WO2016110177A1 - Alkaline antibacterial peptide and targeting design and use thereof - Google Patents

Abstract

Disclosed is an alkaline antibacterial peptide consisting of leucine and lysine. The alkaline antibacterial peptide comprises 12-24 amino acids, wherein the proportion of lysine is more than 33.3%. Also disclosed is a targeted antibacterial peptide formed by the connection of the alkaline antibacterial peptide and the complementarity determining region of an antibody through leucine. The alkaline antibacterial peptide and the targeted antibacterial peptide can be used as an antibacterial and for cancer resistance.

Description

Alkaline antimicrobial peptide and its targeted design and application Technical field

The present invention relates to the field of biotechnology, and more particularly to alkaline antimicrobial peptides and their targeted design and applications.

Background technique

Antibiotics are a class of natural or synthetic compounds that kill bacteria or inhibit the growth of bacteria. With the continuous development of science and technology, the definition of antibiotics has been continuously expanded, and anti-microbial compounds, including antifungal compounds, have been included in the scope of antibiotics.

Since the discovery of the first antibiotic penicillin in 1940 and its application in clinical practice, humans have embarked on a new era of antibiotic treatment. Many infectious diseases that have been seriously endangering human life and health have been effectively controlled by the use of antibiotics, and have greatly reduced the birth mortality rate and post-operative infection rate, and the average life expectancy of humans has been extended by 15 to 20 years. Therefore, a variety of antibiotics have become essential drugs in the treatment of most diseases.

However, it is followed by a serious problem caused by the abuse of antibiotics: antibiotic resistance (Drug resistance). Clinically, drug resistance refers to a decrease in sensitivity to chemotherapeutic drugs such as pathogens and cancer cells. The antibiotic resistance mainly refers to the phenomenon that when the microorganism is exposed to the antibiotic environment, it can still survive and reproduce. The reason for the emergence of drug resistance is that under the pressure of natural selection, the strain with the resistance gene will survive as the dominant strain. These resistance genes are usually present in the plasmid, and for microorganisms (especially bacteria), the resistance gene can be transferred and rapidly replicated by transformation, transduction, etc., so that one colony can quickly acquire resistance.

Studies have shown that it takes about a decade or more to develop a new type of antibiotic, while bacteria produce resistance for less than two years. The speed of development of new drugs is far behind the rate of bacterial resistance. Once there is a "superbug" with multiple resistance genes, people will have no medicine available. As early as 1976, Streptococcus pneumoniae was found to be resistant to penicillin. In the new superbug NDM-1 discovered in South Asia in 2010, all existing antibiotics have no effect on it. So far, the disease caused by NDM-1 has not found an effective treatment, and it has a tendency to spread and has attracted widespread attention.

Therefore, there is an urgent need for research and development of high-efficiency, low-toxicity, high-selective broad-spectrum antibacterial drugs to protect human health. Studies have shown that many organisms also have genes encoding antibiotics in their genomes. These genes encode mostly short peptides called antimicrobial peptides. Antibacterial peptides generally carry a positive charge, which has a The bacteria have strong activity and are not easy to produce drug resistance. Antibacterial peptides are generally 10 to 40 amino acids in length, often form membrane channels and often have hemolytic and toxic properties, and lack of targeted antibacterial and anticancer properties. If artificially designed antibacterial peptides, new antibiotic resources can be developed to effectively solve the current medical antibiotic resistance problem.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects of hemolytic, membrane permeability and toxicity which the conventional antimicrobial peptide often has, and to provide an alkaline antimicrobial peptide.

A second object of the present invention is to provide a targeted design method for the above basic antimicrobial peptide.

A third object of the present invention is to provide a targeted antimicrobial peptide designed from the above alkaline antimicrobial peptide, which can be targeted against cancer.

A fourth object of the present invention is to provide an application of the above basic antimicrobial peptide or a targeted antimicrobial peptide.

The object of the present invention is achieved by the following technical solutions:

An alkaline antimicrobial peptide consisting of leucine and lysine, the basic antimicrobial peptide having 12 to 24 amino acids, wherein the proportion of lysine is greater than 33.3% .

