CN113150071B - Marine brevibacillus brevis antitumor active polypeptide and medicine and application thereof - Google Patents

Abstract

The invention discloses a polypeptide PBN3 produced by Brevibacillus sp.N189, wherein the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018662, wherein the molecular weight of the polypeptide is 759.44Da, and the amino acid sequence of the polypeptide is Leu-Thr-Ala-His-Tyr-Arg. The invention also discloses a pharmaceutical application of the polypeptide and an anti-lung cancer medicament containing the polypeptide.

Description

Marine Brevibacillus antitumor active polypeptide and its medicine and application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a marine Brevibacillus antitumor active polypeptide and its medicine and application.

Background technique

New estimates of the global burden of cancer show that global cancer cases have risen to 19.3 million in 2020, with 10 million cancer deaths, according to the International Agency for Research on Cancer (IARC). Lung cancer remains the leading cause of cancer death. Due to the large ocean area and diverse marine environments, the development of the ocean is in a process of continuous recognition. At the same time, the number of microorganisms in the ocean is 10 ⁶ to 10 ⁹ /mL, which has a huge number in the ocean, and due to the Antarctic Ocean The existence of special environmental conditions such as low temperature makes it easier for the microorganisms living in it to metabolize new metabolites, and it also brings more possibilities for the development of biomedicine. Biological small molecule active substances have the advantages of small molecular weight, easy absorption, and low toxicity and side effects, and have become a hot spot in the development of biomedicine. Through microbial fermentation, extract natural small molecule active substances that can inhibit the proliferation of tumor cells from the metabolites of marine microorganisms, and continuously promote the development and utilization of marine active substances.

Contents of the invention

The object of the present invention is to provide an antitumor active ingredient produced by novel brevibacillus N189 and its medicine and application.

The present invention firstly provides a polypeptide PBN3 produced by Brevibacillus sp.N189. The strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2018662. The molecular weight of the polypeptide is 759.44Da. The amino acid sequence of the polypeptide is Leu-Thr-Ala-His-Tyr-Arg (SEQ ID No.2).

The second aspect of the present invention provides the use of the above polypeptide PBN3 in the preparation of a drug for preventing or treating tumors, the tumors are selected from lung cancer, liver cancer, pancreatic cancer, breast cancer or cervical cancer.

The present invention also provides an antitumor drug comprising the above-mentioned polypeptide PBN3, and the tumor is selected from lung cancer, liver cancer, pancreatic cancer, breast cancer or cervical cancer.

Details of various aspects of the invention are set forth in the ensuing sections. The features, objects and advantages of the present invention will be more apparent from the following description and claims.

Description of drawings

Figure 1 The active component obtained by water-saturated n-butanol extraction through Q anion exchange chromatography column

Figure 2 C18 HPLC chromatogram of active peak Q1

Figure 3 Molecular mass diagram of anti-tumor active peptide PBN3

Fig. 4 Amino acid sequence diagram of anti-tumor active peptide PBN3

Figure 5 PBN3 inhibits proliferation and inhibits different tumor cells

Detailed ways

The novel marine brevibacillus N189 provided by the present invention is the marine brevibacillus N189 that can produce anti-tumor substances and is isolated from Antarctic seawater. It constitutes a branch alone and is determined to be a new type of strain, named as marine brevibacillus N189. (Brevibacillus sp. N189). The strain was deposited in the China Center for Type Culture Collection (Address: China. Wuhan. Wuhan University) on October 9, 2018, with the preservation number CCTCC NO: M2018662.

The marine Brevibacillus N189 provided by the present invention is a Gram-positive bacterium, an aerobic bacterium, has good salt tolerance, and grows well in 6.5% NaCl (w/v) culture solution; the growth pH range is 5.5-8.5 ; The growth temperature range is wide, and it can grow well under the environment of 12°C-37°C.

The 16S rDNA gene of the strain N189 was amplified by PCR and detected by agarose gel electrophoresis, and the molecular weight of the amplified product was about 1.5kb. Sequence determination was carried out on the 16S rDNA gene PCR result of strain N189, and the measured sequence length was 1438bp (SEQ ID No.1). Referring to "Berger's Bacteria Identification Manual (Ninth Edition)", combined with the results of similarity analysis of 16S rDNA gene sequence and physiological and biochemical analysis results, the strain N189 was determined to be a new strain of Brevibacillus sp. N189 .

