CN110295132A - One plant has the active bacillus CCPM7645 of powerful anticancer and its application - Google Patents
One plant has the active bacillus CCPM7645 of powerful anticancer and its application Download PDFInfo
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- CN110295132A CN110295132A CN201910707075.6A CN201910707075A CN110295132A CN 110295132 A CN110295132 A CN 110295132A CN 201910707075 A CN201910707075 A CN 201910707075A CN 110295132 A CN110295132 A CN 110295132A
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Abstract
The present invention relates to one plant to have the active bacillus CCPM7645 of powerful anticancer and its application, belongs to biopharmaceutical technology.To solve the problems, such as that existing anticancer drug side effect is big, the present invention provides one plant to have the active bacillus CCPM7645 of powerful anticancer, belong to bacillus (Bacillus), represent a new bacterial species, it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number is CGMCC No.17887, the bacterium derives from human intestine, it is a kind of bacillus of new sources, anticancer activity metabolite can be generated, its metabolite treating cancer can be utilized.Since it is found in human body intestinal canal, or nothing very low for the side effect of human body solves the problems such as current anticancer drug side effect is big, and patient compliance is poor, and is expected to achieve the purpose that pre- anti-cancer by adjusting the balance of intestinal flora.
Description
Technical field
The invention belongs to biopharmaceutical technologies more particularly to one plant to have the active bacillus of powerful anticancer
CCPM7645 and its application.
Background technique
Cancer is all the difficulty that world medical circle is difficult to capture all the time as the first factor being detrimental to health.
The adverse changes of modern life mode, newly-increased emerging one after another for risk factor all promote the generation and development of cancer.Tradition
It mainly include radiotherapy chemotherapy to cancer treatment means, the drug resistance of cancer cell and be all this kind of hand to wrecking on body & mind
The shortcomings that Duan Xianzhu.Even if immunotherapy is risen in recent years, but still immature, there are many unsolved problems, carry out more
It is unable to do what one wishes.It is extremely urgent to find new anti-cancer methods.
Summary of the invention
To solve the problems, such as that existing anticancer drug side effect is big, the present invention provides one plant to have the active bud of powerful anticancer
Spore bacillus CCPM7645 and its application.
Technical solution of the present invention:
One plant has the active bacillus CCPM7645 of powerful anticancer, it belongs to bacillus (Bacillus), generation
One, table bacterial species (species) unknown before this, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers preservation, deposit number are CGMCC No.17887, and the deposit date is on 06 03rd, 2019, preservation address was Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1.
Bacillus CCPM7645 strain morphology feature: Fig. 1 is bacterium obtained by tri- ride culture of bacillus CCPM7645
Fall aspect graph.
The molecular biological characteristics of bacillus CCPM7645: the characteristic sequence of bacillus CCPM7645 such as SEQ ID
Shown in No:1, it is compared by having delivered bacterial sequences in BLAST and GenBank, the bacterium consistency with bacillus
Highest, as shown in Fig. 2 chadogram.The whole genome sequence of streptomycete CCPM7645 has been surveyed complete, and characteristic sequence is from streptomycete
The sequence fragment with clear identification meaning stood out from one's fellows in CCPM7645 whole genome sequence out.And bacillus CCPM7645
It with distinct biological property, is of great significance in bacterial species judgement, therefore is classified as bacillus
(Bacillus), bacillus CCPM7645 represents the bacillus new species without report before this.
One plant provided by the invention has the active bacillus CCPM7645 of powerful anticancer in terms of preparing anticancer drug
Application.
Further, the anticancer drug includes for treating lung cancer, liver cancer, breast cancer, cervix cancer, oophoroma, knot
The drug of the carcinoma of the rectum and/or leukaemia.
Further, the application is application of the fermentation liquid of bacillus CCPM7645 in terms of preparing anticancer drug.
Further, the application is metabolite the answering in terms of preparing anticancer drug of bacillus CCPM7645
With.
