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 brevis antitumor active polypeptide and medicine and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a marine brevibacillus antineoplastic activity polypeptide, a medicine and an application thereof.

Background

According to the international agency for research on cancer (IARC), a new estimate of the global cancer burden has been made, and it has been shown that by 2020, the number of global cancer cases has risen to 1930 ten thousand, and the number of cancer deaths is 1000 ten thousand. Lung cancer remains the leading cause of cancer death. Because of the large ocean area and the diverse ocean environments, the development of the ocean is in a continuous cognitive process, and the number of microorganisms in the ocean is 10 ⁶ ～10 ⁹ The number per mL of the microorganism is huge in the ocean, and because of the special environmental conditions of low temperature and the like in the Antarctic ocean, the microorganism living in the ocean is easier to metabolize into new generationsThe metabolite also brings more possibility for the development of biomedicine. The biological small molecular active substance has the advantages of small molecular weight, easy absorption, small toxic and side effects and the like, and becomes a hot spot for developing biological medicines. Through a microbial fermentation mode, natural small molecular active matters capable of inhibiting tumor cell proliferation are extracted from metabolic products of marine microorganisms, and the development and utilization of the marine active matters are continuously promoted.

Disclosure of Invention

The invention aims to provide an anti-tumor active component produced by novel brevibacillus brevis N189, and a medicament and application thereof.

The invention firstly provides a polypeptide PBN3 produced by Brevibacillus sp.N189, the strain is preserved in China center for type culture Collection, and the preservation number is 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).

The second aspect of the invention provides an application of the polypeptide PBN3 in preparing a medicament for preventing or treating tumors, wherein the tumors are selected from lung cancer, liver cancer, pancreatic cancer, breast cancer or cervical cancer.

The invention also provides an anti-tumor medicament which comprises the polypeptide PBN3, wherein the tumor is selected from lung cancer, liver cancer, pancreatic cancer, breast cancer or cervical cancer.

The details of various aspects of the invention are set forth in subsequent sections. The features, objects, and advantages of the invention will be apparent from the description and from the claims.

Drawings

FIG. 1 chromatogram of active ingredient obtained by extracting water-saturated n-butanol through Q anion exchange chromatography column

FIG. 2C 18 high pressure liquid chromatography of active peak Q1

FIG. 3 molecular mass diagram of anti-tumor active peptide PBN3

FIG. 4 amino acid sequence diagram of anti-tumor active peptide PBN3

FIG. 5 inhibitory effect of PBN3 on proliferation inhibition of various tumor cells

Detailed Description

The novel marine Brevibacillus brevis N189 is obtained by culturing and separating marine Brevibacillus brevis N189 capable of producing anti-tumor substances from Antarctic seawater, and is determined as a novel strain named as marine Brevibacillus brevis N189 (Brevibacillus sp. N189). The strain is preserved in China center for type culture Collection (address: wuhan, china university) in 2018, 10 months and 9 days, and the preservation number is CCTCC NO: and M2018662.

The marine brevibacillus brevis N189 is a gram-positive bacterium and an aerobic bacterium, has better salt tolerance, and grows well in a 6.5 percent NaCl (w/v) culture solution; the growth pH range is 5.5-8.5; the growth temperature range is wide, and the growth can be well carried out in the environment of 12-37 ℃.

The 16S rDNA gene of the strain N189 is amplified by PCR, and agarose gel electrophoresis detection is carried out, and the molecular weight of an amplification product is about 1.5 kb. The 16S rDNA gene PCR result of the strain N189 is subjected to sequence determination, and the sequence length is 1438bp (SEQ ID No. 1). With reference to Bergey bacteria identification manual (ninth edition), the result of similarity analysis of 16S rDNA gene sequences and the result of physiological and biochemical analysis were combined to determine that strain N189 is a novel strain of Brevibacillus sp.N189, which is named Brevibacillus sp.N189.

The polypeptide PBN3 with the antitumor activity provided by the invention can be separated from a metabolite of the strain N189. The molecular weight was 759.44Da by MALDI-TOF mass spectrometry. The amino acid sequence of the polypeptide is LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (SEQ ID No. 2).

