CN106282050A - Actinomycetes BI87 that can produce active anticancer metabolite that one strain separates from human body intestinal canal and application thereof - Google Patents
Actinomycetes BI87 that can produce active anticancer metabolite that one strain separates from human body intestinal canal and application thereof Download PDFInfo
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- CN106282050A CN106282050A CN201610644214.1A CN201610644214A CN106282050A CN 106282050 A CN106282050 A CN 106282050A CN 201610644214 A CN201610644214 A CN 201610644214A CN 106282050 A CN106282050 A CN 106282050A
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Abstract
The invention discloses actinomycetes BI87 that can produce active anticancer metabolite that a strain separates from human body intestinal canal and application thereof.The present invention is the actinomycetes that can produce active anticancer metabolite of isolated, named BI87 from human body intestinal canal, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is: CGMCC No.12598.Research finds that the metabolite of this actinomycetes BI87 has the effect of anticancer.Owing to being the metabolite of antibacterial in human body intestinal canal, therefore low to human body side effect, the problems such as it is big that the proposition of the present invention solves current cancer therapy drug side effect, patient compliance difference, and it is expected to be reached the purpose of prophylaxis of cancer by the balance of regulating intestinal canal flora.
Description
Technical field
The present invention relates to strain actinomycetes, be separated to have from human body intestinal canal particularly to a strain and can produce anticancer work
The actinomycetes of property metabolite, the invention belongs to field of biological pharmacy.
Background technology
Cancer has become as the main reason that the middle and low income country people are dead at present, particularly with middle income country
For cause medical science applied progress due to expanding economy, other cause the sending out of disease of death simultaneously to cause aged tendency of population
Raw rate declines rapidly, and the relative fatality rate making cancer increases.And China is as the most developing country's situation of population in the world
Particularly severe.From last century, China's pathogenesis of cancer number increases the most day by day, nearly 30 years, China's cancer composition in the cause of the death
Ratio is risen to 22.3% by 20 century 70s 10.3%, and mortality rate is also risen to 135.88/10 ten thousand by 73.99/10 ten thousand.City
Area cancer is first cause of the death, and rural area is second cause of the death, and the principal element of cancer mortality includes aged tendency of population, smoking,
Dietary habit, fat etc. relevant, and by China's resident's nutrition and health survey being found the overweight rate of China resident during the decade
38.6% and 80.6% is risen respectively with obesity rates.Meanwhile, the diagnosis of cancer, treatment all can give patient, and country causes huge
Economic pressures.
The measure of reply cancer at present includes: prevention, early diagnosis, treatment.Wherein prevention includes smoking cessation prevention pulmonary carcinoma
Incidence rate, vaccinates the generation of the hepatocarcinoma preventing HCV infection thus prevent viral source；Treatment for cancer is led at present
Drug therapy to be concentrated on, operative treatment and emerging immunization therapy.Operative treatment can be able to reach sometimes with removal of lesions
Cure the purpose of cancer completely, but for cannot be carried out the end-stage patients of operation, control effective for cancer, extend as far as possible
Life span ensures that life quality becomes very important target simultaneously.Relative to the high medical expense of immunization therapy, pass
The Drug therapy of system becomes the first-selection of patient, and it is same that the antitumor drug applied the most clinically all also exists certain side effect
Time also result in drug resistance due to prolonged application, the most emerging antitumor drug is not sayed for the important function of clinical anticancer
And explain.
The mankind apply the material extraction in nature to obtain medicine has had the history of thousand of years to treat disease, from micro-
Biology obtains product to treat the cancer history the most more than 100 years.Therefore the medicine in natural product source is the newest
The important sources of medicine.The most microbe-derived natural product is most widely used with penicillin, lifts foot in drug discovery history
Weight.Wherein natural product abundant species produced by actinomycetes in microorganism, is medically being widely used, can produce multiple
Bioactive substance, antibacterial, antiviral, the aspect such as anticancer has application.
Actinomycetes are class G+ bacterium, and G+C% rich content has at nature and is distributed widely, mainly with spore or
Mycelia state is present in soil, air and water, especially enriches with Organic substance, in most in neutral or subalkaline soil,
Due in recent years for marine resources research deeply, it was found that the actinomycetes in ocean, enrich actinomycetic metabolite, also
Add the chance finding anti-cancer active matter.Along with the rise of the plan of microorganism group in recent years, the weight to people's internal microorganism
Visual range degree is continuously increased, and while research human microorganism with disease relationship, we can not ignore the microorganism in human body
Metabolite is also the very important source of bioactive substance, owing to the living environment of people's internal microorganism is special, therefore its
It is expected to very much become the new source of microbial metabolic products, is the most just very likely to produce new cancer-resisting substance.