Further, the metabolite of the bacillus CCPM7645 the preparation method comprises the following steps:
From taking bacillus CCPM7645 single bacterium to fall in 5mL GRC001 fluid nutrient medium on plate, in certain culture temperature
Amplification cultivation certain time under degree and revolving speed, then 5mL bacterium solution is transferred in 100mL GRC001 fluid nutrient medium, in certain temperature
Bacterium solution, is finally centrifuged, takes supernatant by massive amplification culture certain time under degree and revolving speed.
Further, the formula of the GRC001 fluid nutrient medium are as follows:
Starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O
0.01g, distilled water are settled to 1L, high pressure steam sterilization.
Further, the temperature of the liquid amplification cultivation of the bacillus CCPM7645 is 14-39 DEG C, revolving speed 120
Rev/min, incubation time is 2-10 days.
Further, the temperature of the massive amplification culture of the bacillus CCPM7645 is 14-39 DEG C, revolving speed 120
Rev/min, incubation time is 2-10 days.
Further, the centrifugal condition is that 8000rpm/min is centrifuged 10min.
Beneficial effects of the present invention:
The present invention passes through the macro genome high flux examination combination conventional bacteria and the micro- life of molecule of intestinal microbiota
Object and genetic method, separation and identification have the active bacterium of powerful anticancer.It is provided by the invention to have powerful anticancer living
Property bacillus CCPM7645 derive from human intestine, be a kind of bacillus of new sources, have ocean, soil etc. other
The different property of the bacillus in source can generate the metabolite with very strong anticancer activity.The metabolite has wide spectrum
Anticancer activity, can be used for the treatment of cancer, including be used to prepare treatment lung cancer, liver cancer, breast cancer, cervix cancer, oophoroma, knot
The drug of the carcinoma of the rectum and/or leukaemia.
Since bacillus CCPM7645 is found in human body intestinal canal, after anticancer drug is made in its metabolite
For the very low or nothing of side effect meeting of human body, had no toxic side effect by zoopery confirmation to experimental animal.
Influence of the intestinal flora to health is gradually found and payes attention to, and suffers from very close pass with various diseases
System, therefore the research in intestinal flora field is carried out, there is profound significance on exploring efficiency natural anticancer new method.Pass through tune
The difference for saving the state of human body intestinal canal microorganism or the ratio of quantity, to achieve the purpose that pre- anti-cancer.
Detailed description of the invention
Fig. 1 is colonial morphology figure obtained by tri- ride culture of bacillus CCPM7645；
Fig. 2 is bacillus (Bacillus) CCPM7645 and nearly chadogram of the edge bacterium based on whole genome sequence；
Fig. 3 is morphological contrast map of the human ovarian cancer ES2 cell after the effect of the metabolite of bacillus CCPM7645,
A figure is that human ovarian cancer ES2 cell, the B figure after CCPM7645 effect are control；
Fig. 4 is the CCK8 experiment knot that human ovarian cancer ES2 cell acts on 6h by the metabolite of bacillus CCPM7645
Fruit figure；
Fig. 5 is bacillus CCPM7645 anticancer effect zoopery-mouse inoculation human ovarian cancer ES2 cell line.
Specific embodiment
Below with reference to embodiment, the following further describes the technical solution of the present invention, and however, it is not limited to this, all right
Technical solution of the present invention is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be contained
Lid is within the protection scope of the present invention.
Embodiment 1
One plant has the active bacillus CCPM7645 of powerful anticancer, it belongs to bacillus (Bacillus), generation
One, table bacterial species (species) unknown before this, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers preservation, deposit number are CGMCC No.17887, and the deposit date is on 06 03rd, 2019, preservation address was Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1.
Embodiment 2
Present embodiments provide the screening technique of bacillus CCPM7645 a kind of.
One, screening and culturing medium and the preparation method in relation to solution:
(1) GRC001 solid culture based formulas (1L):
Starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O
0.01g, agar 20g；First dissolution or suspension starch, are then added other compositions, shake up after high pressure.Starch in the present embodiment comes
From potato or maize or soybean or millet or other cereal or other plant.