The anti-tumor active polypeptide PBN3 provided by the invention can be isolated from the metabolites of bacterial strain N189. MALDI-TOF mass spectrometry showed that its molecular weight was 759.44Da. The amino acid sequence of the polypeptide is LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (SEQ ID No. 2).

The antitumor drug provided by the present invention comprises the above-mentioned polypeptide PBN3, which is effective against human non-small cell lung cancer cell A549, human large cell lung cancer cell NCI-H460, human liver cancer cell HepG2, human pancreatic cancer cell Panc 28, breast cancer MCF-7 and human cervical cancer Cancer cell Hela has a certain inhibitory effect on its proliferation.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Screening and Identification of Example 1 Bacterial Strain N189

1.1. Isolation and purification of strain N189

The Antarctic surface seawater samples were suction-filtered through a 0.22 μm filter membrane, and then the bacterial filter membrane was stored in a bottle containing 25% glycerin and stored in a -80°C refrigerator. Transfer the bacterial filter membrane to a sterile workbench, crush it, redissolve it in the sample seawater, and shake it moderately to form a bacterial liquid. Under normal pressure conditions, take an appropriate amount of bacterial liquid and spread it on the 2216E seawater separation medium, and place it at 25°C for static culture. When the diameter of a single colony is about 1mm, pick different single colonies to smear and purify. The purified bacteria were inoculated into the corresponding liquid medium, cultured with proper shaking, and stored in sterile glycerol tubes containing 25% until the logarithmic growth phase, and stored at -80°C.

1.2. Fermentation of strain N189

Preparation of medium: peptone 5g/L, yeast powder 1.0g/L, ferric phosphate 0.008g/L, prepared with aged sea water, sterilized at 121°C for 15min.

Take a 100μL glycerol-preserved strain N189, inoculate it into 30mL medium, and culture it in a shaker at 16°C and 200r/min for 24h at a constant temperature until it reaches the logarithmic growth phase, which is the seed fermentation broth. For subsequent Gram stain detection.

The seed fermentation liquid was inoculated into 300 mL medium according to the inoculation amount of 4%, and continued to cultivate for 36 hours under the same conditions, which was the strain fermentation liquid.

Preparation of solid medium: peptone 5g/L, yeast powder 1.0g/L, ferric phosphate 0.008g/L, agar powder 2%, add aged sea water to prepare, sterilize at 121°C for 15min under high pressure steam. Prepare a solid medium plate, spread the seed fermentation liquid, grow for 3 days, and form a single colony for physiological and biochemical experiments.

1.3 Identification of strain N189.

Gram staining: Take the bacterial liquid to make a smear, dry and fix it. Then stain with ammonium oxalate crystal violet for 1 min, and wash the dye with distilled water. Cover with iodine solution for 1 min, rinse with water, and absorb excess water with absorbent paper. Decolorize with 95% ethanol for 20s, wash with water immediately and blot dry with filter paper. Safranin staining solution was counter-stained for 1 min, washed with water, blotted dry, and microscopically examined. The staining result was purple, indicating that it was a Gram-positive bacterium. Aerobic bacteria have good salt tolerance and grow well in 6.5% NaCl (w/v) culture solution; the pH range of growth is 5.5-8.5; Grows well.

Microscope morphology observation found that the morphological characteristics of the solid medium changed with different growth periods. In the lag phase, the bacteria were in the shape of long and thin rods; in the logarithmic growth phase, the bacteria were in the shape of short and thick rods; In the stationary phase, the bacteria are elongated and rod-shaped, and occasionally oval spores can be observed; peri-flagellate, aerobic; colonies are milky white, raised, and the edges are not smooth; opaque, no pigmentation.

Pick the purified single colony on the solid medium plate into a sterile water test tube, grind it into a bacterial suspension with a concentration of 0.5 McFarland, add it to the physiological and biochemical identification tube, and conduct the test according to the instructions of the microbial physiological and biochemical kit API20E. The physiological and biochemical detection results of strain N189 were obtained (Table 1).