The anti-tumor medicament provided by the invention contains the polypeptide PBN3, and has certain effect of inhibiting the proliferation of human non-small cell lung cancer cells A549, human large cell lung cancer cells NCI-H460, human liver cancer cells HepG2, human pancreatic cancer cells Panc 28, breast cancer MCF-7 and human cervical cancer cells Hela.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

EXAMPLE 1 screening and identification of Strain N189

1.1. Isolation and purification of Strain N189

And (3) filtering the south pole surface layer seawater sample through a 0.22-micron filter membrane, then storing the bacteria-attached filter membrane in a bottle containing 25% of glycerol, and storing the bottle in a refrigerator at the temperature of-80 ℃. Transferring the bacterial filtering membrane to a sterile workbench, re-dissolving the bacterial filtering membrane in sample seawater after crushing, and moderately shaking to form bacterial liquid. Coating proper amount of the bacterial liquid on 2216E seawater separation culture medium under normal pressure, and standing at 25 deg.C for culture. When the diameter of the single colony is about 1mm, different single colonies are picked, coated, purified and cultured. Inoculating the purified bacteria to corresponding liquid culture medium, culturing under shaking, storing in sterile glycerol tube containing 25% of bacteria at logarithmic growth phase, and storing at-80 deg.C.

1.2. Fermentation culture of Strain N189

Preparation of a culture medium: 5g/L of peptone, 1.0g/L of yeast powder and 0.008g/L of high-iron phosphate, and sterilizing at 121 ℃ for 15min.

Taking 1 piece of 100 mu L glycerol-preserved strain N189, inoculating to 30mL culture medium, and culturing in a shaking table at 16 ℃ and 200r/min at constant temperature for 24h until logarithmic phase to obtain seed fermentation liquid. For subsequent gram stain detection.

Inoculating the seed fermentation liquor into 300mL of culture medium according to the inoculation amount of 4%, and continuously culturing for 36h under the same condition to obtain the strain fermentation liquor.

Preparing a solid culture medium: 5g/L of peptone, 1.0g/L of yeast powder, 0.008g/L of ferric phosphate and 2% of agar powder, adding aged seawater for preparation, and sterilizing at 121 ℃ for 15min by high-pressure steam. Preparing a solid culture medium plate, coating seed fermentation liquor, growing for 3 days to form a single colony, and using the colony for physiological and biochemical experiments.

1.3 identification of the strain of strain N189.

Gram staining: preparing smear with bacterial liquid, drying and fixing. Then, the dye is dyed by ammonium oxalate crystal violet for 1min, and the dye is washed by distilled water. Adding iodine solution, covering for 1min, washing with water, and removing excessive water with absorbent paper. Decolorized with 95% ethanol for 20s, immediately washed with water and blotted dry with filter paper. And re-dyeing with the tomato red dyeing solution for 1min, washing with water, blotting, and performing microscopic examination to obtain a purple dyeing result, which indicates that the bacteria are gram-positive bacteria. Aerobic bacteria, which have good salt tolerance and grow well in 6.5% NaCl (w/v) culture; the growth pH range is 5.5-8.5; the growth temperature range is wide, and the growth can be well carried out in the environment of 12-37 ℃.

The morphological observation of a microscope shows that the morphological characteristics of the solid culture medium change with different growth periods, and the thalli are in a slender rod shape in a delay period; in the logarithmic growth phase, the thalli are in a short and thick rod shape; during the stationary phase, the thalli are in a slender rod shape, and occasionally ovate spores can be observed; perigenesis flagellum and aerobic; the colony is milky white, convex and has unsmooth edges; opaque, no pigment produced.

The purified single colony is picked from a solid medium plate and placed into a sterile water test tube, is ground into bacterial suspension with the concentration of 0.5 McLee, is added into a physiological and biochemical identification tube, and is tested according to the instruction of a microbial physiological and biochemical kit API20E, so as to obtain the physiological and biochemical detection result of the strain N189 (Table 1).

TABLE 1

"+": the reaction is positive or can grow and utilize: "-": reaction is negative or can not grow and utilize

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

1.4. 16S rDNA sequencing and analysis of Strain N189

1.4.1. Extraction of genomic DNA of Strain N189

The Genomic DNA of the strain N189 is extracted by an Invitrogen Genomic DNA mini Kit, and the detailed method is as follows:

the temperature of the water bath was set to 55 ℃ and 37 ℃ respectively. 200. Mu.L of lysozyme buffer was added with fresh lysozyme to a final lysozyme concentration of 20mg/mL. 2X 10 Collection by centrifugation ⁹ Gram positive cells, with 180 u L containing lysozyme buffer heavy suspension cells, vortex vibration, 37 degrees C were cultured for 30min. Add 20. Mu.L protease and vortex. Then 200 mu L of PureLink is added ^TM The genome lysis/binding buffer was vortexed and mixed well and incubated at 55 ℃ for 30min. Adding 200 μ L96-100% ethanol solution into the lysate, and mixing by vortex. The adsorption column was placed in a collection tube, 640. Mu.L of a lysate solution was added thereto, and centrifugation was carried out at 10000 Xg for 1min at room temperature. The collection tube was discarded, the adsorption column was placed in a new collection tube, 500. Mu.L of elution buffer 1 was added, and centrifugation was carried out at 10000 Xg for 1min at room temperature. The collection tube was discarded, the adsorption column was placed in a new collection tube, 500. Mu.L of elution buffer 2 was added, and centrifugation was carried out at maximum speed for 3min at room temperature. Placing the adsorption column into a 1.5 μ L centrifuge tube, adding 25-2100 μ L genome elution buffer solution, waiting for 1min at room temperature, and centrifuging at maximum speed for 1min. The purified DNA was collected in tubes and stored at-20 ℃.

1.4.2. Primer design for 16S rDNA of strain N189

Is synthesized by Shanghai by adopting 16S rDNA universal primer.

Forward primer 27F: 5-;

reverse primer 1492R:5 'and ggttaccttgttacgactt-3' (SEQ ID No. 4);

1.4.3. amplification of 16S rDNA of Strain N189

PCR amplification System for 16S rDNA sequence of Strain N189: taking genomic DNA extracted from strain N189 as a template, 2. Mu.L of primer (see 1.4.2) was added; 0.25. Mu.L of Taq DNA polymerase (5U. ML) ^-1 ) (ii) a5 μ L of 10 XPCR reaction buffer; 4 μ L dNTP MasterMix; ddH20 to 50. Mu.L.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4min;30 cycles (94 ℃,30s,55 ℃, 40s,72 ℃,90 s); final extension at 72 ℃ for 10min.

1.4.4. Agarose electrophoresis detection of PCR products

The 16S rDNA sequence amplification product of the strain N189 is added into 2 percent agarose gel, and 2,000bp of Marker is used as a contrast for electrophoresis detection. A bright single band exists around 1.5kb of Marker, and the success of 16S rDNA gene sequence amplification is proved. And (3) sending the PCR product successfully amplified to Shanghai life worker, performing gene sequencing, and measuring the sequence length to be 1438bp (SEQ ID No. 1). With reference to Bergey handbook of identification of bacteria (ninth edition), combined with the results of analysis of sequence similarity of 16S rDNA genes and physiological and biochemical analysis, strain N189 was determined to be a novel strain of Mycobacteria, bacillales, bacillaceae, brevibacillus, named Brevibacillus sp.N189.

EXAMPLE 2 fermentation culture of Bacillus brevis N189

Inoculating a single colony of the south-pole marine Brevibacillus sp.N189 (Latin name is Brevibacillus sp.N189) cultured by a plate on a seed culture medium, wherein the components of the seed culture medium are as follows: beef extract 3g/L, peptone 1g/L, sodium chloride 5g/L, pH7.0 (agar 20g/L was added to the plate medium), and the medium was maintained at 1.05kg/cm ² And sterilizing at 121 ℃ for 20 min. Culturing at 25 deg.C and 180r/min until the bacterial liquid is turbid. The seed solution was inoculated into a 250mL flask containing 50mL of amplification medium at an inoculum size of 4%, and cultured at 25 ℃ at 180r/min until the suspension became turbid. Then inoculating the bacterial liquid into a 2L culture bottle containing 500mL of fermentation medium according to the inoculation amount of 4%, and fermenting for 48h under the conditions of 25 ℃ and 160 r/min. Collecting fermentation liquid, centrifuging at 8000r/min at 20min and 4 deg.C, and collecting supernatant.

EXAMPLE 3 preparation of Bacillus brevis N189 polypeptide PBN3

(1) Concentrating by ultrafiltration

Filtering and concentrating the fermentation supernatant of the strain N189 by using an ultrafiltration membrane of 0.3KDa to obtain a component less than 0.3KDa and a component more than 0.3KDa, wherein the total number of the components is 2. And (3) carrying out anti-tumor activity measurement on the two components, and determining that the active component is positioned in the concentrated solution part, namely the component with the KDa of more than 0.3.