The present invention utilizes the actinomycetic metabolite treatment cancer in Healthy People intestinal, by entering the sample collected
Row separation and Culture obtains actinomycetes, and to actinomycetes amplification culture, obtains its active metabolite, joins the cancer of Secondary Culture
In cell, cultivate 6 hours, in Microscopic observation cancerous cell form, find that pyknosis occurs in cancerous cell, dead.Illustrate to derive from strong
The HE actinomycetic metabolite of health has anticancer activity.
Summary of the invention
It is an object of the invention to the actinomycetes that can produce active anticancer metabolite that a strain separates from human body intestinal canal
BI87 and metabolite thereof and purposes.
For reaching above-mentioned purpose, present invention employs techniques below means:
(1) present invention is with Healthy People feces as sample, therefrom separation and Culture actinomycetes detect its active anticancer.
(2) for the actinomycetes amplification culture of isolated, it is thus achieved that a large amount of metabolites.
(3) checking of metabolite active anticancer is carried out with the cancerous cell line including HeLa cell, NB4 cell etc..
(4) metamorphosis of cancerous cell, and record are observed.
Inventor is separated and active anticancer screening final from human body intestinal canal isolated one strain can produce
The actinomycetes of raw active anticancer metabolite, named BI87, Classification And Nomenclature is Cyprinus carpio streptomycete (Streptomyces
Carpaticus), being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is exposed to the sun in Beijing
North Star West Road, district 1 No. 3 Institute of Microorganism, Academia Sinica of institute, its deposit number is: CGMCC No.12598, the preservation time
It it is on June 6th, 2016.
Further, the invention allows for a kind of metabolite with active anticancer, it is put by of the present invention
Line bacterium BI87 obtains after cultivating.
In the present invention, it is preferred to, the described metabolite with active anticancer is by actinomycetes of the present invention
The mono-colony inoculation of BI87 is cultivated in Gause I fluid medium, cultivates 5-7 days for 0-40 DEG C, and rotating speed is 150-200rpm,
After cultivation terminates, it is transferred to bacterium solution in Gause I fluid medium be enlarged cultivating, cultivates 8-12 days for 0-40 DEG C, rotating speed
For 150-200rpm, it is centrifuged, takes supernatant, be actinomycetic metabolite.
Further, the invention allows for described actinomycetes and metabolite thereof the use in preparing cancer therapy drug
On the way.
Compared to prior art, it is an advantage of the current invention that:
(1) actinomycetes derive from human body intestinal canal, are the actinomycetes in a kind of new source, possess the actinomycetes of other soil sources
Different character.
(2) being to find in healthy human body intestinal due to it, the side effect for human body is the lowest or nothing.
(3) by the regulation state of human body intestinal canal microorganism or the difference of the ratio of quantity, thus prophylaxis of cancer is reached
Purpose.
(4) convenient for production, cheap, reduce the financial burden of cancer patient.
Accompanying drawing explanation
Fig. 1 is the HeLa cell result through BI87 metabolite effect 6h；
Fig. 2 is the NB4 cell result through BI87 metabolite effect 6h；
Fig. 3 is that ZYZ (being located away from the blood of leukaemic in clinic) leukaemia is through BI87 metabolite effect
The result of 6h；
Fig. 4 is the evolution position of BI87；
Fig. 5 is the NB4 cell result through BI87 metabolite effect 24h, 48h, 72hMTT of variable concentrations.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.But these embodiments are only exemplary, the scope of the present invention is not constituted any restriction.People in the art
Member it should be understood that to enter the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention
Row amendment or replacement, but these amendments and replacement each fall within protection scope of the present invention.
The separation of embodiment 1 actinomycetes BI87 and qualification
(1) with Healthy People feces as sample, therefrom separation and Culture actinomycetes.
1, sampling explanation:
We will give every feces supplier one sampling bag, including following article:
50mL centrifuge tube x 1
Spoon x 1
PE glove x 2
Paper disc x 1
Informed Consent Form x 1
X 1 is said in sampling explanation
2, operating instruction:
(1) reading Informed Consent Form in detail, feces supplier signature also fills in essential information in detail.
(2) please try one's best and feces is discharged in disposable paper disc.
(3) PE glove on band, take feces core with spoon, and about 10g (reaches about centrifuge tube 10mL
Graduation mark at), put in 50mL centrifuge tube, after tightening centrifuge tube lid, put back in valve bag.
(4) whole operating process is avoided touching other uncorrelated article as far as possible.
(5) ensure that sample is fresh as far as possible.
(6) centrifuge tube and the Informed Consent Form that please will be equipped with feces load valve bag, lab assistant centralized collection return
Come.