Table 1

"+": The reaction is positive or can be grown and utilized: "-": The reaction is negative or cannot be grown and utilized

It can be seen from Table 1 that the physiological and biochemical detection results of strain N189 are positive for URE, VP, and GEL, and negative for ONPG, ADH, LDC, ODC, H ₂ S, TDA, and IND; the results of fermentation experiments show that: GLU, SAC, MEL and ARA were positive, MAN, INO, SOR, RHA and AMY were positive; the results of assimilation test showed that CIT was negative.

1.4. Determination and analysis of 16S rDNA sequence of strain N189

1.4.1. Extraction of Genomic DNA from Strain N189

Genomic DNA of strain N189 was extracted using Invitrogen Genomic DNA mini Kit, the detailed method is as follows:

The temperatures of the water baths were set at 55°C and 37°C, respectively. Take 200 μL of lysozyme buffer and add fresh lysozyme to make the final lysozyme concentration 20 mg/mL. Collect 2×10 ⁹ Gram-positive cells by centrifugation, resuspend the cells with 180 μL of buffer containing lysozyme, vortex, and incubate at 37°C for 30 minutes. Add 20 μL of protease and vortex. Then add 200 μL PureLink ^TM genome lysis/binding buffer, vortex to mix evenly, and incubate at 55°C for 30min. Add 200 μL of 96-100% ethanol solution to the lysate, and vortex to mix evenly. Put the adsorption column into the collection tube, add 640 μL of lysate to it, and centrifuge at 10000×g for 1 min at room temperature. Discard the collection tube, put the adsorption column into a new collection tube, add 500 μL of elution buffer 1, and centrifuge at 10,000×g for 1 min at room temperature. Discard the collection tube, put the adsorption column into a new collection tube, add 500 μL of elution buffer 2, and centrifuge at the maximum speed for 3 min at room temperature. Put the adsorption column into a 1.5 μL centrifuge tube, add 25-2100 μL genome elution buffer, wait for 1 min at room temperature, and then centrifuge at the maximum speed for 1 min. Collect the purified DNA in the tube and store at -20°C.

1.4.2. Primer design for 16S rDNA of strain N189

16S rDNA universal primers were used, synthesized by Shanghai Sangong.

Forward primer 27F: 5'-agagtttgatcctggctca-3' (SEQ ID No.3);

Reverse primer 1492R: 5'-ggttacccttgttacgactt-3' (SEQ ID No.4);

1.4.3. Amplification of 16S rDNA from strain N189

PCR amplification system for the 16S rDNA sequence of strain N189: use the genomic DNA extracted from strain N189 as a template, add 2 μL of primers (see 1.4.2); 0.25 μL of Taq DNA polymerase (5U·mL ^-1 ); 5 μL of 10x PCR Reaction Buffer; 4 μL dNTP MasterMix; ddH20 make up to 50 μL.

PCR reaction conditions: pre-denaturation at 95°C for 4 minutes; 30 cycles (94°C for 30s, 55°C for 40s, 72°C for 90s); final extension at 72°C for 10 minutes.

1.4.4. Detection of PCR products by agarose electrophoresis

The 16S rDNA sequence amplification product of strain N189 was added to a 2% agarose gel, and a 2,000 bp marker was used as a control for electrophoresis detection. There is a bright single band at around 1.5kb of the Marker, which proves that the 16S rDNA gene sequence was successfully amplified. The successfully amplified PCR product was sent to Shanghai Sangong for gene sequencing, and the measured sequence length was 1438bp (SEQ ID No.1). Referring to "Berger's Bacteria Identification Manual (Ninth Edition)", combined with the results of 16S rDNA gene sequence similarity analysis, and the results of physiological and biochemical analysis, it was determined that the strain N189 was Firmicutes, Bacillus, Bacillus, Bacillus, A novel strain of the genus Brevibacillus, named Brevibacillus sp. N189.

