CN103224898B - Marine bacillus and its polypeptide with antitumor activity - Google Patents
Marine bacillus and its polypeptide with antitumor activity Download PDFInfo
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- CN103224898B CN103224898B CN201310128152.5A CN201310128152A CN103224898B CN 103224898 B CN103224898 B CN 103224898B CN 201310128152 A CN201310128152 A CN 201310128152A CN 103224898 B CN103224898 B CN 103224898B
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Abstract
The invention relates to a marine bacillus and its polypeptide with antitumor activity. The strain is preserved at China Center for Type Culture Collection in Wuhan, and its preservation number is CCTCC M 2013063. A novel marine bacillus polypeptide with anti-tumor activity is obtained from a fermentation product of the strain. By means of an MTT method, the invention finds that the polypeptide has an obvious proliferation inhibition effect on human hepatoma carcinoma cell BEL-7402, human breast cancer cell MCF-7, human glioma cell U251, human non-small-cell lung cancer cell A549 and other tumor cells, has relatively small cytotoxicity on human fibroblast HFL1, and can be applied in drugs treating human liver cancer, gliomas, lung cancer and breast cancer.
Description
Technical field
The invention belongs to marine microorganism field, relate to particularly a kind of bacillus marinus of anti-tumour active polypeptide and polypeptide with anti-tumor activity of generation thereof produced.
Background technology
Society, the mankind's life and health in malignant tumour serious threat, is the first killer of human society.Mostly treating malignant tumor means are to adopt composite treatment clinically, combine with excision, radiotherapy, chemotherapy and immunotherapy.Though the antitumor drug having used at present has certain curative effect to most of tumours, but still exist that treated effect is low, poor selectivity, toxic side effect obviously, easily produce the problems such as cells resistance.Therefore, find efficient, low toxicity, the clear and definite antitumor drug of action target spot and be still the study hotspot in biology, medical research field.
Due to geography, weather and the environmental quality of ocean uniqueness, the novelty that marine organisms have and diversity, all have important value at the aspect such as scientific research, application and development.Therefore ocean is considered to potential, an important Biological resources storehouse, may be the potential provenance that produces novel bioactive material and lead compound, brings new opportunity and breakthrough will to the screening of new type natural medicine and discovery.Marine microorganism contains the antitumor metabolites of abundant novel structure, and they mostly are alkaloids, terpene, macrolides compound, is mainly derived from marine actinomycete and thalassiomycetes.It is relatively less that but the antitumor lead compound that derives from marine bacteria is studied, and awaits doing in this respect deep research.
Summary of the invention
The technical problem that will solve of the present invention is to provide a kind of bacillus marinus N16(Bacillus sp.N16 that produces anti-tumor active substance); This bacterial strain can produce the polypeptide with anti-tumor activity.
A kind of bacillus marinus, it is the bacillus marinus that culture of isolated obtains producing new antitumoral material from seawater, called after bacillus marinus N16(Bacillus sp.N16), this bacillus marinus N16 is preserved in Wuhan, China typical case's culture collection center on March 4th, 2013, and preserving number is CCTCC M2013063.
The present invention also provides a kind of bacillus marinus N16 polypeptide with anti-tumor activity, and this polypeptide molecular weight is 1015Da, is made up of 9 amino acid, and aminoacid sequence is Arg-Cys-Phe-Ser-Ile-Met-Ser-Asp-Arg.