(2) actinomycetic separation and Culture:
1. sampling, numbering: 1,2,3,4 ....Centrifugal: 8000rpm, 30min.Collect supernatant.
2. sample pellet is mixed with the sand after autoclaving.Outdoor air-dried one week.
3. pulverize, 80 DEG C of xeothermic 1h of constant temperature.
4. take the sample that 1g processed and be dissolved in 10mL PBS vibration 30min.
5. stand 30s.
6. take supernatant and carry out 10 times, 100 times, 1000 times of dilutions.
7. take 10-2, 10-3Diluent 100 μ l in dilution tube is coated with flat board (interpolation potassium dichromate) respectively.
8.0-40 DEG C cultivation 5-10 days.
9. take the line of single bacterium colony three road, cultivate 5-10 days for 0-40 DEG C.
10. from the flat board of three road line, take single close painting of bacterium colony, cultivate 5-10 days for 0-40 DEG C.
11. deposit bacterium: scrape a certain amount of thalline to after mixing in 500 μ l Gause I fluid mediums, add 500 μ l
50% glycerol, deposits bacterium for-80 DEG C.
12. inclined-planes: Inoculating needle picking colony inserts inclined-plane, is repeatedly inserted into, more repeatedly rules on inclined-plane, 0-40 DEG C of training
Support, standby.
Preparation of reagents:
1) Gause I solid medium (1L)
2) in culture medium, potassium dichromate concentration is 50mg/L (during screening use, other need not).
Storing liquid concentration in process for preparation is 5g/L.
3)FeSO4·7H2O mother solution: 0.01g/mL
By 1g FeSO4·7H2O is dissolved in 100mLH2It is female that O, 1000mL Gause I agar culture medium adds 1mL ferrous sulfate
Liquid.
4) PBS (1L)
(3) for the actinomycetes amplification culture of isolated:
It is enlarged cultivating in 3mL Gause I fluid medium by actinomycetic single colony inoculation, cultivates 6 days for 28 DEG C,
Rotating speed is 160rpm.After cultivation terminates, it is transferred to 3mL bacterium solution in 100mL Gause I fluid medium be enlarged cultivating,
Cultivating 10 days for 28 DEG C, rotating speed is 160rpm, centrifugal, takes supernatant, is actinomycetic metabolite.
Cultivate HeLa cell, Secondary Culture and bed board, every hole 100 μ l, be added thereto to put after HeLa cell attachment
The metabolite 5 μ l of line bacterium, observes after being further cultured for six hours.Microscopic observation Hela cellular morphology.The training method of NB4 cell
Identical with HeLa.Wherein, HeLa cell result after BI87 metabolite effect 6h is as shown in Figure 1；NB4 cell passes through
The result of BI87 metabolite effect 6h is as shown in Figure 2.As can be seen from the figure, after BI87 metabolite effect, cell goes out
Showed become round, pyknosis and death.
(4) from clinical leukemia patient blood sample, leukaemia is separated
1) transferring samples to 15ml centrifuge tube, 1500rpm is centrifuged 10min.
2) sucking-off blood plasma is placed in-80 DEG C and preserves the 1640 basis trainings adding equimultiple blood cell volume in backward 15ml centrifuge tube
Support base mixing.
3) in another one 15ml centrifuge tube, add the lymphocyte separation medium of blood cell volume 2 times, and will be with 1640
The hemocyte of mixing is slowly added to separate on liquid liquid level, and 2000rpm is centrifuged 20min.
4), after 1640 basal mediums of the superiors being absorbed, it is buffy coat in cloud layer under it, lymph is thin
Adding 5-6ml PBS after born of the same parents' sucking-off, 1500rpm is centrifuged 10min.
5) cell after being centrifuged adds in 1640 culture medium with serum to be cultivated, by named for this cell line ZYZ.
Treat that the density of ZYZ leukaemia reaches 1x105During/mL, add in the ZYZ leukaemia 100 μ L cultivated
Actinomycetic metabolite 5 μ L, observes after cultivating six hours.ZYZ leukaemia is after BI87 metabolite effect 6h
Result as shown in Figure 3.As can be seen from the figure leukaemia occurs in that pyknosis after the metabolite effect of BI87
And death.
(5) separation of actinomycetes BI87 and preservation
Separated and active anticancer screening has finally given a strain and can produce the unwrapping wire of relatively high anti-cancer activity metabolite
Bacterium, named BI87, Classification And Nomenclature is Cyprinus carpio streptomycete (Streptomyces carpaticus), is deposited in China Microbiological bacterium
Planting preservation administration committee common micro-organisms center, address is micro-in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Biological study institute, its deposit number is: CGMCC No.12598, and the preservation time is on June 6th, 2016.
