CN114395509A - Bacillus WYJ-E14 separated from Curcuma wenyujin and its application in preparing antineoplastic agent - Google Patents

Bacillus WYJ-E14 separated from Curcuma wenyujin and its application in preparing antineoplastic agent Download PDF

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CN114395509A
CN114395509A CN202210096208.2A CN202210096208A CN114395509A CN 114395509 A CN114395509 A CN 114395509A CN 202210096208 A CN202210096208 A CN 202210096208A CN 114395509 A CN114395509 A CN 114395509A
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CN114395509B (en
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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 a bacillus WYJ-E14 separated from curcuma wenyujin and application thereof in preparing anti-tumor drugs. The invention relates to an endophyte separated from a rhizome part of curcuma wenyujin, which belongs to the following categories: bacillus belgii. Bacillus belgii is widely distributed in nature, has the good characteristics of rapid growth and stable heredity, is harmless to human beings and animals, and does not pollute the environment. The metabolite is rich, has broad-spectrum antibacterial activity and stronger anti-stress capability, and plays an increasingly important role in many fields such as agriculture, environment, fermentation industry and the like. In the invention, the Bacillus belgii WYJ-E14 has extremely obvious anti-tumor cell effect and has high application value in the development of biological anti-tumor drugs.

Description

Bacillus WYJ-E14 separated from Curcuma wenyujin and its application in preparing antineoplastic agent
Technical Field
The invention belongs to the technical field of biology, and relates to a plant endophyte and application thereof in preparation of an anti-tumor medicament, in particular to a bacillus WYJ-E14 separated from curcuma wenyujin and application thereof in preparation of an anti-tumor medicament.
Background
Curcuma wenyujin Y.H.Chen et C.Ling, which is a plant of Curcuma of Zingiberaceae, is mainly distributed in Wenzhou area of Zhejiang province, not only can be used as an ornamental flower, but also the whole plant can be used for medicine. The tuber of the medicinal material, namely the Wen radix curcumae, is one of famous medicinal materials, namely Zhe eight flavors, in Zhejiang province.
Nearly one hundred chemical components have been reported to be isolated from Curcuma wenyujin, the main types being monoterpenes, sesquiterpenes, diterpenes, alkaloids, etc. Modern pharmacological studies show that the curcuma wenyujin has obvious curative effect on anti-inflammation and analgesia, and also has the effects of protecting the liver, resisting melancholy, resisting thrombus, improving blood circulation and the like. In recent years, the research shows that the curcuma aromatica contains active substances for resisting tumors, including zedoary root cyclodiene, beta-elemene, delta-elemene and the like. Zedoary turmeric diene, as one of the main components of Curcuma wenyujin rhizome volatile oil, has inhibitory effect on tumor cells. Especially, researches prove that the beta-elemene has the function 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, stomach cancer, bone metastasis cancer and the like. Accordingly, antitumor drugs have been developed.
However, in the production practice, the zedoary root cyclic diolefine, the beta-elemene and the delta-elemene are all volatile substances and have extremely low content in the medicinal plant curcuma wenyujin, so that the direct extraction of the medicinal components from the curcuma wenyujin is difficult. Although the beta-elemene liposome can be synthesized by a chemical way, the process is complex, the preparation process is expensive, and the price of the medicine is high.
On the other hand, a plurality of endophytes exist in the plant body, secondary metabolites of the endophytes form a reciprocal relationship with the host plant, 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 on the anti-tumor effect of endophytes at home and abroad are available.
Disclosure of Invention
The invention aims to provide a bacillus WYJ-E14 separated from curcuma wenyujin aiming at the defects of the prior art, which is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 15 days of 2021 with the preservation number of CGMCC No. 24104; the classification is named as Bacillus velezensis.