The fermentation culture of embodiment 2 brevibacillus N189

Inoculate a single colony of Antarctic marine Brevibacillus sp.N189 (named Brevibacillus sp.N189 in Latin) cultured on the plate into the seed medium. The composition of the seed medium is: beef extract 3g/L, peptone 1g/L, sodium chloride 5g /L, pH7.0 (add agar 20g/L to the plate medium), and sterilize the medium at 1.05kg/cm ² , 121°C, 20min. Cultivate at 25°C and 180r/min until the bacterial solution becomes turbid. Inoculate the seed solution into a 250mL culture bottle containing 50mL expansion medium according to the inoculation amount of 4%, and cultivate it at 25°C and 180r/min until the bacterial solution is turbid. Then inoculate the bacterial liquid into a 2L culture bottle containing 500mL of fermentation medium according to the inoculum amount of 4%, and ferment for 48h at 25°C and 160r/min. The fermentation broth was collected, centrifuged at 8000r/min, 20min, and 4°C, and the supernatant was collected.

Embodiment 3 Preparation of Bacillus brevis N189 polypeptide PBN3

(1) Concentration by ultrafiltration

The fermentation supernatant of strain N189 was filtered and concentrated with a 0.3KDa ultrafiltration membrane to obtain a fraction less than 0.3KDa and a fraction greater than 0.3KDa, a total of 2 fractions. The anti-tumor activity of the two components was determined, and it was confirmed that the active component was located in the concentrated solution part, that is, the component greater than 0.3KDa.

(2) Butanol extraction

Fully mix the above-mentioned active components with 2 times the volume of water-saturated n-butanol, extract for 4 hours, then place it in a separatory funnel and let it stand until the layers are completely separated, take out the n-butanol part of the upper layer, and repeat the fermentation broth of the lower layer Extract once and combine the n-butanol extracts. Under the conditions of 44mbar, 100r/min, and 50°C, the n-butanol component was rotary evaporated to dryness, and the residual n-butanol was removed overnight at room temperature. Dissolve the spin-evaporated components with 20 mM Tris-HCl buffer solution, pH 7.0, and centrifuge at 10,000 r/min, 15 min, and 4°C to obtain the supernatant. The anti-tumor activity of the supernatant was determined, and it was found that the water-saturated n-butanol extraction fraction had a very obvious effect of inhibiting the proliferation of tumor cells.

(3) Q anion exchange chromatography

The active components obtained by extraction with water-saturated n-butanol were passed through a 0.22 μm sterile microporous membrane, and separated and purified by Q anion exchange chromatography column (Figure 1). The sample volume is 3 mL, the injection buffer is 20 mM tris hydrochloride solution at pH 7.0, and the eluent is 20 mM tris hydrochloride at pH 7.0 containing 1 mol/L NaCl Salt solution, the eluent was eluted at a concentration of 30% (v/v), the flow rate was 3mL/min, the detection wavelength was 254nm, and the breakthrough peak Q1, breakthrough peak Q2, elution peak Q3 and elution peak were collected. Peak Q4, the anti-tumor activity of the components corresponding to each peak was detected by MTT method, and the results showed that breakthrough peak Q1 and breakthrough peak Q2 had anti-tumor activity, and the anti-tumor activity of the component corresponding to breakthrough peak Q1 was the best, and further The active component Q1 is separated and purified.

(4) C18 high pressure liquid chromatography

Dissolve 1 g of active component Q1 in 1 mL of 5% acetonitrile containing 0.1% trifluoroacetic acid (TFA), filter the sample with an organic filter, and perform separation and purification by high performance liquid chromatography (Figure 2). WATERS 2489 preparative chromatography, chromatographic conditions: preparative chromatographic column: WATERS SunFire C18 preparative, flow rate: 3mL/min; temperature: 24°C; injection volume: 1mL; detection wavelength: 254nm; mobile phase A: 0.1% trifluoro Aqueous solution of acetic acid (water:TFA=100:0.1, v/v), mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (acetonitrile:TFA=100:0.1, v/v). HPLC chromatographic gradient elution program: equilibrium buffer is 95% A + 5% B; gradient elution: 0 ~ 5min, 33% B; 5 ~ 35min, 38% B; 35 ~ 40min, 100% B; 40 ~ 45min, 5% B); Obtain multiple components, wherein the retention times (min) of several main peaks at the detection wavelength are 11.98 (N1), 12.50 (N2), 14.05 (N3) and 15.33 (N4) respectively , collect each elution peak separately, and the part other than the elution peak is collected according to time and gradient. After freeze-drying, 20mM, pH8.0, Tris-HCl redissolved to measure the activity, N3 activity is the best.