(2) potassium bichromate: the final concentration of 50mg/L of solution.Storage liquid concentration is 5g/L, and solvent is water, and matching while using is kept away
Light, filtration sterilization.It is toxic, it is sure not to contact skin or sucking；When preparing 100mL GRC001 solid medium, 1mL weight chromium is added
Sour potassium stores liquid, is added when culture medium is not too hot, mixes.
(3)FeSO4·7H2O mother liquor: 0.01g/mL.1g FeSO4·7H2O is dissolved in 100mLH2In O, every 1000mL
1mL ferrous sulfate mother liquor is added in GRC001 solid medium.
(4) PBS buffer solution formula (1L): KH2PO40.27g, Na2HPO41.42g, NaCl 8g, KCl 0.2g.
Two, the screening technique of bacillus CCPM7645:
It is taken from the bacterium of human faecal mass sample, the specific method is as follows:
(1) one sampling packet of excrement supplier, including following article: 50mL centrifuge tube x 1 sampling explanation: are given；Meal is used
Soupspoon x 1；PE gloves x 2；Meal paper disc x 1；Informed consent form x 1；Sample specification x 1.
(2) operating instruction: reading informed consent form in detail fills in excrement supplier essential information in detail and signs；By excrement
Just it is discharged in disposable meal paper disc；PE gloves are taken, dining soupspoon takes excrement central part about 10g, is put into 50mL
In centrifuge tube, after tightening centrifuge tube lid, put back in valve bag；A operating process avoids touching other uncorrelated articles；It will dress
The centrifuge tube and informed consent form for having excrement are packed into valve bag, are returned by lab assistant centralized collection.
(3) sample treatment and bacterium are separately cultured: sampling, and number is suspended in the PBS buffer solution of 10 times of volumes, takes 100
μ l applies 90 millimeters of plates of diameter, in plate containing containing potassium bichromate+GRC001 solid medium, 14~39 DEG C are cultivated；
14-39 DEG C of three ride of single colonie culture, is shown in Fig. 1.
(4) deposit bacterium :+500 μ l GRC001 fluid nutrient medium of 500 μ l, 50% glycerol deposits bacterium after mixing.
Embodiment 3
Present embodiments provide the amplification method of bacillus CCPM7645 a kind of:
From taking single bacterium to fall in 5mL GRC001 fluid nutrient medium on plate, 120 revs/min, 14-39 DEG C, 2-10 is expanded
After it, then 5mL bacterium solution is transferred in 100mL GRC001 fluid nutrient medium, 120 revs/min, 14-39 DEG C, massive amplification 2-
10 days, finally bacterium solution is centrifuged, 8000rpm/min is centrifuged 10min, and supernatant is taken to do subsequent anticancer experiment.
Embodiment 4
Present embodiments provide a kind of bacillus CCPM7645 anticancer activity confirmatory experiment:
(1) human ovarian cancer ES2 cell culture: bed board, every 100 μ l of hole, cell density 1x105/ mL, to human ovarian cancer
The 5 μ l of metabolite of bacillus CCPM7645 is added after ES2 cell is adherent thereto, control group is isometric GRC001 liquid
Body culture medium is observed after being further cultured for 6-24 hours.Microscopic observation human ovarian cancer ES2 cellular morphology.Human ovarian cancer ES2 cell becomes
Justify, fall off, is dead, seeing Fig. 3.
(2) the metabolite preparation of bacillus CCPM7645: single bacterium is taken to fall on the training of 5mL GRC001 liquid from plate
It supports in base, 120 revs/min, 14-39 DEG C, bacterium solution 8000rpm/min is centrifuged 10min after amplification, supernatant is taken to do subsequent anticancer
Cell experiment.
