CN110295132B - A strain of Bacillus CCPM7645 with strong anticancer activity and its application - Google Patents
A strain of Bacillus CCPM7645 with strong anticancer activity and its application Download PDFInfo
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Abstract
本发明涉及一株具有强力抗癌活性的芽孢杆菌CCPM7645及其应用，属于生物制药技术领域。为解决现有抗癌药物副作用大的问题，本发明提供了一株具有强力抗癌活性的芽孢杆菌CCPM7645，属于芽孢杆菌属(Bacillus)，代表一个新的细菌物种，已保藏于中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.17887，该菌来源于人类肠道，是一种新来源的芽孢杆菌，能产生抗癌活性代谢产物，可利用其代谢产物治疗癌症。由于其是在人体肠道内发现，因此对于人体的副作用很低或无，解决了目前抗癌药物副作用大，病人顺应性差等问题，并且有望可以通过调节肠道菌群的平衡达到预防癌症的目的。
The present invention relates to a strain of Bacillus CCPM7645 with strong anticancer activity and its application, belonging to the field of biopharmaceutical technology. In order to solve the problem of large side effects of existing anticancer drugs, the present invention provides a strain of Bacillus CCPM7645 with strong anticancer activity, belonging to the genus Bacillus (Bacillus), representing a new bacterial species, which has been preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the preservation number is CGMCC No.17887. The bacteria originates from the human intestine and is a new source of Bacillus, which can produce anticancer active metabolites, and its metabolites can be used to treat cancer. Since it is found in the human intestine, it has very low or no side effects on the human body, which solves the problems of large side effects of current anticancer drugs and poor patient compliance, and is expected to achieve the purpose of preventing cancer by regulating the balance of intestinal flora.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to bacillus CCPM7645 with strong anticancer activity and application thereof.
Background
Cancer, the first factor that is harmful to human health, has long been a difficult challenge for the world medical community. The adverse changes of the living modes of modern people and the ever-growing danger factors promote the occurrence and development of cancers. Traditional treatments for cancer, including mainly radiotherapy and chemotherapy, resistance to cancer cells and physical and mental disabilities, are significant drawbacks of these approaches. Even though immunotherapy has emerged in recent years, it is still immature, and many unsolved problems exist, which are not very practical. It is urgent to find new anticancer methods.
Disclosure of Invention
In order to solve the problem of large side effect of the existing anticancer drugs, the invention provides bacillus CCPM7645 with strong anticancer activity and application thereof.
The technical scheme of the invention is as follows:
A Bacillus CCPM7645 with strong anticancer activity belongs to Bacillus and represents a previously unknown bacterial species (species) which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.17887, the preservation date of 2019 of 06 month 03 and the preservation address of North Star Xila No.1, 3 in the Korean region of Beijing.
Morphological characteristics of bacillus CCPM7645 strain: FIG. 1 is a diagram showing colony morphology obtained by three-compartment streak culture of Bacillus CCPM 7645.
Molecular biological characteristics of bacillus CCPM 7645: the characteristic sequence of bacillus CCPM7645 is shown as SEQ ID No:1, the identity with bacteria of the genus bacillus is highest by BLAST compared to published bacterial sequences in GenBank, as shown in the evolutionary tree of fig. 2. The whole genome sequence of bacillus CCPM7645 has been detected, and the characteristic sequence is a sequence fragment which has clear identification meaning and is extracted from the whole genome sequence of bacillus CCPM 7645. Moreover, bacillus CCPM7645 has clear biological characteristics and is of great importance in determining bacterial species, and therefore is classified as Bacillus, and Bacillus CCPM7645 represents a novel species of Bacillus that has not been reported before.
The bacillus CCPM7645 with strong anticancer activity provided by the invention is applied to the preparation of anticancer drugs.
Further, the anticancer drugs include drugs for treating lung cancer, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer and/or leukemia.
Further, the application is the application of the fermentation broth of bacillus CCPM7645 in preparing anticancer drugs.
Further, the application is the application of the metabolite of bacillus CCPM7645 in preparing anticancer drugs.
Further, the preparation method of the metabolite of bacillus CCPM7645 comprises the following steps:
And (3) taking bacillus CCPM7645 single colony from a flat plate, amplifying and culturing in 5mL GRC001 liquid culture medium at a certain culture temperature and rotating speed for a certain time, transferring 5mL bacterial liquid into 100mL GRC001 liquid culture medium, amplifying and culturing for a certain time at a certain temperature and rotating speed in a large quantity, and finally centrifuging the bacterial liquid, and taking supernatant.