The basic amino acid (lysine) has a positive charge. Generally, it can bind negative ions such as chloride ions, and some hydrophobic amino acids can bind to the cell membrane, destroy the integrity of the cell membrane, and produce antibacterial and bactericidal effects by changing the membrane permeability. . There are also antibacterial peptides obtained by studying basic amino acids (lysine) and hydrophobic amino acids (leucine) in the prior art, but the design of the antibacterial peptide usually requires consideration of the secondary structure of the peptide, and the secondary structure is also It will affect the activity of the finally synthesized alkaline antimicrobial peptide, which not only increases the synthesis cost of the antimicrobial peptide, but also affects the practical application of the antimicrobial peptide; in addition, the alkaline antimicrobial peptide synthesized by the prior art usually forms a membrane channel, and more It is hemolytic and is not safe to the human body.

The ratio of the basic amino acid of the antimicrobial peptide obtained by the invention is greater than 33.3%; at the same time, the alkaline antimicrobial peptide of the present invention is a non-high permeability type peptide (usually low permeability type) and does not cleave human red blood cells. , no hemolysis activity, generally does not form a high-efficiency membrane channel, so it is safe for the human body.

Applicants have found through extensive screening and research that when the hydrophobic amino acid is placed at one end of the antimicrobial peptide, the antibacterial effect of the alkaline antimicrobial peptide is very good; specifically, the hydrophobic amino acid is located at one end of the alkaline antimicrobial peptide, and the hydrophobic amino acid thereof The number is 3-8.

The basic antibacterial peptide of the invention has the composition of trillions, and the design of the antibacterial peptide does not need to consider the secondary structure and physicochemical properties of the amino acid, as long as the amino acid composition and the high basic amino acid ratio are maintained, the activity can be formed high. Alkaline antimicrobial peptide.

Preferably, the amino acid sequence of the basic antimicrobial peptide is as shown in SEQ ID NOS: 1 to 23.

More preferably, the sequence of the basic antimicrobial peptide is L _m (L/K) _n , (L/K) _n L _m , wherein 12 ≤ _n ≤ 24, m ≤ 8.

Preferably, the amino acid in the antimicrobial peptide may be D-form or L-form, and may have both L-form and D-form.

A targeted antimicrobial peptide obtained by linking any one of the above basic antimicrobial peptides to an antibody complementarity determining region through a plurality of leucine; specifically, the targeted antimicrobial peptide is composed of an antimicrobial peptide -Lo-antibody complementarity determining region; wherein o is the number of leucine, o ≥ 4.

Preferably, the sequence of the targeted antimicrobial peptide is set forth in SEQ ID NOS: 25-31.

It should be noted that only a few antibacterial peptides attached to certain antibody complementarity determining regions become targeted antibacterial peptides, and the antibacterial peptides that do not cleave human red blood cells become targeted antibacterial peptides for lysing human red blood cells. Therefore, appropriate screening should be carried out when developing antibacterial peptide drugs.

In addition, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have anti-Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multiple resistance Staphylococcus aureus Y5 (Zhang Ying et al., Molecular typing of Staphylococcus aureus in a food poisoning event, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al. A combinatorial yeast overlay Method for the isolation of antibacterial oligopeptides, Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 2014, 84(4): 1069-1075), the effect of Saccharomyces cerevisiae (INVSc1) and the like.

Therefore, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention can be used for bacteriostatic, and can also be used for preparing bacteriostatic drugs. Among the above antimicrobial peptides, when the number of lysines is equal to or exceeds 50% of the peptide, it is effective against Escherichia coli. Above or below 50%, it is effective against both Staphylococcus aureus and Pseudomonas aeruginosa; antimicrobial peptides between 16 and 24 amino acids in length are effective against the above three bacteria.

Meanwhile, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention also have an anticancer effect, and the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have activity against lung cancer cell A549 and immortalization of SV40 virus. The killing effect of bronchial epithelial cell line 16HBE14o-; the targeted antibacterial peptide has stronger anticancer activity than the control antibacterial peptide at a lower concentration, especially the antibacterial peptide numbered 59, which kills lung cancer cells in 0.1 mM The efficiency is as high as 100.3%; therefore, the targeted antimicrobial peptide of the present invention can be used for targeting bacteriostatic or directed anticancer, and can also be used for preparing bacteriostatic or anticancer drugs.

