CN114395509B - Bacillus WYJ-E14 separated from radix curcumae and application thereof in preparation of antitumor drugs - Google Patents

Bacillus WYJ-E14 separated from radix curcumae and application thereof in preparation of antitumor drugs Download PDF

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CN114395509B
CN114395509B CN202210096208.2A CN202210096208A CN114395509B CN 114395509 B CN114395509 B CN 114395509B CN 202210096208 A CN202210096208 A CN 202210096208A CN 114395509 B CN114395509 B CN 114395509B
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向太和
黄小平
曾章慧
陈哲皓
王利琳
庞基良
童霞秀
李亚菲
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Hangzhou Normal University
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Abstract

The invention discloses bacillus WYJ-E14 separated from radix curcumae and application thereof in preparation of antitumor drugs. The endophyte separated from the rhizome part of the curcuma wenyujin belongs to the following parts: bacillus bailii. Bacillus bailii is widely distributed in nature, has good characteristics of rapid growth and genetic stability, is harmless to human and animals, and does not pollute the environment. The product has rich metabolite, broad-spectrum antibacterial activity and strong anti-stress capability, and plays an increasingly important role in many fields such as agriculture, environment, fermentation industry and the like. In the invention, bacillus belicus WYJ-E14 has extremely remarkable anti-tumor cell efficacy, and has high application value in the aspect of development of biological anti-tumor drugs.