(2) Extraction with n-butanol

Mixing the above active components with 2 times volume of water saturated n-butanol, extracting for 4 hr, standing in separating funnel until completely layering, taking out upper n-butanol fraction, extracting lower layer fermentation broth for one time, and mixing n-butanol extract fractions. The n-butanol fraction was rotary evaporated to dryness at 50 ℃ at 44mbar, 100r/min and the residual n-butanol was removed overnight at room temperature. Dissolving rotary evaporation component with 20mM Tris-HCl buffer solution with pH7.0, centrifuging at 10000r/min, 15min and 4 deg.C, and collecting supernatant. The determination of the antitumor activity of the supernatant shows that the water-saturated n-butanol extraction component has very obvious effect of inhibiting the proliferation of tumor cells.

(3) Q anion exchange chromatography

The active component extracted with water saturated n-butanol is filtered through 0.22 μm sterile microporous membrane, and separated and purified by Q anion exchange chromatography column (figure 1). The sample loading amount is 3mL, the sample injection buffer solution is 20mM, pH7.0 trihydroxymethyl aminomethane hydrochloride solution, the eluent is 20mM containing 1mol/L NaCl, pH7.0 trihydroxymethyl aminomethane hydrochloride solution, the eluent is eluted at the concentration of 30% (v/v), the flow rate is 3mL/min, the detection wavelength is 254nm, a penetration peak Q1, a penetration peak Q2, an elution peak Q3 and an elution peak Q4 are collected, the MTT method is used for detecting the antitumor activity of the components corresponding to each peak, the result shows that the penetration peak Q1 and the penetration peak Q2 have the antitumor activity, wherein the antitumor activity of the component corresponding to the penetration peak Q1 is optimal, and the active component Q1 is further separated and purified.

(4) C18 high pressure liquid chromatography

1g of active ingredient Q1 was dissolved in 1mL of 5% acetonitrile containing 0.1% trifluoroacetic acid (TFA), and the sample was filtered through an organic filter and subjected to separation and purification by high performance liquid chromatography (FIG. 2). WATERS 2489 preparative chromatography, chromatography conditions: preparative chromatography column: WATERS SunFire C18 preparation, flow rate: 3mL/min; temperature: 24 ℃; sample injection amount: 1mL; detection wavelength: 254nm; mobile phase A:0.1% trifluoroacetic acid in water (water: TFA =100, 0.1,v/v), mobile phase B:0.1% trifluoroacetic acid in acetonitrile (acetonitrile: TFA = 100. HPLC chromatographic gradient elution procedure: equilibration buffer is 95% of B + 5%; gradient elution: 0 to 5min,33% by volume B; 5-35min, 38 percent B;35 to 40min,100 percent by weight B;40 to 45min,5% by weight B); obtaining a plurality of components, wherein the retention time (min) of several main peaks at the detection wavelength of 254nm is respectively 11.98 (N1), 12.50 (N2), 14.05 (N3) and 15.33 (N4), respectively collecting each elution peak, and collecting the parts except the elution peaks in a gradient manner according to time. After freeze-drying, the activity is determined at 20mM, pH8.0, and Tris-HCl redissolution is performed, and the N3 activity is optimal.

Further subjecting the active ingredient N3 to high pressure liquid phase multiple sample loading separation and purification to obtain a single peak, and freeze-drying to obtain lyophilized powder N3.

EXAMPLE 4 MALDI-TOF determination of molecular weight and amino acid sequence of active peptide PBN3

The study was conducted at the proteomics research center at the university of Compound denier with sample pre-treatments as follows: firstly, dissolving the freeze-dried powder N3 by using 50% acetonitrile water (containing 0.1% trifluoroacetic acid) solution, taking 0.5 mu L of dissolved sample on a MALDI target plate, naturally drying, then dotting 0.5 mu L of 4g/L CHCA solution (solvent, 50% acetonitrile water solution containing 0.1% trifluoroacetic acid), and naturally drying at room temperature. And then performing mass spectrometry and spectrogram analysis. A5800 type series time-of-flight mass spectrometer is adopted for mass spectrometry of a sample, a laser source is an Nd: YAG laser with the wavelength of 355nm, the accelerating voltage is 20kV, and data are acquired in a positive ion mode and an automatic data acquisition mode. The instrument uses myoglobin enzymolysis peptide fragment to carry out external standard correction. The PMF mass scan range for the matrix and sample was 700-3600Da. After MS is carried out, target peptide fragment ions are directly selected for MS/MS analysis. MS adopts Reflector Positive parameter: CID (OFF), mass ranging (700-3600 Da), focus Mass (1600 Da), fixed laser intensity (6000), digitizer: bin Size (0.5 ns). MS/MS adopts 1KV Positive parameter: CID (ON), precursor Mass Windows (Relative 100resolution (FWHM)), fixed laser intensity (7000), digitizer: bin Size (1 ns). The obtained MS/MS spectra were then sequenced De Novo using the own analytical tool De Novo Explorer of the Instrument software 4000 Explorer in combination with manual analysis. And (4) obtaining a sequence, and fragmenting the parent ions such as b and y on an MS/MS icon by using software Data Explorer to obtain the daughter ions.