Further, the formula of the GRC001 liquid culture medium is as follows:
Starch 20g,KNO3 1g,K2HPO4 0.5g,MgSO4·7H2O 0.5g,NaCl 0.5g,FeSO4·7H2O 0.01g, distilled water is fixed to volume to 1L, and high pressure steam sterilization is carried out.
Further, the liquid amplification culture temperature of the bacillus CCPM7645 is 14-39 ℃, the rotating speed is 120 revolutions per minute, and the culture time is 2-10 days.
Further, the temperature of the mass amplification culture of the bacillus CCPM7645 is 14-39 ℃, the rotating speed is 120 rpm, and the culture time is 2-10 days.
Further, the centrifugation conditions were 8000rpm/min for 10min.
The invention has the beneficial effects that:
The invention separates and identifies bacteria with strong anticancer activity by combining the metagenome high throughput screening of intestinal microbiota with traditional bacteriology and molecular microbiology and genetic methods. The bacillus CCPM7645 with strong anticancer activity provided by the invention is derived from human intestinal tracts, is a new source of bacillus, has different properties of other sources of bacillus such as ocean, soil and the like, and can generate metabolites with strong anticancer activity. The metabolite has broad spectrum anticancer activity, and can be used for treating cancer, including lung cancer, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer and/or leukemia.
Because bacillus CCPM7645 is found in human intestinal tracts, the side effects of the metabolites of bacillus CCPM7645 on human bodies after being prepared into anticancer drugs are very low or no, and animal experiments prove that the bacillus CCPM7645 has no toxic or side effects on experimental animals.
The influence of intestinal flora on health is gradually discovered and emphasized, and the intestinal flora has very close relation with various diseases, so that the research on the field of intestinal flora is carried out, and the method has profound significance in exploring a novel natural efficient anticancer method. The aim of preventing cancer is achieved by adjusting the different states or the different quantity ratios of the intestinal microorganisms of the human body.
Drawings
FIG. 1 is a diagram showing colony morphology obtained by three-compartment streak culture of Bacillus CCPM 7645;
FIG. 2 is a phylogenetic tree of Bacillus (Bacillus) CCPM7645 and related bacteria based on whole genome sequences;
FIG. 3 is a diagram showing the comparison of the forms of human ovarian cancer ES2 cells after the action of the metabolites of Bacillus CCPM7645, wherein FIG. A shows the human ovarian cancer ES2 cells after the action of CCPM7645, and FIG. B shows the control;
FIG. 4 is a graph showing the results of CCK8 experiments in which human ovarian cancer ES2 cells were subjected to the metabolite action of Bacillus CCPM7645 for 6h;
FIG. 5 shows the Bacillus CCPM7645 anticancer effect animal experiment-mice were inoculated with the human ovarian carcinoma ES2 cell line.
Detailed Description
The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention.
Example 1
A Bacillus CCPM7645 with strong anticancer activity belongs to Bacillus and represents a previously unknown bacterial species (species) which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.17887, the preservation date of 2019 of 06 month 03 and the preservation address of North Star Xila No.1, 3 in the Korean region of Beijing.
Example 2
The present example provides a screening method for bacillus CCPM 7645.
1. Screening culture medium and preparation method of relevant solution:
(1) GRC001 solid medium formulation (1L):
Starch 20g,KNO3 1g,K2HPO4 0.5g,MgSO4·7H2O 0.5g,NaCl 0.5g,FeSO4·7H2O 0.01g, agar 20g; the starch is dissolved or suspended first, then other components are added, and the mixture is shaken up after high pressure. The starch in this embodiment is derived from potato or maize or soybean or millet or other grains or other plants.
(2) Potassium dichromate: the final concentration of the solution was 50mg/L. The concentration of the storage solution is 5g/L, the solvent is water, the preparation is carried out in situ, the light is prevented, and the filtration and the sterilization are carried out. Toxic, do not contact skin or inhale; when 100mL of GRC001 solid medium is prepared, 1mL of potassium dichromate stock solution is added, and when the medium is not too hot, the mixture is uniformly mixed.
(3) FeSO 4·7H2 O mother liquor: 0.01g/mL.1g of FeSO 4·7H2 O was dissolved in 100mLH 2 O, and 1mL of ferrous sulfate mother liquor was added per 1000mL of GRC001 solid medium.