Description

Bacillus WYJ-E14 separated from radix curcumae and application thereof in preparation of antitumor drugs
Technical Field
The invention belongs to the technical field of biology, relates to endophytes and application thereof in preparing antitumor drugs, and in particular relates to bacillus WYJ-E14 separated from radix curcumae and application thereof in preparing antitumor drugs.
Background
Curcuma wenyujin Y.H.Chen et C.Ling is a plant of Curcuma of Zingiberaceae, and is mainly distributed in Wenzhou region of Zhejiang province, and can be used as ornamental flower, and can be used for medicinal use. The tuber of the Chinese medicinal material "Wenyujin" is one of the famous genuine medicinal materials "Zhejiang eight ingredients" in Zhejiang province.
It has been reported that nearly hundred chemical components have been isolated from curcuma wenyujin, and the main types are monoterpenes, sesquiterpenes, diterpenes, alkaloids, etc. Modern pharmacological researches show that the curcuma wenyujin has remarkable curative effect in anti-inflammatory and analgesic aspects, and also has the effects of protecting liver, resisting depression, resisting thrombus, improving blood circulation and the like. Recent researches show that the curcuma aromatica contains antitumor active substances including furanodiene, beta-elemene, delta-elemene and the like. The furanodiene is used as one of the main components of Curcuma wenyujin Y.H.Chen et C.Ling rhizome volatile oil, and has tumor cell inhibiting effect. Particularly, the research proves that the beta-elemene has the effect of increasing and inducing apoptosis, shows high-efficiency and low-toxicity anti-tumor activity, and is safe and effective for treating various cancers such as lung cancer, liver cancer, brain cancer, esophagus cancer, gastric cancer, bone metastasis cancer and the like. Accordingly, antitumor drugs have been developed.
However, in the production practice, since the furanodiene, the beta-elemene and the delta-elemene are volatile substances and the content of the furanodiene in the medical plant curcuma aromatica is extremely low, the direct extraction of the medical components from the curcuma aromatica is difficult. Although the beta-elemene liposome can be synthesized by chemical approach, the process is complex, the preparation process has high cost and the medicine has high price.
On the other hand, various endophytes exist in the plant body, secondary metabolites of the endophytes and host plants form a reciprocal relationship, and the endophytes not only influence the growth and development of the host, but also have a plurality of important applications in biological control of plant diseases and insect pests. At present, few reports about the anti-tumor effect of endophytes at home and abroad are provided.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides bacillus WYJ-E14 separated from curcuma wenyujin, which is preserved in China general microbiological culture collection center (CGMCC) in 12 months 15 of 2021 with a preservation number of CGMCC No.24104; the classification was designated bacillus belicus Bacillus velezensis.
The biological characteristics of Bacillus WYJ-E14 are as follows: the bacterial colony has the advantages of yellowish white color, smooth surface and round and regular bacterial colony characters. The whole genome contains 1 circular chromosome, and the whole chromosome length is: 4020075bp, wherein the GC content is: 46.15% of the strain is plasmid-free.
The second object of the invention is to provide the application of the bacillus WYJ-E14 in preparing antitumor drugs.
Preferably, the tumor is melanoma or glioma.
The third object of the invention is to provide a medicine or health care product for treating or preventing tumor, comprising bacillus WYJ-E14.
Preferably, a pharmaceutically acceptable carrier is also included.
The beneficial effects of the invention are as follows:
the endophyte separated from the rhizome part of the curcuma wenyujin belongs to the following parts: bacillus bailii. Bacillus bailii is widely distributed in nature, has good characteristics of rapid growth and genetic stability, is harmless to human and animals, and does not pollute the environment. The product has rich metabolite, broad-spectrum antibacterial activity and strong anti-stress capability, and plays an increasingly important role in many fields such as agriculture, environment, fermentation industry and the like. In the invention, bacillus belicus WYJ-E14 has extremely remarkable anti-tumor cell efficacy, and has high application value in the aspect of development of biological anti-tumor drugs.
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FIG. 1 single colony of WYJ-E14 isolated and purified;
FIG. 2 is an electrophoretogram of PCR amplification of WYJ-E14 genomic DNA using universal primers 27F and 1492R.
Wherein, the left side is DS2000 DNAmarker; right PCR product.
FIG. 3 inhibition of tumor cells by WYJ-E14 metabolites; wherein Melanoma is Melanoma cells, glioma is Glioma cells, 1 to 14 in the abscissa are experimental groups of endophytes WYJ-E1 to WYJ-E14 isolated from Curcuma wenyujin, respectively, and the ordinate is the survival rate of tumor cells, con: 25 μl of LB liquid medium is added, 25 μl: mu.l of metabolite of different bacteria, 50. Mu.l were added: mu.l of the metabolite of the different bacteria was added.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1: isolated culture of bacillus WYJ-E14.
1. Materials and methods
1. Experimental materials:
cultivars of Curcuma wenyujin: curcuma wenyujin No.1 was given away by Tianhe biotechnology Co., wittig.of Zhejiang. The radix curcumae is planted in a glass pot of a university of Hangzhou, a college of life and environmental science.
2. Culture medium for experiments:
