claims 2 to 7, characterized in that: The fusion antimicrobial peptide is used for non-therapeutic intracellular or extracellular killing of Mycobacterium tuberculosis.

9. The use of the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, wherein the fusion antimicrobial peptide is prepared by the method according to any one of claims 2 to 7, characterized in that: The fusion antimicrobial peptide is used for non-therapeutic extracellular promotion of the proliferation of macrophages or A549 cells, or for promoting the expression of IL-2 and IL-10 in macrophages, and inhibiting the expression of inflammatory factors TNF-α and IP-10.

10. The use of the fusion antimicrobial peptide for inhibiting Mycobacterium tuberculosis according to claim 1, wherein the fusion antimicrobial peptide is prepared by the method according to any one of claims 2 to 7, characterized in that: The fusion antimicrobial peptide is used for non-therapeutic intracellular inhibition of the expression of TNF-αmRNA and IL-6mRNA.

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