(4) PBS buffer formulation (1L): KH 2PO4 0.27g,Na2HPO4 1.42g,NaCl 8g,KCl 0.2g.
2. Screening method of bacillus CCPM 7645:
bacteria from human faeces samples are obtained by the following method:
(1) Sampling and description: a sampling package for delivery to a fecal provider, comprising the following items: 50mL centrifuge tube x 1; spoon x1 for dining; PE glove x 2; meal tray x 1; informed consent x 1; sample instruction x 1.
(2) Description of operation: reading the informed consent in detail, filling in the basic information of the fecal provider in detail and signing; discharging the feces into a disposable dining paper tray; taking about 10g of the center part of the excrement by using a spoon for dining, putting the excrement into a 50mL centrifuge tube, screwing the cover of the centrifuge tube, and then putting the centrifuge tube back into a self-sealing bag; the individual operation processes avoid touching other irrelevant articles; centrifuge tubes containing feces and informed consent were filled into self-sealing bags and collected back by laboratory personnel.
(3) Sample treatment and bacterial isolation culture: sampling, numbering, suspending in PBS buffer solution with 10 times of volume, taking 100 mu l of a plate with the diameter of 90 mm, and culturing the plate at 14-39 ℃ by using GRC001 solid culture medium containing potassium dichromate+; single colony three-zone line 14-39deg.C culture, see FIG. 1.
(4) And (3) bacterial storage: mu.l of 50% glycerol+500. Mu.l of GRC001 liquid medium were mixed and stored.
Example 3
The embodiment provides a bacillus CCPM7645 amplification method:
Single colony is taken from a flat plate and put into 5mL of GRC001 liquid culture medium, the temperature is 120 rpm, the temperature is 14-39 ℃, after amplification is carried out for 2-10 days, 5mL of bacterial liquid is transferred into 100mL of GRC001 liquid culture medium, the temperature is 120 rpm, the temperature is 14-39 ℃, mass amplification is carried out for 2-10 days, finally, the bacterial liquid is centrifuged, the centrifugation is carried out for 10 minutes at 8000rpm, and the supernatant is taken for the subsequent anticancer experiment.
Example 4
The present example provides a bacillus CCPM7645 anticancer activity verification experiment:
(1) Human ovarian carcinoma ES2 cell culture: and (3) plating 100 μl of each well, wherein the cell density is 1x10 5/mL, adding 5 μl of the metabolite of bacillus CCPM7645 after the human ovarian cancer ES2 cells are attached, culturing the control group with an equal volume of GRC001 liquid culture medium for 6-24 hours, and observing. The morphology of human ovarian cancer ES2 cells was observed under a microscope. Human ovarian carcinoma ES2 cells round, shed and die, see figure 3.
(2) Preparation of a metabolite of bacillus CCPM 7645: single colony is taken from the flat plate and put into 5mL GRC001 liquid culture medium, 120 revolutions per minute and 14-39 ℃, bacterial liquid is centrifuged at 8000rpm per minute for 10min after amplification, and supernatant is taken for subsequent anticancer cell experiments.
(3) CCK8 assay: human ovarian cancer ES2 cells were plated at 100. Mu.l per well at a cell density of 1X10 5/mL, and 5. Mu.l, 2.5. Mu.l, 1.25. Mu.l, 0.625. Mu.l, 0.313. Mu.l, and 0.156. Mu.l of the metabolite of Bacillus CCPM7645 were added to the cells after the human ovarian cancer ES2 cells had adhered to the walls, respectively, and the control group was observed after culturing for 6 to 24 hours without adding the metabolite of the strain, with 4 replicate wells each. After adding 10. Mu.l of CCK8 solution to each well and continuing to culture for 0.5-1h, the absorbance of each well was measured at OD450nm of the ELISA. As shown in FIG. 4, the metabolic products of Bacillus CCPM7645 exhibited a gradient in inhibition and concentration of human ovarian cancer ES2 cells.
Finally, the bacillus CCPM7645 screened from human intestinal tracts has obvious inhibitory activity on human ovarian cancer ES2 cells.