Further, the active component N3 was separated and purified by high-pressure liquid phase loading for multiple times to obtain a single peak, which was lyophilized to obtain lyophilized powder N3.

Example 4 MALDI-TOF Determination of Molecular Weight and Amino Acid Sequence of Active Peptide PBN3

The research was carried out at the Proteome Research Center of Fudan University, and the sample pretreatment was as follows: First, the lyophilized powder N3 was dissolved in 50% acetonitrile water (containing 0.1% trifluoroacetic acid) solution, and 0.5 μL of the dissolved sample was placed on the MALDI target plate After natural drying, 0.5 μL of 4g/L CHCA solution (solvent, 50% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid) was added, and dried naturally at room temperature. Mass spectrometry and spectral analysis were then performed. The samples were analyzed by mass spectrometry with a 5800 tandem time-of-flight mass spectrometer. The laser source was a Nd:YAG laser with a wavelength of 355nm, and the accelerating voltage was 20kV. The positive ion mode and the mode of automatic data acquisition were used to collect data. The instrument first uses the myoglobin enzymatic peptide for external standard calibration. The PMF mass scan range of matrix and sample is 700-3600 Da. After MS, the target peptide ions were directly selected for MS/MS analysis. MS adopts Reflector Positive parameters: CID (OFF), mass rang (700-3600Da), Focus Mass (1600Da), Fixed laser intensity (6000), Digitizer: Bin Size (0.5ns). MS/MS adopts 1KV Positive parameters: CID (ON), Precursor Mass Windows (Relative 100resolution (FWHM)), Fixed laser intensity (7000), Digitizer: Bin Size (1ns). The obtained MS/MS spectra were de novo sequenced using the analysis tool De Novo Explorer that comes with the instrument software 4000Explorer combined with manual analysis. After obtaining the sequence, use the software Data Explorer to display the product ions obtained by fragmenting parent ions such as b and y on the MS/MS icon.

result:

① Molecular weight determination of peptides

It can be seen from Fig. 3 that the molecular ion peak [M+H] ⁺¹ obtained after MALDI-TOF-TOF MS analysis is 759.44Da (Fig. 3).

② Peptide sequence verification analysis

In order to calculate its sequence, the molecular ion peak [M+H] ⁺¹ is 759.44 for MS/MS analysis, and then the software is used for comparison analysis, and the peptide sequence with the molecular ion peak [M+H] ⁺¹ of 759.44 is LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (Figure 4).

The molecular weight of the active peptide PBN3 determined by MALDI-TOF mass spectrometry was 759.44Da, and its amino acid sequence composition was LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (SEQ ID No.2).

Example 5 Verification of the anti-tumor cell activity of PBN3

(1) BCA protein concentration determination of active component protein concentration

The TIANGEN brand BCA protein concentration assay kit was used in the research, and the detailed operation is as follows:

①Mix the BCA reagent and Cu ²⁺ reagent at a ratio of 50:1 (v/v) thoroughly to obtain the BCA working solution.

② Add the newly prepared BSA standard solution (Table 2) and the sample to be tested into the 96-well flat-bottomed plate, 25 μL each, and set up 3 parallels. Add 200 μL of BCA working solution to each well, cover and place in a microplate reader for shaking and mixing;

③ Place the 96-well plate in a 37°C constant temperature biochemical incubator and incubate for 30 minutes;

④ Cool the sample to room temperature, and detect its absorbance with a microplate reader, λ=562nm;

⑤ Draw a standard curve according to the absorbance value and concentration of BSA, and calculate the protein concentration in the sample through the standard curve.

Using the BCA protein concentration assay method, set λ=562nm, measure the absorbance value of different concentrations of BSA, and make 3 parallels for each concentration. Calculate the average value and draw a standard curve of BSA protein concentration using the absorbance and standard protein concentration.

The active PBN3 freeze-dried sample was formulated with 0.01M PBS buffer solution into a certain concentration of mother solution, and the protein concentration of the mother solution was determined by BCA method to determine the activity of PBN3.