The biological characteristics of Bacillus WYJ-E14 are as follows: the colony color is yellow white, the surface is smooth, and the colony character is round and regular. The whole genome contains 1 circular chromosome, and the whole chromosome length is: 4020075bp, wherein the GC content is: 46.15%, the strain is plasmid-free.
The second purpose of the invention is to provide the application of the bacillus WYJ-E14 in preparing anti-tumor drugs.
Preferably, the tumor is melanoma or brain glioma.
The third purpose of the invention is to provide a medicine or health care product for treating or preventing tumor, which comprises bacillus WYJ-E14.
Preferably, the composition further comprises a pharmaceutically acceptable carrier.
The invention has the beneficial effects that:
the invention relates to an endophyte separated from a rhizome part of common turmeric, which belongs to the following categories: bacillus belgii. Bacillus belgii is widely distributed in nature, has the good characteristics of rapid growth and stable heredity, is harmless to human beings and animals, and does not pollute the environment. The metabolite is rich, has broad-spectrum antibacterial activity and stronger anti-stress capability, and plays an increasingly important role in many fields such as agriculture, environment, fermentation industry and the like. In the invention, the Bacillus belgii WYJ-E14 has extremely obvious anti-tumor cell effect and has high application value in the development of biological anti-tumor drugs.
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A single colony of purified WYJ-E14 isolated in fig. 1;
FIG. 2 is an electrophoretogram of the PCR amplification of WYJ-E14 genomic DNA using universal primers 27F and 1492R.
Wherein, the left side is DS2000 DNAmarker; on the right, PCR products.
FIG. 3 inhibition of tumor cells by the WYJ-E14 metabolite; wherein Melanoma is Melanoma cell, Glioma is Glioma cell, 1 to 14 in abscissa are endophyte WYJ-E1 to WYJ-E14 experimental groups separated from Curcuma wenyujin respectively, the ordinate is survival rate of tumor cell, con is that 25 mul LB liquid culture medium is added, 25 mul: 25 μ l of metabolites of different bacteria were added, 50 μ l: 50 μ l of metabolites from different bacteria were 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: and (3) carrying out isolated culture on the bacillus WYJ-E14.
Materials and methods
1. Experimental materials:
the cultivar of the curcuma wenyujin: wen Yu jin No.1, which is a gift from Tianhe Biotechnology GmbH, Wenzhou, Zhejiang. The Curcuma wenyujin is planted in a glass room basin of the institute of Life and environmental science of Hangzhou university.
2. Experimental media:
the PDA liquid culture medium comprises the following components: peeling potato 200g, cutting into small pieces, adding 1000mL water, cooking, filtering with two layers of gauze to obtain filtrate, adding glucose 20g/L, agar 15g/L, and pH 7.0.
The MS basic culture medium comprises: NH (NH)4NO31.65 g/L、KNO31.9 g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O0.37 g/L, KH2PO40.17 g/L, KI 0.83.83 mg/L, H3BO35.2 mg/L, MgSO 4.7H 2O 22.3.3 mg/L, ZnSO 4.7H 2O 3.6.6 mg/L, Na2MoO 4.2H 2O 0.25mg/L, CuSO 4.5H 2O 0.025mg/L, CoCl 2.6H 2O 0.025mg/L, Na2EDTA37.3 mg/L, FeSO 4.7H 2O 27.8.8 mg/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, the solvent is water, and the pH is 5.8.
3. Sterilization of isolated endophyte material and isolation of WYJ-E14 endophytes
(1) Growing Curcuma wenyujin seedlings for 5 months in a pot, taking tubers with shoot tips about 2cm in diameter, soaking in an aqueous solution containing detergent for 10min, washing with tap water, soaking in 75% alcohol for 1min, sterilizing with 0.1% mercuric chloride for 15min, and washing with sterile water for 3 times.