(4) Tumor animal model test: after nude mice (SPF grade) were purchased, they were subjected to adaptive breeding for one week at a temperature (25.+ -. 2) C and humidity maintained at 45% to 50%. Taking ES-2-ovarian cancer cells which have good growth state and are in logarithmic growth phase, digesting the cells with 0.25% pancreatin in a biosafety cabinet, collecting the cells, centrifuging the cells at 5000r/min for 5 minutes, preparing cell suspension by using cold PBS, counting the number of living cells by a cell counter, regulating the cell concentration to 1X 10 6 cells/mL, and placing the cell suspension on ice for standby. The skin of the injection site of nude mice was sterilized, 50. Mu.L of the cell suspension was aspirated by a syringe, and inoculated subcutaneously into nude mice to form a hillock. The injected nude mice are marked by an ear puncher, a marker pen and the like, and the weight and the tumor volume of the animals are measured. Following tumor development, the following indices were recorded every other day: animal weight, tumor volume, animal status, and the like. Tumor volumes were recorded starting from about 10 days when tumors were seen to protrude subcutaneously, and dosing was performed according to the experimental design, specifically CCPM7645 metabolite (experimental group) and PBS buffer (control group) were injected into the tumors of the right hind limb of CCPM 7645-dosed and PBS-controlled animals, respectively, and the experimental results are shown in fig. 4. Animals were sacrificed 22 days later and body weight (g), tumor volume (v=0.5 ab 2, a long diameter, b short diameter), spleen weight (g) were recorded in detail. The success rate of the tumor formation of the ES-2 ovarian cancer cell animal is 100%. As the experiment progresses, it can be observed that the tumor volume of CCPM 7645-dosed group is significantly smaller than that of control animals compared to PBS group. Animal experiment results prove that the bacillus CCPM7645 has obvious inhibition activity on tumor cells and obvious anticancer effect.
SEQUENCE LISTING
<110> Harbin Tianqi human second genome technology development and application technology Co., ltd
<120> Bacillus CCPM7645 with powerful anticancer activity and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1464
<212> DNA
<213> Bacillus sp.)
<400> 1
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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
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gcattattta agttgattaa tttagttgta gaacaaatga taaccgaaaa aaaagaaata 660
caagcaattt atgatctaat tcctaaagat aaactagacg ggatacaaaa tcgagatcat 720
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attggattat ctttgccgca acaagtatct gcccaagaaa ctcaaagtaa tagtcctaaa 840
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ggagtagtat cttctgagac tcaggaaaag cttttggaca agcttgctaa aggagaagcc 960
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Claims (6)
1. The application of a metabolite of Bacillus CCPM7645 with anticancer activity in preparing anticancer drugs for treating ovarian cancer is characterized in that the anticancer drugs have remarkable inhibitory activity on human ovarian cancer ES2 cells, the Bacillus CCPM7645 belongs to Bacillus (Bacillus), the Bacillus CCPM7645 is found in human intestinal tracts, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17887, the preservation date of 2019 of 06 months 03, and the preservation address of North Star Xway No. 1, the Korean area of Beijing, and the 3 rd.
2. The use of a metabolite of bacillus CCPM7645 with anticancer activity for preparing an anticancer drug according to claim 1, wherein the preparation method of the metabolite of bacillus CCPM7645 is as follows:
the bacillus CCPM7645 single colony is taken from a flat plate to 5 mL of GRC001 liquid culture medium, amplified and cultured for a certain time at a certain culture temperature and rotating speed, then 5 mL bacterial liquid is transferred to 100mL of GRC001 liquid culture medium, amplified and cultured for a certain time at a certain temperature and rotating speed, and finally bacterial liquid is centrifuged, and supernatant fluid is taken.
3. The use of a metabolite of bacillus CCPM7645 with anticancer activity for preparing an anticancer drug according to claim 2, wherein the formulation of the GRC001 liquid medium is:
Starch 20g,KNO3 1g,K2HPO4 0.5g,MgSO4·7H2O 0.5g,NaCl 0.5g,FeSO4·7H2O 0.01g.
4. The use of a metabolite of bacillus CCPM7645 with anticancer activity for preparing anticancer drugs according to claim 2, wherein the temperature of the amplification culture of bacillus CCPM7645 single colony in 5 mL of grc001 liquid medium is 14-39 ℃, the rotation speed is 120 rpm, and the culture time is 2-10 days.
5. The use of a metabolite of bacillus CCPM7645 with anticancer activity for preparing anticancer drugs according to claim 2, wherein the temperature of the mass amplification culture of bacillus CCPM7645 is 14-39 ℃, the rotation speed is 120 rpm, and the culture time is 2-10 days.
6. The use of a metabolite of bacillus CCPM7645 with anticancer activity according to claim 2, wherein said centrifugation condition is 8000 rpm centrifugation 10 min.
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