CN107384819B

CN107384819B - Enteromorpha bacillus Y15-8 and anti-tumor active protein thereof

Info

Publication number: CN107384819B
Application number: CN201710606370.3A
Authority: CN
Inventors: 郑兰红; 苑志欣; 朱祥杰; 王震; 康道乐
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2017-07-19
Filing date: 2017-07-24
Publication date: 2020-05-22
Anticipated expiration: 2037-07-24
Also published as: CN107384819A

Abstract

An enteromorpha bacillus Y15-8 and an anti-tumor active protein thereof belong to the technical field of marine biological resources and biological medicines, the enteromorpha bacillus Y15-8 is preserved in the Wuhan type culture Collection in China at 2016, 12 months and 12 days, and the preservation number is CCTCC M2016744. The strain and the enteromorpha are epiphytic, and the produced metabolite active protein has cytotoxicity on various tumor cells, and has good research value and application prospect.

Description

Enteromorpha bacillus Y15-8 and anti-tumor active protein thereof

Technical Field

The invention belongs to the technical field of marine biological resources and biological medicines, and particularly relates to enteromorpha bacillus Y15-8 and an anti-tumor active protein thereof.

Background

In the present society, malignant tumor is a first killer in China and even in the whole human society, and is a serious threat to human life and health. At present, the clinical means for treating malignant tumor mostly adopts a comprehensive therapy, and combines surgical excision, radiotherapy, chemotherapy and immunotherapy. In the field of cancer chemotherapy, although natural products from a plurality of microorganisms have strong drug effects, the natural products cannot be continuously applied due to factors such as large molecular weight, low water solubility, strong toxicity, large side effects and the like, so that the search for effective antitumor drugs and methods is a global research hotspot.

The research of natural antitumor medicine from marine microbe has gained great results and excellent development advantages, the ① microbe produces great amount of secondary metabolite with unique and complicated chemical structure and difficult artificial synthesis, and the ② microbe metabolite has small molecular weight, high water solubility and low toxicity, may be used directly as medicine, has latent treating activity and clinical pharmacokinetic attribute, and the ③ microbe has short growth period, easy control of metabolic process and easy selection of strain, and may be used in industrial production.

As an important marine resource, the enteromorpha prolifera is rich in carbohydrate, amino acid, trace elements, vitamins, fatty acid and other nutrient substances, and has the effects of reducing blood sugar, resisting bacteria, diminishing inflammation, resisting tumor activity and improving immunity. The enteromorpha resource in China is very rich, the outbreak amount is over 50 ten thousand tons only in 2016 years in the coastal area of the Qingdao, the enteromorpha is researched more mature at present, but the large-scale utilization is not realized mostly, the overall utilization rate is not high, and the large waste of the enteromorpha resource is caused. Meanwhile, the enteromorpha also contains some co-attached microorganisms, but the microorganisms and metabolites thereof are not fully excavated and utilized so far.

Disclosure of Invention

The invention aims to provide enteromorpha bacillus Y15-8 and anti-tumor active protein thereof, overcomes the defects of the prior art, and improves the utilization rate of ocean resources.

An Enteromorpha Bacillus Y15-8(Bacillus sp.Y15-8) is preserved in the Wuhan type culture Collection in China at 2016, 12 months and 12 days, with the preservation number of CCTCC M2016744.

The invention also provides enteromorpha bacillus protein produced by fermenting the enteromorpha bacillus Y15-8, and the protein has anti-tumor activity; the fermentation culture conditions of the enteromorpha bacillus Y15-8 are as follows: the final concentration of each component in the fermentation liquor is 2-6 g/L of beef extract, 7-12 g/L of peptone, 2-6 g/L of sodium chloride, pH is 6.0-9.0, and the concentration is 1.05kg/cm²Sterilizing at 115-121 ℃ for 15-35 min, and culturing in a constant-temperature shaking table at 150-250 r/min and 25-37 ℃ for 48-80 h.