The present invention also provides a kind of screening method of bacillus marinus N16 polypeptide, the steps include:
(1) fermentation culture
By bacillus marinus N16 fermentation, fermentation condition: in fermented liquid, the final concentration of each composition is extractum carnis 4-6g/L, peptone 8-12g/L, sodium-chlor 4-7g/L, pH7.0-7.6, sterilizing 1.05kg/cm
2, 20-35min cultivates 20-33h under 150-250r/min, the condition of 25 DEG C in rotary shaker;
(2) ultra-filtration and separation
By the fermented liquid of bacillus marinus N16, centrifugal in batches, 8000-10000g, 5-10min, collecting supernatant liquor, is the ultra-filtration membrane ultrafiltration of 0.3kDa, 5kDa and 50kDa with molecular weight, obtains the different components of molecular weight 0.3-5kDa and 5-50kDa, detect the anti-tumor activity of each component, result shows anti-tumor activity the best of molecular weight 0.3-5kDa component;
(3) diethylaminoethyl-(DEAE) FF anion-exchange chromatography
The component with best anti-tumor activity that ultrafiltration is obtained, cross DEAE FF anion-exchange chromatography prepacked column, sample-loading buffer is 3-6mmol/L pH8.0-9.5 Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid, each applied sample amount is 5-20ml, elutriant is the 3-6mmol/L pH8.0-9.5Tris-HCl containing 0.5-2mol/L NaCl, elutriant carries out gradient elution with the concentration of 0-100% (v/v), flow velocity is 3-6mL/min, detection wavelength is 280nm, collect each peak, detect the anti-tumor activity of each peak component, the component desalination final vacuum lyophilize with better anti-tumor activity peak is preserved,
(4) Superdex30 gel permeation chromatography
Get the component with better anti-tumor activity peak that DEAE collects, cross Superdex30 gel-filtration prepacked column, each applied sample amount is 1-10ml, moving phase is ultrapure water, flow velocity is 1-5ml/min, collects each peak, detects the anti-tumor activity of each peak component, the component desalination final vacuum lyophilize with better anti-tumor activity peak is preserved, be bacillus marinus N16 polypeptide.
Further, the detection method of described anti-tumor activity, for taking liver cancer cell BEL-7402 as target cell, adopts mtt assay to follow the trail of antitumor component.
Further, described mtt assay: the liver cancer cell BEL-7402 in the vegetative period of taking the logarithm, with after trysinization, be inoculated in 96 orifice plates with the density in 4 × 104/hole, every hole 180 μ L, at 37 DEG C, cultivate 24h, then application of sample, sample solution should first pass through filtering with microporous membrane degerming, diluted sample is become to 5 concentration gradients with nutrient solution, every hole adds 20 μ L, 4 parallel holes, be placed in cell culture incubator and continue to cultivate 48h, then, add 5mg/mL MTT20 μ L, put 37 DEG C of CO2 incubators and cultivate 4h; Nutrient solution in reject hole, then every hole adds DMSO150 μ L, and 37 DEG C of constant temperature oscillation 30min are fully to dissolve the crystallization of first a ceremonial jade-ladle, used in libation, and microplate reader detects each hole at 570nm wavelength place absorbance, and experiment at least repeats 3 times; Cell inhibitory rate=［ (A contrast-A sample)/A contrast ］ × 100%, wherein A is absorbance; Adopt Excel analysis software calculation of half inhibitory concentration IC50.
Further, in the elutriant in above-mentioned steps (3), the concentration of NaCl is preferably 1mol/L.
Further, the flow velocity of the elutriant in above-mentioned steps (3) is preferably 5.0mL/min.
Further, in above-mentioned steps (3), each applied sample amount is preferably 10mL.
Further, in above-mentioned steps (4), each applied sample amount is preferably 5mL.
Further, in above-mentioned steps (4), moving phase is ultrapure water, and flow velocity is preferably 2.6mL/min.
The present invention also provides a kind of antitumor drug, and this pharmaceutical pack is containing bacillus marinus N16 polypeptide.
The present invention also provides the application of a kind of bacillus marinus N16 polypeptide in anti-tumor medicine.
The present invention's beneficial effect compared with prior art:
This bacterial strain can be stable the polypeptide of expression anti-tumor activity, this polypeptide has good soda acid and thermostability, and kinds of tumor cells is had to cytotoxicity, relatively little to the cell toxicant of human normal cell line, has good research and using value.
Brief description of the drawings
The electron scanning micrograph of Fig. 1 bacterial strain N16
The DEAE FF anion chromatography collection of illustrative plates of Fig. 2 crude extract
The superdex30 gel permeation chromatography collection of illustrative plates of Fig. 3 ion-exchange active ingredient
Fig. 4 MALDI-TOF-TOF measures the aminoacid sequence of bacillus marinus polypeptide
Fig. 5 bacillus marinus N16 polypeptide to the different tumour cells of people and Normocellular cell toxicant
Bacillus marinus N16, Latin name is called Bacillus sp.N16, is preserved in Luo Jia Shan, wuchang, wuhan Wuhan University Wuhan, China typical case's culture collection center on March 4th, 2013, and preserving number is CCTCC M2013063.