(2) Experimental groups tubers were cut into 5mm pieces or shoot tips of about 2-3mm length were cut from tubers and placed on streptomycin-containing Str (50mg/L) Potato Dextrose Agar (PDA) medium with the cut facing the medium and 4 pieces placed per dish. Control group the experimental materials treated with the same sterilization were directly rolled on a control dish without cutting for testing the surface sterilization effect of the materials, and 1 open-lid PDA plate was placed in a clean bench to test the cleanliness of the operating environment. The experimental group and the control group were cultured at 28 ℃ in the dark for 3 days.
(3) When bacterial spots grow from the cut surface of the tuber or the bud tip section of the common turmeric, inoculating the bacterial spots to a new PDA culture medium containing Str (50mg/L) by using an inoculating loop streak line for continuous propagation and culture, and repeating for 3 times until separated and purified bacterial strains are obtained.
4. Extraction of genomic DNA of strains and molecular characterization of strain classification
(1) Extraction of bacterial genomic DNA
Single colonies were cultured overnight on PDA + Str 50mg/L liquid medium and collected in 1.0X 10 cells using 2ml centrifuge tubes9(1ml of bacterial suspension OD600 is 1-1.5), 12,000r/min, centrifugation for 30s, and discarding the supernatant. Genomic DNA was extracted using AxyPrep bacterial genomic DNA miniprep kit. The specific operation is carried out according to the kit instruction.
(2) PCR amplification
The primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SQE No. 1; 1492R: 5'-CTACGGCTACCTTGTTACGA-3', as shown in SQE No. 2. The PCR reaction was performed in a total volume of 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 program is as follows: 95 deg.C for 5 min; 35 cycles of 95 deg.C, 30s, 58 deg.C, 30s, 72 deg.C, 90 s; finally, the temperature is 72 ℃ for 7 min. After the reaction was completed, 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 kit instruction.
(4) Sequence determination and analysis
And taking the PCR product after each strain purification, and carrying out DNA sequencing by using a sequencer ABI 3730-XL. And 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 similarity to the sequence of the species to be detected.
5. Determination and assembly of WYJ-E14 whole genome sequence
A Whole Genome Shotgun strategy (white Genome Shotgun, WGS) was used to construct a library of different inserts, namely: preparing an experimental flow (Illumina TruSeq DNA Sample Preparation Guide) by adopting a standard Illumina TruSeq Nano DNA LT library to construct a required genome on-board library; the desired genomic on-board library was constructed Using the standard Pacbio Template Prep Kit 1.0 library Preparation protocol (20kb Template Preparation Using BluePiplin Size Selection). Using a Next-Generation Sequencing technology (NGS) based on illumina novaseq Sequencing platform; meanwhile, the constructed libraries are respectively sequenced on the basis of a PacBio sequence sequencing platform by utilizing a third-generation single-molecule sequencing technology.
And finally, carrying out genome sequence assembly: and assembling the off-line data obtained by the third generation sequencing Pacbio by using HGAP and CANU software to obtain a contig sequence. And (3) correcting the third generation contig result by using the second generation high-quality data through pilot software, and finally splicing to obtain a complete genome sequence.
6. Isolation of metabolite of endophyte strain and analysis of antitumor efficacy
A single colony of a strain of endophyte is subjected to suspension culture on an LB + Str 50mg/L liquid culture medium overnight at 120r/min, a bacterial liquid in a logarithmic phase is taken, the bacterial liquid is centrifuged at 11000r/min for 5min, and a supernatant, namely a separated metabolite (fermentation liquid) of the 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 the north nano benth of china, BeNa Culture Collection (BNCC). Tumor cells were grown in DMEM medium supplemented with 1 XPcillin/streptomycin, 10% fetal bovine serum, at 37 deg.C and 5% CO2Is maintained in the incubator.
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, cells were treated with a metabolite of the strain of the endophyte, control 25. mu.l LB medium and at 37 ℃ and 5% CO2And culturing for 72 hours. To each well was added 10. mu.l of CCK8 reagent and incubated at 37 ℃ for 1 hour. Absorbance was quantified using a microplate reader at a wavelength of 450 nm. Each group of cells was repeated 3 times more. The effect of the metabolite (fermentation broth) on the viability and growth of the tumor cells was observed.