As a result:

(1) molecular weight determination of peptide fragments

As can be seen from FIG. 3, the molecular ion peak value [ M + H ] was obtained by MS analysis using MALDI-TOF-TOF] ⁺¹ Is 759.44Da (FIG. 3).

(2) Peptide fragment sequence verification analysis

To calculate its sequence, the molecular ion peak [ M + H ] was assigned] ⁺¹ MS/MS analysis is carried out for 759.44, and then comparison analysis is carried out by adopting software to obtain a molecular ion peak value [ M + H ]] ⁺¹ The sequence of the polypeptide of 759.44 is LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (FIG. 4).

The molecular weight of the active peptide PBN3 is measured to be 759.44Da by using MALDI-TOF mass spectrometry technology, and the amino acid sequence of the active peptide PBN3 is LTAHYR (Leu-Thr-Ala-His-Tyr-Arg) (SEQ ID No. 2).

Example 5 verification of antitumor cell Activity of PBN3

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

The TIANGEN brand BCA protein concentration determination kit is applied in the research, and the detailed operation is as follows:

(1) mixing BCA reagent and Cu ²⁺ And (3) fully and uniformly mixing the reagents according to a ratio of 50 (v/v) to 1 (v/v) to obtain the BCA working solution.

(2) Freshly prepared BSA standard (Table 2) and test samples were added to a 96-well flat-bottom plate, 25. Mu.L each, and 3 replicates were set. Adding 200 mu L of BCA working solution into each hole, covering the hole, placing the hole in an enzyme labeling instrument, and uniformly mixing the hole and the plate by oscillation;

(3) placing the 96-well plate in a constant-temperature biochemical incubator at 37 ℃, and incubating for 30min;

(4) cooling the sample to room temperature, and detecting the absorbance of the sample by using an enzyme-linked immunosorbent assay (lambda =562 nm);

(5) and drawing a standard curve according to the BSA light absorption value and the concentration, and calculating the protein concentration in the sample through the standard curve.

Using BCA protein concentration assay, set λ =562nm, absorbance was determined for different concentrations of BSA, 3 replicates for each concentration. Calculating the average value, and drawing a BSA protein concentration standard curve by using the absorbance and the concentration of the standard protein.

The active PBN3 freeze-dried sample is prepared into a mother solution with a certain concentration by using 0.01M PBS buffer solution, and the protein concentration of the mother solution is measured by using a BCA method to measure the activity of the PBN 3.

TABLE 2 BSA Standard concentration preparation Table

(2) MTT method for detecting inhibition effect of PBN3 on proliferation of different tumor cells

In the experiment, the anti-tumor activity is detected by the non-small cell lung cancer cell A549 and an MTT method. For determining the IC of the active component on tumor cells ₅₀ Values, 6 gradients (with blank control) were set up in total and the loading volume was 20. Mu.L. The specific operation method comprises the following steps: (1) taking human non-small cell lung cancer cells A549 in logarithmic growth phase, discarding culture solution, and washing with 3-4mL of PBS. Adding 800 μ L of pancreatin digestive juice, digesting for 40-60s, sucking pancreatin, adding 5% of CO ₂ Incubating for 60-80s in a constant-temperature cell incubator at 37 ℃, observing under a microscope, and stopping reaction when about 80% of cells become round and shrink; (2) adding proper amount of RPMI-1640 cell culture solution into the digested cells, and blowing and beating the cells into single cell suspension by using a sterile pipette. Measuring the number of suspension cells with a cell counter, adding appropriate amount of cell suspension into fresh 18mL RPMI-1640 cell culture solution to make the concentration of new cell suspension be 3-4 × 10 ³ Per mL; (3) adding the cell suspension into a 96-well plate, adding 180 mu L of the cell suspension into each well, and culturing for 16-20h in a constant-temperature incubator; (4) adding 20 μ L of sample into each well, filtering with 0.22 μm sterile filter membrane before adding, and setting 3 parallel wells in each group; (5) then, the culture was continued in the incubator for 48 hours, and 20. Mu.L of MTT (5 mg/mL, i.e., 0.5% MTT) was added to each sample well, and the mixture was placed in the incubator for further 4 hours; (6) terminating the culture reaction, removing liquid in the holes, washing the holes for 2 times by using RPMI-1640 culture medium, adding 150 mu L DMSO into each hole, and oscillating the multifunctional microplate reader for 10min to fully dissolve the formazan crystal; (7) measuring the absorbance value at OD 570nm by using a microplate reader, and calculating the cell inhibition rate by using the following formula: cell inhibition (%) = ((OD control-OD experimental)/OD control) × 100%. The drug concentration when the inhibition rate reaches 50% is IC ₅₀ Value, experiment repetition 3 times resultsExpressed as mean ± SD.