the PDA liquid culture medium consists of: 200g of peeled potatoes are cut into small pieces, 1000mL of water is added for boiling, two layers of gauze are used for filtering to obtain filtrate, 20g/L of glucose and 15g/L of agar are added, and the pH value is 7.0.
The MS minimal medium consists of: NH (NH) 4 NO 3 1.65 g/L、KNO 3 1.9 g/L、CaCl 2 ·2H 2 O 0.44g/L、MgSO 4 ·7H 2 0.37g/L of O, KH2PO40.17 g/L, KI 0.83.83 mg/L, H3BO35.2 mg/L, mgSO 4.7H2O 22.3mg/L, znSO 4.7H2O 3.6mg/L, na MoO4.2H2O 0.25mg/L, cuSO 4.5H2O 0.025mg/L, coCl 2.6H2O 0.025mg/L, na2EDTA37.3 mg/L, feSO 4.7H2O 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, sucrose 30g/L, agar powder 10g/L, and the solvent is water with pH of 5.8.
3. Disinfection of isolated endophyte material and isolation of WYJ-E14 endophytes
(1) The seedlings of the curcuma wenyujin are grown in a pot for 5 months, tubers with bud tips and diameters of about 2cm are taken, soaked in an aqueous solution containing detergent for 10min, washed clean by tap water, soaked in 75% alcohol for 1min, sterilized by 0.1% mercuric chloride for 15min, and finally washed by sterile water for 3 times.
(2) The experimental group cut the tubers into small pieces of 5mm, or cut pieces of bud tips about 2-3mm long from the tubers, and place them on Potato Dextrose Agar (PDA) medium containing streptomycin Str (50 mg/L), with the cuts facing the medium, and place 4 pieces per dish. The control group directly rolls the test materials subjected to the same disinfection treatment on a control dish without cutting, is used for detecting the surface disinfection effect of the materials, and simultaneously places 1 PDA dish with an open cover in an ultra clean bench to detect the cleanliness of the operation environment. Both the experimental group and the control group were incubated at 28℃for 3 days in the dark at constant temperature.
(3) When bacterial plaque grows out from the incision surface of the tuber or bud tip segment of the curcuma wenyujin, the bacterial plaque is streaked and inoculated on a new PDA culture medium containing Str (50 mg/L) by an inoculating loop, and the propagation culture is continued, and the steps are repeated for 3 times until the isolated and purified bacterial strain is obtained.
4. Extraction of genomic DNA from strains and molecular characterization of strain classification
(1) Extraction of bacterial genomic DNA
Single colonies were grown overnight on PDA+Str 50mg/L liquid medium and collected 1.0X10 s with a 2ml centrifuge tube 9 (1 ml bacterial culture with OD600 of 1-1.5), 12,000r/min, centrifuged for 30s, and the supernatant discarded. Genomic DNA was extracted using AxyPrep bacterial genomic DNA minikit. The specific operation is carried out according to the instruction of the kit.
(2) PCR amplification
The primers were 27F:5'-AGAGTTTGATCCTGGCTCAG-3' as shown in SQE NO. 1; 1492R:5'-CTACGGCTACCTTGTTACGA-3' as shown in SQE No. 2. The total volume of the PCR reaction was 50. Mu.l, and the following ingredients were added to a 0.2ml centrifuge tube:
PCR amplification reaction system
Figure BDA0003491088700000041
The PCR amplification reaction procedure was: 95 ℃ for 5min; 35 cycles were performed at 95℃for 30s,58℃for 30s,72℃for 90 s; finally, the temperature is 72 ℃ for 7min. After completion of the reaction, 3. Mu.l of the PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR amplified fragment.
(3) Recovery of PCR products
The PCR product was recovered using AxyPrep DNA gel recovery kit. The specific operation is carried out according to the instruction of the kit.
(4) Sequencing and analysis
The purified PCR products of each strain were taken and subjected to DNA sequencing using a sequencer ABI 3730-XL. And (3) comparing the spliced sequence file with data in a 16S database in GenBank by using an NCBI (www.ncbi.nlm.nih.gov) online Blast program to obtain species information with the maximum sequence similarity with the species to be detected.
5. Determination and assembly of WYJ-E14 whole genome sequence
A whole genome shotgun (Whole Genome Shotgun, WGS) strategy was used to construct a library of different inserts, namely: the standard Illumina TruSeq Nano DNA LT library preparation experimental procedure (Illumina TruSeq DNA Sample Preparation Guide) was used to construct the desired on-press genomic library; standard Pacbio Template Prep Kit 1.0.1.0 library preparation Experimental procedure (20 kb Template Preparation Using BluePippin Size Selection) was used to construct the desired genomic library. Using a second generation sequencing technology (Next-Generation Sequencing, NGS) based on IlluminaNovaSeq sequencing platform; meanwhile, the constructed libraries were sequenced separately based on the PacBio sequence sequencing platform using third generation single molecule sequencing technology.
Finally, genome sequence assembly is carried out: and assembling the off-machine data obtained by the third-generation sequencing Pacbio by using HGAP and CANU software to obtain a contig sequence. And correcting the third generation contig result by using pilot software on the second generation high-quality data, and finally splicing to obtain the complete genome sequence.
6. Isolation of endophyte strain metabolites and analysis of antitumor efficacy
Single colony of endophyte strain is suspended and cultured overnight on LB+Str50mg/L liquid culture medium, 120r/min, bacterial liquid in logarithmic phase is taken, 11000r/min is centrifuged for 5min, and supernatant, namely metabolite (fermentation liquor) of the separated strain is taken.
Tumor cell culture: cell lines a375 (human melanoma), a875 (human melanoma) and a2058 (human melanoma), ln229 (human glioblastoma), SHG44 (human glioblastoma), U138 (human glioblastoma) were purchased from north nano-organism BeNa Culture Collection (BNCC) in china. Tumor cells were grown in DMEM medium, supplemented with 1 Xpenicillin/streptomycin, 10% foetal calf serum, at 37℃and 5% CO 2 Is maintained in the incubator of (a).