For targeted antimicrobial peptides (numbered 49, 50, 59, 60), the number of intermediate leucines was 4. These peptides have stronger carcinostatic activity than the control antimicrobial peptide 2 or the control antimicrobial peptide 11 at a low concentration, and the antibody complementarity determining region is the complementarity determining region 3 and 2 of the antibody of the CD47 protein (Yasufumi Kikuchi, Shinsuke) Uno, Yasuko Kinoshita, et al. HUMANIZED ANTI-C D47ANTIBODY. European Patent Application EP1693385), the sequences are: ARGGYYTYDDWG and YIYPYNDGTKYNEKFKD, respectively. Human cancer cells tested by scientists all express CD47, which is usually expressed at higher levels (on average about three times higher) than normal cells. The antimicrobial peptide of the present invention is linked to the antibody complementarity determining region of the CD47 protein, and the antimicrobial peptide can be brought to the cancer cell site.

The antibody complementarity determining region of the antibacterial peptide of the present invention and the CD47 protein must be linked by a leucine phase, and the inventors have found through experiments that the target formed by the complementarity determining region of the antibody directly linked to the CD47 protein by the basic antimicrobial peptide numbered 2 The antimicrobial peptide (No. 63) completely lost its antibacterial activity.

Accordingly, the present invention also provides the use of any of the above alkaline antimicrobial peptides for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or Kill immortalized cells.

The present invention also provides the use of the targeted antimicrobial peptide for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or kill immortalization cell.

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

The present invention provides an alkaline antimicrobial peptide composed of leucine and lysine, and the number of amino acids of the basic antimicrobial peptide is 12 to 24, wherein lysine The ratio is greater than 33.3%; the alkaline antimicrobial peptide of the present invention does not consider the secondary structure and physicochemical properties of the amino acid, and the antimicrobial peptide can be arbitrarily combined; it is a non-high permeability type peptide (usually low permeability type) and does not cleave the human body. Red blood cells, no hemolytic activity, generally do not form a high-efficiency membrane channel, so it is safe for the human body; at the same time, it has a broad spectrum for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, multi-drug resistant Staphylococcus aureus, Saccharomyces cerevisiae, etc. The bacteriostasis effect; the above-mentioned alkaline antimicrobial peptide is made into a targeted antimicrobial peptide, and the efficiency of the targeted antimicrobial peptide in the 0.1 mM lung cancer-killing cell is as high as 100.3%; the antimicrobial peptide or the targeted antimicrobial peptide of the present invention can be used for inhibiting Bacteria and anti-cancer, can be widely used in medical, agricultural, food preservatives and other fields.

DRAWINGS

Figure 1 shows the hemolytic activity of antimicrobial peptides and targeted antimicrobial peptides.

Figure 2 is the rate at which 80 μM different antimicrobial peptides hydrolyze ONPG.

Figure 3 is a graph showing the rate at which 80 μM of different antimicrobial peptides hydrolyze ONPG.

Figure 4 shows the results of antibacterial and antimicrobial peptides against multi-drug resistant S. aureus Y5.

Figure 5 shows the results of antibacterial activity of antibacterial peptides and targeted antibacterial peptides against Pseudomonas aeruginosa.

Figure 6 shows the results of an antibacterial peptide and a targeted antimicrobial peptide against S. cerevisiae INVSc1 inhibition zone.

Figure 7 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on lung cancer cell line A549.

Figure 8 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on the human bronchial epithelial cell line 16HBE14o– immortalized with SV40 virus.

Figure 9 is a graph showing the killing effect of a targeted antimicrobial peptide on the human bronchial epithelial cell line 16HBE14o- immortalized with the SV40 virus.

detailed description

The contents of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but are not to be construed as limiting. Modifications or substitutions of the methods, procedures, or conditions of the present invention are intended to be within the scope of the present invention without departing from the spirit and scope of the invention; Conventional means well known.

Example 1 Determination of minimum inhibitory concentration (MIC)

According to the polypeptide sequence of Table 1, various peptides were synthesized by Shanghai Botai Biotechnology Co., Ltd. by fmoc solid phase synthesis, and purified by RP-HPLC; the polypeptide includes an antimicrobial peptide and a targeted antimicrobial peptide, wherein the targeted antimicrobial peptide is The anti-peptide and antibody complementarity determining regions are obtained by several leucine linkages.