(3) CCK8 is tested: human ovarian cancer ES2 plating cells, every 100 μ l of hole, cell density 1x105/ mL, to people's ovary
5 μ l of metabolite, 2.5 μ l, 1.25 μ l, 0.625 of bacillus CCPM7645 are separately added into after cancer ES2 cell is adherent thereto
The metabolite of bacterial strain is not added in μ l, 0.313 μ l, 0.156 μ l, control group, respectively sets 4 repeating holes, sees after being further cultured for 6-24 hours
It examines.10 μ l CCK8 solution are added in every hole, continue to measure each hole at enzyme-linked immunosorbent assay instrument OD450nm after cultivating 0.5-1h
Light absorption value.As a result as shown in figure 4, the metabolite of bacillus CCPM7645 for human ovarian cancer ES2 cell inhibiting effect
Gradient is presented with concentration.
It is significant finally to confirm that the bacillus CCPM7645 screened from people's enteron aisle has human ovarian cancer ES2 cell
Inhibitory activity.
(4) animal model for tumour is tested: after (SPF grades) of nude mice purchases, adaptive feeding one week, temperature (25 ± 2) DEG C was wet
Degree is maintained at 45% to 50%.It takes growth conditions good and is in logarithmic growth phase ES-2- ovarian cancer cell, in Biohazard Safety Equipment
Interior 0.25% pancreatin digests, and collects cell, and 5000r/min is centrifuged 5 minutes, and cell suspension, cytometer is made with cold PBS
Number instrument meter number viable count, and cell concentration is adjusted to 1 × 106Cell suspension is placed in stand-by on ice by a/mL.It sterilizes naked
Mouse injection site skin draws 50 μ L of cell suspension with syringe, is inoculated in nude mice by subcutaneous, forms small skin mound.What injection terminated
Nude mice applies ear's punch, and marking pen etc. is marked animal, measures to the weight of animals and tumor formation volume.Kind tumor
Afterwards, the next day, records following indexs: the weight of animals, knurl product, animal state etc..Visible knurl on the about 10th protrudes from subcutaneously, starts
Knurl product is recorded, and is administered according to experimental design, specially in CCPM7645 administration group and PBS control group right hind paw tumour
CCPM7645 metabolite (experimental group) and PBS buffer solution (control group) are injected respectively, and experimental result is as shown in Figure 4.After 22 days
Animal is put to death, in detail record weight (g), tumor weight (g), knurl product (V=0.5ab2, a major diameter, b minor axis), spleen weight amount (g).
The success rate that ES-2 ovarian cancer cell animal tumor is formed is 100%.As the process of experiment can be observed, compared with PBS group,
The gross tumor volume of CCPM7645 administration group is significantly less than control animals.Bacillus can be proved by results of animal
CCPM7645 has significant inhibitory activity to tumour cell, and anticancer effect is significant.
SEQUENCE LISTING
<110>the neat mankind's the second genome-based technologies development and application science and technology limited Company in Harbin day
<120>one plants have the active bacillus CCPM7645 of powerful anticancer and its application
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1464
<212> DNA
<213>bacillus (Bacillus sp.)