Embodiment
Further illustrate technical scheme of the present invention below by embodiment, but protection scope of the present invention is not subject to any type of restriction of embodiment.Be not limited to the following example:
Embodiment 1
The screening and separating process of bacillus marinus N16
(1) active primary dcreening operation
Adopt respectively the different culture media such as seawater base basal culture medium 2216E and beef-protein medium NRG to carry out optionally separation and Culture to seawater sample, obtain altogether 47 strain list bacterium.This 47 strain microbial fermentation product is carried out to anti-tumor activity preliminary screening, found that, the inhibiting rate of liver cancer cell BEL-7402 is reached to more than 60% 1 strain that has; Inhibiting rate reaches 1 strain that has more than 50-60%.
(2) bacterial strain sieves again
Measure above-mentioned 2 kinds of active bacterial strain fermented liquids to other tumour cells, comprise the increment restraining effect of human breast cancer cell MCF-7, human glioma cells U251 and Non-small cell lung carcinoma cell A549.Result shows, wherein there is the fermented liquid of a bacterial strain N16 all to there is good cell inhibitory effect effect to above tumour cell, wherein human breast cancer cell MCF-7, human glioma cells U251 and Non-small cell lung carcinoma cell A549 people's inhibiting rate is respectively to 68.1%, 59.8%, 63.1%.
Embodiment 2
Bacillus marinus N16 morphological specificity and physiological and biochemical property
(1) morphological specificity
Bacterial strain is carried out to gramstaining, is gram-positive microorganism.Bacterial strain N16 has bacillus form and produces the key character bacillus of these two kinds of bacillus of gemma ability, adopts plate inserted sheet method to cultivate, and with the thalli morphology of scanning electron microscopic observation bacterial strain N16,0.8-2.0 μ m is long, 0.2-0.6 μ m wide (Fig. 1).At 25 DEG C, on seawater base basal culture medium 2216E, cultivate after 24h, bacterium colony ellipse, Quan Yuan, flat, diameter 0.5 is to 1.5mm, smooth tarnish, mucus shape outward appearance, oyster white.
This bacterium is aerobic bacteria, is 7.5 in pH value, and culture temperature is 25 DEG C, NaCl in medium 6%(w/v) time well-grown.
(2) biochemical reactions feature
Main according to the method for " general bacteriology method handbook " and " common bacteria system identification handbook ", determination part divides physiological and biochemical property, negative: oxidase test, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylation, urase, gelatinase, EC 1.4.1.19, indoles experiment, the positive: nitrate reductase, VP experiment, semi-lactosi formula enzyme, beta-galactosidase enzymes, citric acid utilization, H
2s produces; Fermentation utilizes: negative findings, N.F,USP MANNITOL, sucrose, melibiose, inositol, sorbyl alcohol, positive findings, glucose, pectinose; Assimilation experiment is negative: seminose, N.F,USP MANNITOL, N-acetyl-glucosamine, maltose, gluconate, oxysuccinic acid, citric acid.
Embodiment 3
Preparation bacterial genomes DNA in a small amount, by pcr amplification 16S rDNA, adopting agarose gel electrophoresis to detect PCR product is after single band, directly to serve Hai Shenggong order-checking.Survey two reactions, survey logical.Sequence total length is 1542bp, and 16S rDNA sequence is shown in sequence table.
This 16SrDNA sequence is carried out to BLAST comparison in NCBI website, carry out homologous sequence retrieval, found that this bacterium is bacillus (Bacillus), called after bacillus marinus N16(Bacillus sp.N16).This bacillus marinus N16 is preserved in Wuhan, China typical case's culture collection center on March 4th, 2013, and preserving number is CCTCC M2013063.The probability that 16S rDNA gene order is undergone mutation in some site is different, but kind, show the conservative property of structure and function in genus level, have the reputation of " bacterial fossil ", be the timing register of organic evolution history.Adopt 16S rDNA as molecular indexes, can realize to microorganism carry out fast, accurately, trace, easy classification identify.