The bacteria used were Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multi-drug resistant Staphylococcus aureus Y5 (Zhang Ying et al, together with food Molecular typing of Staphylococcus aureus in poisoning events, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al. A combinatorial yeast overlay method for the isolation of antibacterial oligopeptides, Proceedings of the National Academy Of Sciences, India Section B: Biological Sciences, 2014, 84(4): 1069–1075),

The minimum inhibitory concentration (MIC) determination process is as follows:

(1) Bacterial single colonies were inoculated in a conventional LB liquid medium, cultured at 37 ° C, shaking at 200 rpm for 11 hours overnight, and colony forming units were determined.

(2) Each peptide (antibacterial peptide and targeted antimicrobial peptide) was set to four final concentrations: 40 μM, 80 μ. M, 160 μM and 320 μM; each peptide was set to 3 parallels per concentration.

(3) Dilute the culture solution overnight with a conventional LB liquid medium to obtain a bacterial solution using 10 ⁶ colony forming units; experimental group: 50 μl of each peptide and bacterial solution, and 1:100 μl of sterile culture in the control group. Base 2; control group 2: 50 μl of sterile medium and bacterial solution; control group 3: 50 μl of bacterial solution + corresponding amount of DMSO and medium corresponding to the sample; control group 4: 50 μl Bacterial solution + corresponding amount of water and medium corresponding to the sample.

(4) After culturing the 96-well plate with the sample in the incubator at 37 ° C for 20 hours, the OD value was measured at 492 nm and 620 nm with a microplate reader.

(5) Data analysis

Calculation formula: Calculated using the OD value at 492 nm.

Inhibition rate%=100×[1-(peptide and bacterial liquid experimental group - no bacterial culture control group) / (water or DMSO and bacterial liquid control group - no bacterial culture control group)]

When the inhibition rate reached 79.5%, it was recorded as MIC (minimum inhibitory concentration), and the results are shown in Table 1.

Table 1 Minimum inhibitory concentration of antimicrobial peptides against various bacteria

*- indicates no determination; the numbered parentheses indicate DMSO, indicating the peptide dissolved in DMSO. Not labeled as water-soluble peptide; no activity: all four concentrations of inhibition rate less than 15%; promote growth: all four concentrations of inhibition rate is less than -20%; brackets expressed as the maximum inhibition rate. The amino acid is lowercased to D type. a: ARGGYYTYDDWG (complementarity determining region 3 of the antibody of CD47 protein). b: YIYPYNDGTKYNEKFKD (complementarity determining region 2 of the antibody of CD47 protein).

Example 2 Hemolysis Experiment

Blood treatment

(1) Collect 5 ml of fresh human blood into a tube containing 0.5 ml of 3.8% sodium citrate, and blow the blood in the anticoagulant tube to mix well with the anticoagulant.

(2) The above blood was centrifuged at 2000 rpm for 8 minutes, and the supernatant was removed. The blood was rinsed with 10 mM, pH=7.4 PBS, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the operation was repeated until the red blood cell suspension was clear and free of impurities such as serum, and the supernatant was discarded. It was then dissolved in PBS at 5% (v/v) to give a red blood cell suspension.

2. Antibacterial peptide treatment: The antimicrobial peptide was dissolved in PBS, and finally an antibacterial peptide solution having a final concentration of 40 μM, 80 μM, 160 μM, and 320 μM was obtained.

3. System mixing

Pipette 50 μl of the treated antimicrobial peptide solution into a 96-well plate at a concentration of 3 parallels per peptide. Then, use a lance to draw the red blood cell suspension poured onto the culture dish, and add 50 μl per well to the 96-well. In the plate, after the addition was completed, the 96-well plate was sealed and placed in a shaker at 37 ° C for 2 hours at 200 rpm.

20 μM, 40 μM, 80 μM and 160 μM cecropin (CecA for short) were mixed with the red blood cell suspension according to the above method, the positive control was 1% Triton X-100; the negative control was PBS solution.

4. Data analysis

The 96-well plate after shaking culture was centrifuged at 2000 rpm for 8 minutes, and 60 μl of the supernatant was aspirated and transferred to a new 96-well plate, and the OD value at a wavelength of 540 nm was detected by a microplate reader.