Cell viability was determined using cell counting kit-8 (CCK 8): all cells were seeded at a density of 5000 cells/well in 96-well culture plates. 12 hours after inoculation, the cells were treated with endophyte strain metabolite, control 25. Mu.l LB medium, and at 37℃and 5% CO 2 Incubate for 72 hours. Mu.l of CCK8 reagent was added to each well and incubated at 37℃for 1 hour. Absorbance was quantified at a wavelength of 450nm using a microplate reader. Each group of cells was repeated more than 3 times. The effect of the metabolite (fermentation broth) on the viability and growth of the tumor cells was observed.
2. Results
1. Obtaining endophyte WYJ-E14 strain
In the present invention, 13 endophytes (named WYJ-E1 to WYJ-E13, respectively) were isolated from tubers. In addition, the base of the bud tip segment of about 2-3mm length cut from the tuber of Curcuma wenyujin (i.e., at the root cut of the bud tip of about 2-3mm length cut from the tuber) was isolated to 1 endophyte. The endophyte is purified to obtain single colony. The colony had a yellowish white color, a smooth surface and a round and regular colony character (FIG. 1). The strain grows fast and has stable morphological characters, and is named as: WYJ-E14.
WYJ-E14 has been deposited in: the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) has a collection date of: 2021, 12 and 15 days, the preservation number is CGMCC No.24104, and the preservation address is: beijing, chaoyang area, north Chenxi Lu No.1, 3, postal code 100101.
2. Molecular classification identification of strains
PCR amplification using primers 27F and 1492R gave a distinct single band of about 1500bp (FIG. 2). Through sequencing, the length of a PCR band is 1470bp, and the specific rDNA sequence is as follows: 5'-CCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGAATTAAAA-3' as shown in SQE No. 3.
Blast comparison is carried out with GenBank international nucleic acid database, and the biological classification of the strain is determined to belong to: bacillus belgium (Bacillus velezensis) of the genus Bacillus.
3. Sequences of WYJ-E14 Whole genome
Whole genome sequencing was performed on WYJ-E14, wherein 7723036 high quality reads (reads) containing 1147602121bp were obtained by second generation sequencing. After three generations of sequencing, 479777 sequences, containing 458948982bp, are obtained. And finally confirming that the WYJ-E14 whole genome contains 1 circular chromosome through proofreading and splicing, wherein the total length of the chromosome is as follows: 4020075bp, wherein the GC content is: 46.15%. The strain is plasmid-free.
4. Inhibition of tumor growth by the Strain WYJ-E14 metabolite
The effect of metabolites of 14 endophytes on the growth of 3 different tumor cell lines each of melanoma and glioma was examined by the cck8 method, respectively. Of these, WYJ-E14 extremely significantly inhibited the viability and growth of tumor cells in all cell lines (fig. 3).
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and falls within the scope of the present invention as long as the present invention meets the requirements.
Sequence listing
<110> Hangzhou university of education
<120> a bacillus WYJ-E14 isolated from Curcuma wenyujin and its application in preparing antitumor drugs
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<170> SIPOSequenceListing 1.0
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agagtttgat cctggctcag 20
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ctacggctac cttgttacga 20
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<213> Bacillus bailii (Bacillus velezensis)
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ccttcggcgg ctggctccat aaaggttacc tcaccgactt cgggtgttac aaactctcgt 60
ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc 120
gattactagc gattccagct tcacgcagtc gagttgcaga ctgcgatccg aactgagaac 180
agatttgtgg gattggctta acctcgcggt ttcgctgccc tttgttctgt ccattgtagc 240
acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg 300
gtttgtcacc ggcagtcacc ttagagtgcc caactgaatg ctggcaacta agatcaaggg 360
ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca 420
ccacctgtca ctctgccccc gaaggggacg tcctatctct aggattgtca gaggatgtca 480
agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt 600
aatgcgttag ctgcagcact aaggggcgga aaccccctaa cacttagcac tcatcgttta 660
cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt 720
cagttacaga ccagagagtc gccttcgcca ctggtgttcc tccacatctc tacgcatttc 780
accgctacac gtggaattcc actctcctct tctgcactca agttccccag tttccaatga 840
ccctccccgg ttgagccggg ggctttcaca tcagacttaa gaaaccgcct gcgagccctt 900
tacgcccaat aattccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt 960
agttagccgt ggctttctgg ttaggtaccg tcaaggtgcc gccctatttg aacggcactt 1020
gttcttccct aacaacagag ctttacgatc cgaaaacctt catcactcac gcggcgttgc 1080
tccgtcagac tttcgtccat tgcggaagat tccctactgc tgcctcccgt aggagtctgg 1140
gccgtgtctc agtcccagtg tggccgatca ccctctcagg tcggctacgc atcgtcgcct 1200
tggtgagccg ttacctcacc aactagctaa tgcgccgcgg gtccatctgt aagtggtagc 1260
cgaagccacc ttttatgtct gaaccatgcg gttcaaacaa ccatccggta ttagccccgg 1320
tttcccggag ttatcccagt cttacaggca ggttacccac gtgttactca cccgtccgcc 1380
gctaacatca gggagcaagc tcccatctgt ccgctcgact tgcatgtatt aggcacgccg 1440
ccagcgttcg tcctgagcca gaattaaaa 1469

Claims (4)

1. The bacillus WYJ-E14 separated from curcuma wenyujin is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24104 in the 12 th month 15 of 2021; the classification is named as bacillus bailiiBacillus velezensis。
2. The use of bacillus WYJ-E14 isolated from curcuma wenyujin in preparing antitumor drugs according to claim 1, wherein the tumors are melanoma and glioma.
3. A medicament for treating tumors, comprising bacillus WYJ-E14 isolated from curcuma aromatica according to claim 1, wherein the tumors are melanoma and glioma.
4. A medicament for the treatment of tumors as claimed in claim 3, further comprising a pharmaceutically acceptable carrier.
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