<400> 1
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gaagaagcta agcgtcgcaa aacaacggga ttccttgata acttaactgc tttaagtgtg 240
tgtacccatt gtaataagcc ggctatctgg atagatgaaa aattagtata tcctaaaaat 300
atttctgtag aacctccaaa ttctgatatg ccggaggacg ttaaaaaact ttatgatgaa 360
gctagagaaa taagtgtact atcacctaga gcctcggcag cattactaag attggcatta 420
gaaaaattac taccttcatt aggagcacag gggaaaagaa taaatgacat gataggtgac 480
ttggtaaaga aaggactttc gccgacaata caacaagctt tggatagctt acgtgtaata 540
ggaaatgaag ctgttcatcc aggttcaatt gatttagatg aggaaaaaga ggttgcgtta 600
gcattattta agttgattaa tttagttgta gaacaaatga taaccgaaaa aaaagaaata 660
caagcaattt atgatctaat tcctaaagat aaactagacg ggatacaaaa tcgagatcat 720
ctgaaaacaa caaaataaat gaagaaatta gttcttttgt ttgtaattgc ttttgcaaca 780
attggattat ctttgccgca acaagtatct gcccaagaaa ctcaaagtaa tagtcctaaa 840
gagcagctcg gttttactaa ggaacagcta gacaaggtga agcaaaatat ggagattgaa 900
ggagtagtat cttctgagac tcaggaaaag cttttggaca agcttgctaa aggagaagcc 960
ctagattctt ttgttttacc ggaagaaaaa gctgttgaca cgattaagaa agaaacggat 1020
aatggttatg aaataactta catttttgcc gatggttcct atacccaagc ggctgtggat 1080
gaacctgatt tctctgtggc tggtgtaggt attaaagatg gaaagtgcca aggtggatca 1140
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aaaaaagaaa ctgatttcaa aaaagccgaa gctagacttt cagctacatt gaatatcggt 1380
ggaggagtag gcagtataac acgaagtgtt tccctcatcg taggtaaaga taaagctaaa 1440
gcccagggca acacttacta ttaa 1464
Claims (10)
1. one plant has the active bacillus CCPM7645 of powerful anticancer, which is characterized in that it belongs to bacillus
(Bacillus), a new bacterial species are represented, China Committee for Culture Collection of Microorganisms's commonly micro- life has been preserved in
The preservation of object center, deposit number are CGMCC No.17887, and the deposit date is on 06 03rd, 2019, preservation address was Beijing
The institute 3 of Chaoyang District North Star West Road 1.
2. one plant as described in claim 1 there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space
The application in face.
3. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space according to claim 2
The application in face, which is characterized in that the anticancer drug include for treat lung cancer, liver cancer, breast cancer, cervix cancer, oophoroma,
The drug of colorectal cancer and/or leukaemia.
4. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen according to Claims 2 or 3
The application in object space face, which is characterized in that the application is the fermentation liquid of bacillus CCPM7645 in terms of preparing anticancer drug
Application.
5. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen according to Claims 2 or 3
The application in object space face, which is characterized in that the application is that the metabolite of bacillus CCPM7645 is preparing anticarcinogen object space
The application in face.
6. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space according to claim 5
The application in face, which is characterized in that the metabolite of the bacillus CCPM7645 the preparation method comprises the following steps:
From taking bacillus CCPM7645 single bacterium to fall in 5mL GRC001 fluid nutrient medium on plate, in certain cultivation temperature and
Amplification cultivation certain time under revolving speed, then 5mL bacterium solution is transferred in 100mL GRC001 fluid nutrient medium, in certain temperature and
Bacterium solution, is finally centrifuged by massive amplification culture certain time under revolving speed, takes supernatant.
7. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space according to claim 6
The application in face, which is characterized in that the formula of the GRC001 fluid nutrient medium are as follows:
Starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g steams
Distilled water is settled to 1L, high pressure steam sterilization.
8. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space according to claim 6
The application in face, which is characterized in that the temperature of the liquid amplification cultivation of the bacillus CCPM7645 is 14-39 DEG C, and revolving speed is
120 revs/min, incubation time is 2-10 days.
9. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticarcinogen object space according to claim 6
The application in face, which is characterized in that the temperature of the massive amplification culture of the bacillus CCPM7645 is 14-39 DEG C, and revolving speed is
120 revs/min, incubation time is 2-10 days.
10. one plant there is the active bacillus CCPM7645 of powerful anticancer to prepare anticancer drug according to claim 6
The application of aspect, which is characterized in that the centrifugal condition is that 8000rpm/min is centrifuged 10min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852682A (en) * | 2020-08-11 | 2021-05-28 | 刘树林 | Bacterial strain of Thelenota ananas closely related to bacillus and anticancer application thereof |
| WO2023142162A1 (en) * | 2022-01-26 | 2023-08-03 | 杭州师范大学 | Bacillus strain wyj-e14 isolated from curcuma wenyujin y. h. chen & c. ling and use thereof in preparation of anti-tumor drug |
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