Embodiment 4
There is the preparation of the bacillus marinus N16 polypeptide of anti-tumor activity
(1) fermentation culture
Get bacillus marinus N16 bacterial strain one ring of preserving on inclined-plane and be inoculated in seed fermentation liquid, 25 DEG C, 200r/min cultivates 24h, the centrifugal 20min of fermented liquid 4000r/min, and gained supernatant liquor is the thick liquid containing tumor protein p53.In fermented liquid, the final concentration of each composition is: extractum carnis 5g/L, peptone 10g/L, NaCl5g/L, pH7.5.
(2) ultra-filtration and separation
By bacterial strain fermentation liquor, centrifugal in batches, 8000g, 7min, collecting altogether about 5000mL supernatant liquor, is the ultra-filtration membrane ultrafiltration of 0.3kDa, 5kDa and 50kDa with molecular weight, obtains the different components of molecular weight 0.3-5kDa and 5-50kDa, detect the anti-tumor activity of each component, result shows component anti-tumor activity the best of molecular weight 0.3-5kDa.
(3) diethylaminoethyl-(DEAE) FF anion-exchange chromatography
By the best antitumor component that has of ultrafiltration acquisition, cross DEAE FF anion-exchange chromatography prepacked column, sample-loading buffer is 4mmol/L pH8.5Tris-HCl damping fluid, each applied sample amount is 10mL, elutriant is the 4mmol/L pH8.5Tris-HCl containing 1mol/L NaCl, elutriant is first successively with 3% (v/v), 6% (v/v), 9% (v/v), 12% (v/v), 24% (v/v), the concentration of 100% (v/v) is carried out gradient elution, flow velocity is 5mL/min, detection wavelength is 280nm, chromatography collection of illustrative plates (Fig. 2), collect each peak, detect the anti-tumor activity of each peak component, result shows that peak 2 components have best anti-tumor activity, peak 2 component desalination final vacuum lyophilizes are preserved.
(4) Superdex30 gel permeation chromatography
Get the best antitumor component of above-mentioned steps, cross Superdex30 gel-filtration prepacked column, each applied sample amount is 5mL, and moving phase is ultrapure water, and flow velocity is 2.6mL/min, collect each peak, chromatography collection of illustrative plates (Fig. 3), detects the anti-tumor activity of each peak component, and result shows that peak 8 components have anti-tumor activity, peak 8 component desalination final vacuum lyophilizes are preserved, be bacillus marinus N16 polypeptide.
Described each peak component anti-tumor activity analysis, for taking liver cancer cell BEL-7402 as target cell, adopts mtt assay to follow the trail of antitumor component.
Mtt assay: the liver cancer cell BEL-7402 in the vegetative period of taking the logarithm, with after trysinization, be inoculated in 96 orifice plates with the density in 4 × 104/hole, every hole 180 μ L, at 37 DEG C, cultivate 24h.Then application of sample, sample solution should first pass through filtering with microporous membrane degerming, diluted sample is become to 5 concentration gradients with nutrient solution, and every hole adds 20 μ L, and 4 parallel holes are placed in cell culture incubator and continue to cultivate 48h.Then, add MTT(5mg/mL) 20 μ L, put CO
237 DEG C of cultivation 4h of incubator.Nutrient solution in reject hole, then every hole adds DMSO150 μ L, and 37 DEG C of constant temperature oscillation 30min are fully to dissolve the crystallization of first a ceremonial jade-ladle, used in libation.Microplate reader detects each hole in 570nm wavelength place absorbancy (A) value, and experiment at least repeats 3 times.Cell inhibitory rate=［ (A contrast-A sample)/A contrast ］ × 100%.Adopt Excel analysis software calculation of half inhibitory concentration IC50.
Embodiment 5
Bacillus marinus N16 peptide molecule flow measurement and amino acid analysis:
Peak in the Fig. 3 preparing by above flow process 8 antitumor component are carried out to high pressure liquid phase analysis, and result is simple spike.Carry out mass spectroscopy with time-of-flight mass spectrometry instrument (Proteomics Analyzer, TOF/TOF TM), adopt positive ion mode and automatic acquisition data pattern image data.Carry out mass spectrum (Mass Specrometry, MS) analysis, detect that the molecular weight of active ingredient is about 1015Da, formed by 9 amino acid.Further analyze through MS/MS, De Novo Explorer de novo sequencing, sequencing result is shown in Fig. 4, the aminoacid sequence that obtains bacillus marinus N16 polypeptide is Arg-Cys-Phe-Ser-Ile-Met-Ser-Asp-Arg.Carry out protein to protein BLAST comparison in NCBI website, the bioactive peptide matching with it does not detected; Explanation thus, this polypeptide that bacillus marinus N16 produces is a kind of new anti-tumour active polypeptide, called after bacillus marinus N16 polypeptide.