Permeation percentage = (A ₅₄₀ sample - A ₅₄₀ negative control) / (A ₅₄₀ positive control - A ₅₄₀ negative control) x 100%.

The experimental results are shown in Figure 1.

Example 3 Escherichia coli membrane permeability test

--galactosidase is a hydrolase located in the cytoplasm of bacteria that hydrolyzes o-nitrophenyl β-D-galactopyranoside (ONPG) to galactose and o-nitrophenol (yellow). A certain amount of ONPG was added to the system, and the degree of hydrolysis of ONPG was determined by measuring the change in the absorbance of the culture solution at 420 nm to determine whether β-galactosidase hydrolyzes ONPG. In general, since the enzyme is located inside the cell, the ONPG cannot enter the cell. However, once the cell membrane permeability changes, ONPG enters the cell and hydrolyzes, the culture solution rapidly turns yellow, and the A ₄₂₀ value rises rapidly in a short time. Therefore, this method can be used to detect the effect of antimicrobial peptides on cell membrane permeability. High membrane permeability indicates that the antimicrobial peptide can form a membrane channel.

The permeability experiment process of the present invention is as follows:

(1) Pick up E. coli ML-35 single colony overnight culture, and measure its colony forming unit; according to the measured colony forming unit value, take an appropriate volume of the overnight culture in EP tube, centrifuge at 10,000 rpm for 1 minute, and use 10 mM Resuspend in sodium phosphate (containing 0.1 M NaCl) buffer. The reaction was repeated 3 times, and the bacterial body weight was suspended to a colony forming unit = 2 × 10 ⁷ with a sufficient amount of sodium phosphate buffer.

(2) Take 10 μl of 10 mM peptide (including antimicrobial peptide and targeted antimicrobial peptide), add 490 μl of sodium phosphate buffer to obtain 200 μM, 500 μl of peptide dilution; take 10 μl of water or DMSO instead of peptide, add 90 μl of sodium phosphate buffer was used as a control group.

(3) Take 200 μl of peptide dilution, add 50 μl of ONPG solution to obtain peptide-ONPG mixture; add 50 μl of ONPG to the control group; add 96-well plate with peptide-ONPG mixture, 50 per well. Microliters, three replicates per sample; take the bacterial solution stored at 4 °C, and add 50 μl to each well with a lance. According to this method, the final experimental group has a volume of 100 μl per well, a final peptide concentration of 80 μM, a final concentration of ONPG of 1.5 mM, and a bacterial concentration of 1×10 ⁷ colony forming units; and the final concentration of the peptide in the experimental group is 40 μM per well.

After adding the bacterial solution at room temperature, it was quickly transferred to a multi-plate reader, and its absorbance at 420 nm at 37 ° C was measured, and it was measured every 10 minutes from the first measurement. Measured 13 times. The data was recorded and analyzed, and the results are shown in Figures 2 and 3.

Example 4 inhibition zone experiment

Peptide-to-multi-drug resistant S. aureus Y5 and Pseudomonas aeruginosa inhibition zone experiments: Inoculation of bacterial single colonies in conventional LB liquid medium, shaking at 37 ° C, 200 rpm overnight for 11 hours, and determination of colony forming units . When the LB solid medium is cooled to a non-hot but not solidified state, the counted S. aureus Y5 and Pseudomonas aeruginosa (1.2464) bacteria are respectively added, so that the concentration of the bacterial liquid is about 1×10 ⁸ colony forming units/ml. Pour into the plate and cool until completely solidified.

1. Sample processing:

S. aureus plates were filled with 40 microliters of 5 mM peptide per well, the negative control was 40 microliters of DMSO/water, and the positive control was 10 microliters of 5 mg/ml Amp ⁺ , 30 microliters of water.

P. aeruginosa plates were supplemented with 25 microliters of 5 mM peptide per well, the negative control was 25 microliters of water per DMSO, and the positive control was 10 microliters, 5 milligrams per milliliter of Amp ⁺ , 15 microliters of water.

The treated samples were incubated at 37 ° C for 16 hours at constant temperature to record whether there were inhibition zones and their size results (Figures 4, 5 and Table 2).