Then according to the method, respectively paving the breast cancer cell MCF-7, the cervical cancer cell Hela, the pancreatic cancer cell Panc 28, the liver cancer cell HepG2 and the lung cancer cell NCI-H460 in a 96-well plate, adding PBN3 with different concentrations into the 96-well plate with different cells, and repeating 3 groups of the concentrations for observation. The half-lethal inhibition rate of PBN3 on tumor cells after treating different tumor cells for 48 hours is measured by using an MTT method.

The experimental results show that: PBN3 has good effect of inhibiting tumor cell proliferation for human non-small cell lung cancer cell A549, human large cell lung cancer cell NCI-H460, human hepatoma cell HepG2, human pancreatic cancer cell Panc 28, breast cancer MCF-7 and human cervical cancer cell Hela, and IC ₅₀ 38.24. Mu.g/mL, 18.99. Mu.g/mL, 25.92. Mu.g/mL, 32.65. Mu.g/mL, 28.74. Mu.g/mL and 14.34. Mu.g/mL, respectively (FIG. 5). Shows that PBN3 obtained from Brevibacillus sp.N189 has extensive effect of inhibiting tumor cell proliferation in vitro.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Shanghai health medical college

<120> ocean brevibacillus antitumor activity polypeptide and medicine and application thereof

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tgatccgcga ttactagcga ttccgacttc atgtaggcga gttgcagcct acaatccgaa 180

ctgagattgg ttttaagaga ttagcatctt ctcgcgaagt agcatcccgt tgtaccaacc 240

attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccgcc 300

ttcctccgtc ttgtcgacgg cagtctctct agagtgccca actgaatgct ggcaactaaa 360

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ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540

cttgtgcggg cccccgtcaa ttcctttgag tttcactctt gcgagcgtac tccccaggcg 600

gagtgcttat tgcgttagct gcggcactaa gggtattgaa acccctaaca cctagcactc 660

atcgtttacg gcgtggacta ccagggtatc taatcctgtt tgctccccac gctttcgcgc 720

ctcagtgtca gttacaggcc agaaagtcgc cttcgccact ggtgttcctc cacatctcta 780

cgcatttcac cgctacacgt ggaataccac tttcctctcc tgcactcaag ctacacagtt 840

tccaatgcga accgaggttg agccccgggc tttaacatca gacttacata gccacctgcg 900

cgcgctttac gcccaataat tccgggacaa cgcttgccac ctacgtatta ccgcggctgc 960

tggcacgtag tttagccgtg cttctcgtta ggtaccgtca aggtgcctac tctttattta 1020

aatggcactg tttcttcccc taacaacaga cttttacgac ccgaaagccc ttcatcgatc 1080

acgcgcatgc tcatcagact tccgtccatt gtgcaaaatc cttactgctg gctccggtag 1140

aagttctggc ccggtgttct ctccagttcc ccaagtcacc ctctcaggtc ggctacgcat 1200

cgtcgccttg gtgagccttt acctcaccaa ctagctaatg cgccgcaggc ccatctgcaa 1260

gtgatagctt gcgccatctt tccgtttcgc ttcatgcgaa gcaaaactct atccggtatt 1320

agcataagtt tccctatgtt atcccagtct tacaggcagg ttgcctacgt gttactcacc 1380

cgtccgccgc tagggtccga agaccctcgc tcgactgcat gtatagcacg cgcatgcc 1438

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Leu Thr Ala His Tyr Arg

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agagtttgat cctggctca 19

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<213> Artificial Sequence (Artificial Sequence)

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ggttaccttg ttacgactt 19

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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