Embodiment 6
Bacillus marinus N16 polypeptide is to different tumour cells and Normocellular cell toxicant.
Respectively taking tumour cells such as human liver cancer cell BEL-7402, human glioma cells U251, Non-small cell lung carcinoma cell A549 and human breast cancer cell MCF-7 as target cell, adopt mtt assay, detect bacillus marinus N16 polypeptide various tumour cells are had to inhibited proliferation, wherein, the IC to BEL-7402
50value is 5.96 μ mol/L, relatively little to the cytotoxicity of people's lung fibroblast HFL1, as shown in Figure 5.Therefore, bacillus marinus N16 polypeptide of the present invention can be applied in the medicine for the treatment of people liver cancer, glioma, lung cancer, mammary cancer.
Claims (4)
1. a bacillus marinus N16, is characterized in that its Latin name is called Bacillus sp.N16, is preserved in Wuhan, China typical case's culture collection center on March 4th, 2013, and preserving number is CCTCC M2013063.
2. the polypeptide that bacillus marinus N16 claimed in claim 1 produces, this polypeptide molecular weight is 1015Da, is made up of 9 amino acid, aminoacid sequence is Arg-Cys-Phe-Ser-Ile-Met-Ser-Asp-Arg.
3. an antitumor drug, this pharmaceutical pack is containing the polypeptide described in claim 2, described tumour behaviour liver cancer, people's glioma, people's lung cancer and human breast carcinoma.
4. polypeptide claimed in claim 2 is in the application of preparing in anti-tumor medicine, described tumour behaviour liver cancer, people's glioma, people's lung cancer and human breast carcinoma.
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| CN103555622B (en) * | 2013-10-24 | 2016-05-18 | 中国水产科学研究院黄海水产研究所 | The anti-tumour active polypeptide of bacillus marinus S-1 and product thereof |
| CN104473973B (en) * | 2014-12-19 | 2015-08-05 | 山东创新药物研发有限公司 | The application of one strain Clostridium ghonii domestication strain |
| CN104630111B (en) * | 2015-02-09 | 2017-10-27 | 湖南师范大学 | The separation method and application of one bacillus amyloliquefaciens and its active metabolite |
| CN105884871B (en) * | 2016-04-28 | 2019-10-01 | 中国水产科学研究院黄海水产研究所 | A kind of bacillus marinus albumen with anti-tumor activity and its preparation and application |
| CN106474161A (en) * | 2016-10-18 | 2017-03-08 | 桂林洁宇环保科技有限责任公司 | Anaerobic spore-bearing bacilli live body is preparing the application that anticancer is swollen in solid tumor drugs |
| CN107384819B (en) * | 2017-07-19 | 2020-05-22 | 中国水产科学研究院黄海水产研究所 | Enteromorpha bacillus Y15-8 and anti-tumor active protein thereof |
| CN107988105B (en) * | 2017-12-15 | 2021-06-18 | 中国科学院海洋研究所 | Marine bacillus and application thereof |
| CN111593007B (en) * | 2020-06-04 | 2021-10-12 | 上海健康医学院 | Arthrobacter marinum, active components thereof and screening method |
| CN112111435B (en) * | 2020-10-27 | 2022-05-10 | 集美大学 | Bacillus NB-1 and culture method and application thereof |
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| CN101709058A (en) * | 2009-09-30 | 2010-05-19 | 浙江大学 | Polyene macrolides compound, preparation and application thereof |
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| CN101709058A (en) * | 2009-09-30 | 2010-05-19 | 浙江大学 | Polyene macrolides compound, preparation and application thereof |
| CN102061280A (en) * | 2010-11-29 | 2011-05-18 | 西南大学 | Bacillus subtilis SWB8 for producing diosgenin |
| CN102816716A (en) * | 2012-07-31 | 2012-12-12 | 西安交通大学 | Extraction and application of exopolysaccharide metabolite of bacillus amyloliquefaciens strain |
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