(6) species identification of actinomycetes BI87:
The actinomycetes BI87 that can produce anti-cancer active matter is carried out three roads streak culture after, mailing is biological to gold dimension intelligence
Science and Technology Ltd. carries out 16S rRNA order-checking and qualification, and above-mentioned sequence, as shown in SEQ ID NO.1, is uploaded to by sequencing result
In ncbi database, comparison finds that BI87 belongs to Cyprinus carpio streptomycete (Streptomyces carpaticus).The evolution position of BI87
As shown in Figure 4.
The BI87 metabolite of the embodiment 2 variable concentrations MTT experiment to NB4 cell
Cultivating NB4 cell, Secondary Culture and paving 96 plates, every hole 100 μ L, cell density is 1x106/ mL, surrounding paving 100
The PBS of μ L, it is to avoid the liquid evaporation in incubation, the actinomycetes metabolite being separately added into variable concentrations (is implemented
Prepared by example 1), with arsenic trioxide injection (final concentration 0.2 μm ol/L) as positive control, Gause I fluid medium is cloudy
Property comparison, respectively set 6 repeating holes, after being further cultured for 6-72 hour observe.Every hole adds 20 μ L MTT solution, continues to cultivate 4h.Eventually
Only cultivate, carefully suck culture fluid (if herein by purple crystal material sucking-off, final result can be affected) in hole.Every hole
Add 150 μ L dimethyl sulfoxide, put low-speed oscillation 10min on shaking table, make crystalline material fully dissolve.At enzyme-linked immunosorbent assay instrument
The light absorption value in each hole is measured at OD492nm.Result is as it is shown in figure 5, as can be seen from Figure 5 along with incubation time and actinomycetes generation
Thanking to the increase of production concentration, the inhibitory action of NB4 cell is constantly strengthened by BI87 metabolite, and the BI87 that the present invention obtains is described
Metabolite is inhibited to NB4 cell.
Claims (5)
1. the actinomycetes that can produce active anticancer metabolite that a strain separates from human body intestinal canal, named BI87, it is deposited in
China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCCNo.12598.
2. a metabolite with active anticancer, it is characterised in that obtained after cultivating by the actinomycetes described in claim 1
Arrive.
There is the metabolite of active anticancer the most as claimed in claim 1, it is characterised in that be by putting described in claim 1
Line bacterium list colony inoculation is cultivated in Gause I fluid medium, cultivates 5-7 days for 0-40 DEG C, and rotating speed is 150-200rpm,
After cultivation terminates, it is transferred to bacterium solution in Gause I fluid medium be enlarged cultivating, cultivates 8-12 days for 0-40 DEG C, rotating speed
For 150-200rpm, it is centrifuged, takes supernatant, be actinomycetic metabolite.
4. the purposes in preparing cancer therapy drug of the actinomycetes described in claim 1.
5. the purposes in preparing cancer therapy drug of the metabolite described in Claims 2 or 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283762A (en) * | 2019-08-01 | 2019-09-27 | 哈尔滨天齐人类第二基因组技术开发应用科技有限责任公司 | One plant has the active streptomycete CCPM7649 of powerful anticancer and its application |
| CN110373344A (en) * | 2019-04-29 | 2019-10-25 | 广西科学院 | Carp streptomycete and its application |
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| AU2004230489A1 (en) * | 2003-04-15 | 2004-10-28 | Basf Plant Science Gmbh | Plant cells and plants with increased tolerance to environmental stress |
| CN103396957A (en) * | 2013-07-11 | 2013-11-20 | 中国海洋大学 | Streptomyces-carpaticus-derived marine microbial polysaccharide and preparation method thereof |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004230489A1 (en) * | 2003-04-15 | 2004-10-28 | Basf Plant Science Gmbh | Plant cells and plants with increased tolerance to environmental stress |
| CN103396957A (en) * | 2013-07-11 | 2013-11-20 | 中国海洋大学 | Streptomyces-carpaticus-derived marine microbial polysaccharide and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373344A (en) * | 2019-04-29 | 2019-10-25 | 广西科学院 | Carp streptomycete and its application |
| CN110373344B (en) * | 2019-04-29 | 2022-07-29 | 广西科学院 | Streptomyces carpio and application thereof |
| CN110283762A (en) * | 2019-08-01 | 2019-09-27 | 哈尔滨天齐人类第二基因组技术开发应用科技有限责任公司 | One plant has the active streptomycete CCPM7649 of powerful anticancer and its application |
| CN110283762B (en) * | 2019-08-01 | 2020-09-22 | 哈尔滨天齐人类第二基因组技术开发应用科技有限责任公司 | Streptomycete CCPM7649 with strong anticancer activity and application thereof |
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