Table 2 BSA standard concentration preparation table

(2) MTT method to detect the inhibitory effect of PBN3 on the proliferation of different tumor cells

In the experiment, non-small cell lung cancer cells A549 were used to detect anti-tumor activity by MTT method. In order to determine the IC ₅₀ value of the active components on tumor cells, a total of 6 gradients (including blank control) were set up, and the sample volume was 20 μL. The specific operation method is as follows: ① Human non-small cell lung cancer cells A549 in the logarithmic growth phase were collected, the culture medium was discarded, and then washed with 3-4 mL of PBS. Add 800 μL of trypsin digestion solution to digest for 40-60s, suck out the trypsin, and incubate for 60-80s in a 37°C constant temperature cell incubator containing 5% CO ₂ , observe under a microscope, and find that about 80% of the cells become round and shrunk. Stop the reaction; ② Add appropriate amount of RPMI-1640 cell culture medium to the digested cells, and blow them into a single-cell suspension with a sterile pipette. Use a cell counter to measure the number of cells in the suspension, and take an appropriate amount of cell suspension into fresh 18mL RPMI-1640 cell culture medium, so that the concentration of the new cell suspension is 3-4× ¹⁰³ cells/mL; ③ Add the cell suspension to In a 96-well plate, add 180 μL to each well, and incubate in a constant temperature incubator for 16-20 hours; ④ Add 20 μL of sample to each well, and filter the sample with a 0.22 μm sterile filter membrane before adding, and set 3 parallel wells in each group; ⑤ Then continue to cultivate in a constant temperature incubator for 48 hours, add 20 μL of MTT (5 mg/mL, i.e. 0.5% MTT) to each sample well, and place in a constant temperature incubator to continue to cultivate for 4 hours; ⑥ terminate the culture reaction, remove the liquid in the well, and use Wash the RPMI-1640 medium twice, add 150 μL DMSO to each well, and shake for 10 minutes with a multi-functional microplate reader to fully dissolve the crystalline formazan; ⑦ Measure the absorbance at OD 570nm with a microplate reader, and calculate the cell inhibition rate with the following formula : Cell inhibition rate (%)=((OD control group-OD experimental group)/OD control group)×100%. The drug concentration when the inhibition rate reached 50% was the IC ₅₀ value, and the experiment was repeated three times and the results were expressed as mean ± SD.

Then according to the above method, breast cancer cells MCF-7, cervical cancer cells Hela, pancreatic cancer cells Panc28, liver cancer cells HepG2, and lung cancer cells NCI-H460 were spread on 96-well plates, and different concentrations of PBN3 were added to the 96-well plates of different cells. In each concentration, do 3 groups of repetitions and observe. MTT method was used to measure the half-lethal inhibition rate of tumor cells treated with PBN3 for 48 hours.

The experimental results show that: PBN3 has a good effect on human non-small cell lung cancer cell A549, human large cell lung cancer cell NCI-H460, human liver cancer cell HepG2, human pancreatic cancer cell Panc 28, breast cancer MCF-7 and human cervical cancer cell Hela. Inhibition of tumor cell proliferation, IC ₅₀ were 38.24 μg/mL, 18.99 μg/mL, 25.92 μg/mL, 32.65 μg/mL, 28.74 μg/mL and 14.34 μg/mL, respectively (Figure 5). It shows that PBN3 obtained from Brevibacillus sp.N189 has a wide range of anti-tumor cell proliferation effects in vitro.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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Claims (3)

1. Brevibacillus brevisBrevibacillussp.N189-produced polypeptide PBN3, characterized in that the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018662, wherein the molecular weight of the polypeptide is 759.44Da, and the amino acid sequence of the polypeptide is Leu-Thr-Ala-His-Tyr-Arg (SEQ ID No. 2).

2. The use of the polypeptide PBN3 of claim 1 in the preparation of a medicament for the treatment of a tumor selected from the group consisting of lung cancer, liver cancer, pancreatic cancer, breast cancer, or cervical cancer.

3. An antitumor drug comprising the polypeptide PBN3 of claim 1, wherein the tumor is selected from the group consisting of lung cancer, liver cancer, pancreatic cancer, breast cancer and cervical cancer.

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