Table 2: The inhibition zone size of the polypeptide against Staphylococcus aureus and Pseudomonas aeruginosa

Note: The diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2

Example 5 Saccharomyces Cerevisiae INVSc1 inhibition zone experiment

Configure YPD medium: glucose 2%, peptone 2%, yeast extract 1%, solid medium plus agar 2%; streak culture S. cerevisiae INVSc1 single colony; inoculate single colonies at 30 ° C shaker for 24 hours and remove Store at 4 ° C; measure colony forming units.

Melt the YPD solid medium, and add the above-mentioned bacterial colony forming unit when the medium is not too hot. According to the bacterial solution: YPD medium = 1:20, the yeast liquid concentration is 1×10 ⁶ /ml. Pour into a plate of 15 cm in diameter and about 50 ml/plate; after cooling, punch with a 5 mm diameter puncher, add 40 μl of peptide per well, 10 mM peptide, and a negative control of 40 μl of water/DMSO negative control The positive control was 10 microliters, 10 mg/l benomyl + 30 microliters of water. The plate was sealed with a parafilm and placed in a 30 ° C incubator for about 18 hours, photographed and the size of the zone of inhibition was recorded. The results are shown in Figure 6 and Table 3.

Table 3 Saccharomyces cerevisiae inhibition zone size

Note: The diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2

Effect of the peptide of Example 6 on cell viability

The lung cancer cell line A549 is described in the literature (Lieber M, Smith B, Szakal A et al. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer, 2006, 17(1): 62-70). Cell culture methods are as described by the American Type Culture Collection. Immortalized human bronchial epithelial cell line 16HBE14o– cells are found in references (Cozens A L, Yezzi M J, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner W E, Widdicombe J H, and Gruenert D C.” CFTR Expression and chloride secretion in polarized immortal human bronchial epithelial cells. "American Journal of Respiratory Cell and Molecular Biology, 1994, 10 (1): 38-47) cultured in DMEM/F12 medium, A549 cells cultured in DMEM (high sugar) In the medium, 10% fetal calf serum was added to both mediums.

Cell viability was determined by 3–(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT; MBCHAM) colorimetric assay. For specific procedures, see references (Warshamana-Greene GS, Litz J, Buchdunger E et al. The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of Chemotherapy. Clin Cancer Res, 2005, 11(4): 1563-1571). Main steps: 16HBE14o– and A549 cells were cultured in 96-well plates to exponential growth phase. Each well cell is then subjected to the indicated different concentrations of peptide. After 24 hours of treatment, the absorbance at 492 nm was measured by the MTT method. Finally, according to the formula: cell viability inhibition rate (%) = (control group 492 nm absorbance - experimental group 492 nm absorbance value / control group 492 nm absorbance × 00%) to calculate the inhibition rate of various peptides on cell viability The results are shown in Figures 7-9.

Claims (9)

1. An alkaline antimicrobial peptide characterized in that the basic antimicrobial peptide is composed of leucine and lysine, and the number of amino acids of the basic antimicrobial peptide is 12 to 24, wherein lysine The ratio is greater than 33.3%.
2. The alkaline antimicrobial peptide according to claim 1, wherein the amino acid sequence of the basic antimicrobial peptide is as shown in SEQ ID NOS: 1 to 23.
3. The alkaline antimicrobial peptide according to claim 1, wherein the amino acid is D-form or/and L-form.
4. A targeted antimicrobial peptide, characterized in that the structure of the targeted antimicrobial peptide is an antimicrobial peptide-Lo-antibody complementarity determining region; wherein o is the number of leucine, o≥4; the antimicrobial peptide is a right The antimicrobial peptide of any one of 1 to 3 is claimed.
5. The targeted antimicrobial peptide according to claim 4, wherein the sequence of the targeted antimicrobial peptide is as shown in SEQ ID NOS: 25 to 31.
6. Use of the alkaline antimicrobial peptide according to any one of claims 1 to 3 for the preparation of a bacteriostatic or/and anticancer drug.
7. Use of the targeted antimicrobial peptide according to claim 4 or 5 for the preparation of a bacteriostatic or/and anticancer drug.
8. The use according to claim 6, wherein the bacteriostatic action is inhibition of fungi or/and bacteria; the anti-cancer is killing lung cancer cells or killing immortalized cells.
9. The use according to claim 7, wherein the bacteriostatic action is inhibition of fungi or/and bacteria; the anti-cancer is killing lung cancer cells or killing immortalized cells.

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