Second, result in
1. Obtaining the endophyte WYJ-E14 strain
In the present invention, 13 endophytes (named WYJ-E1 to WYJ-E13, respectively) were isolated from tubers. In addition, 1 endophyte was isolated from the base of a shoot tip segment of about 2-3mm in length cut from a Curcuma wenyujin tuber (i.e., at the base cut of a shoot tip of about 2-3mm in length cut from a tuber). The endophyte is purified to obtain a single colony. The colony color is yellow white, the surface is smooth, and the colony character is round and regular (figure 1). The strain has rapid growth and stable morphological characters, and is named as: WYJ-E14.
WYJ-E14 has been deposited at: the China general microbiological culture Collection center has the following preservation dates: 12 and 15 days 2021, the preservation number is CGMCC No.24104, and the preservation address is as follows: xilu No.1 Hospital No.3, Beijing, Chaoyang, North Chen, zip code 100101.
2. Molecular taxonomic identification of strains
A clear single band of about 1500bp in size was amplified by PCR using primers 27F and 1492R (FIG. 2). After sequencing, the length of the PCR band is 1470bp, and the specific rDNA sequence is as follows: 5'-CCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGAATTAAAA-3', as shown in SQE No. 3.
Performing blast comparison with a GenBank international nucleic acid database to determine that the strain belongs to the following biological classifications: bacillus velezensis (Bacillus velezensis) belonging to the genus Bacillus.
3. WYJ-E14 Whole genome sequence
Whole genome sequencing was performed on WYJ-E14, where 7723036 high quality reads (reads) containing 1147602121bp were obtained by second generation sequencing. 479777 sequences containing 458948982bp are obtained by third-generation sequencing. Through proofreading and splicing, the WYJ-E14 whole genome is finally confirmed to contain 1 circular chromosome, and the whole chromosome length is as follows: 4020075bp, wherein the GC content is: 46.15 percent. The strain is plasmid-free.
4. Inhibition of anti-tumor growth by metabolites of strain WYJ-E14
The effect of metabolites of 14 endophytes on the growth of 3 different tumor cell lines each of melanoma and glioma was examined using the cck8 method, respectively. Among them, WYJ-E14 all 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 all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Sequence listing
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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 (7)

1. A Bacillus WYJ-E14 separated from Curcuma wenyujin is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 15 days in 2021 with the preservation number of CGMCC No. 24104; the classification is named as Bacillus velezensis.
2. The isolated Bacillus WYJ-E14 from Curcuma wenyujin Y.H.Chen et C.Ling as claimed in claim 1, wherein the biological characteristics of Bacillus WYJ-E14 are as follows: the colony color is yellow white, the surface is smooth, and the colony character is round and regular; the whole genome contains 1 circular chromosome, and the whole chromosome length is: 4020075bp, wherein the GC content is: 46.15%, the strain is plasmid-free.
3. The use of the bacillus WYJ-E14 isolated from curcuma wenyujin according to any one of claims 1-2 for the preparation of an anti-tumor medicament.
4. Use according to claim 3, characterized in that said tumors are melanomas, brain gliomas.
5. A drug or health product for treating or preventing tumor, comprising the bacillus WYJ-E14 isolated from curcuma wenyujin according to any one of claims 1 to 2.
6. The drug or health product for treating or preventing tumor according to claim 5, further comprising a pharmaceutically acceptable carrier.
7. The drug or health product for treating or preventing tumor according to claim 5 or 6, wherein the tumor is melanoma or brain glioma.
CN202210096208.2A 2022-01-26 2022-01-26 Bacillus WYJ-E14 separated from radix curcumae and application thereof in preparation of antitumor drugs Active CN114395509B (en)

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