The invention also provides a preparation method of the enteromorpha bacillus Y15-8 anti-tumor active protein, which comprises the following steps:

(1) fermentation culture

Fermenting enteromorpha bacillus Y15-8 under the fermentation conditions: the final concentration of each component in the fermentation liquor is 2-6 g/L of beef extract, 7-12 g/L of peptone, 2-6 g/L of sodium chloride, pH6.0-9.0, and is 1.05kg/cm²Sterilizing at 115-121 ℃ for 15-35 min, and culturing in a constant-temperature shaking table at 150-250 r/min and 25-37 ℃ for 48-80 h;

(2) ultrafiltration interception

Centrifuging fermentation liquor obtained by fermenting and culturing enteromorpha bacillus Y15-8 at 8000-10000 rpm for 10-25 min, collecting supernatant, and performing ultrafiltration by using ultrafiltration membranes with molecular weights of 3kDa and 50kDa to obtain components with molecular weights of 3kDa to 50 kDa;

(3) ammonium sulfate precipitation

Gently stirring the active component with the molecular weight of 3-50 kDa in the step (2), slowly adding ammonium sulfate powder to ensure that the mass percentage concentration reaches 60%, controlling the temperature to be 3-6 ℃ in the period, standing the mixture in a refrigerator for 10-12 h after the active component is completely dissolved, centrifuging the mixture under the conditions of 10000-12000 rpm and 25-35 min, collecting supernatant, continuously adding the ammonium sulfate powder to the obtained supernatant to ensure that the mass percentage concentration reaches 75%, controlling the temperature to be 3-6 ℃ in the period, standing the mixture in the refrigerator for 10-12 h after the active component is completely dissolved, centrifuging the mixture under the conditions of 10000-12000 rpm and 25-35 min, collecting precipitate, dissolving the precipitate by using 20-50 mM Tris-HCl with the pH value of 7.5-8.5 and the molecular weight of the precipitate being two times of the volume of the precipitate, and dialyzing the solution for 20-24 h by;

(4) anion exchange chromatography (Q or DEAE)

Passing the fraction with anti-tumor activity obtained in step (3) through a 0.22 μm microporous membrane; passing through a Q or DEAE anion exchange chromatography column, wherein the sample loading amount is 5-50 mL each time, the eluent is Tris-HCl with the concentration of 20-50 mmol/L and the pH value of 7.5-8.5, the eluent is eluted at the concentration of 0-100% (v/v), the flow rate is 3-6mL/min, the detection wavelength is 280nm, a penetration peak Q1 is collected, and the Q1 component is desalted and then is subjected to vacuum freeze drying to obtain the enteromorpha symbiotic microbial active protein, which is named as enteromorpha bacillus protein (Proteinfrombillus sp. Y15-8, PBY15-8 for short).

Further, the concentration of NaCl in the eluate obtained in the above step (4) is preferably 1 mol/L.

Further, the flow rate of the eluent in the step (4) is preferably 2.0 mL/min.

Further, the amount of each sample in the above step (4) is preferably 5 mL.

The invention also provides a medicament for treating or preventing human liver cancer cells HepG2, human lung adenocarcinoma cells A549 or human lung cancer cells NCI-H460, wherein the medicament comprises the enteromorpha bacillus protein.

The invention also provides application of the enteromorpha bacillus protein in medicaments for treating or preventing human liver cancer cells HepG2, human lung adenocarcinoma cells A549 or human lung cancer cells NCI-H460.

Compared with the prior art, the invention has the beneficial effects that: the strain and the enteromorpha are epiphytic, and the produced metabolite active protein has cytotoxicity on various tumor cells, and has good research value and application prospect.

Drawings

FIG. 1Y 15-8 is a morphological feature map on beef extract peptone medium;

FIG. 2 chromatography of ammonium sulfate precipitation actives on Q anion exchange chromatography;

FIG. 3PBY15-8 shows the effect of morphological changes on human hepatoma cells BEL-7402;

FIG. 4PBY15-8 effects on morphological changes in human lung adenocarcinoma cells A549;

FIG. 5PBY15-8 is a graph showing the effect of morphological changes on human large cell lung carcinoma cells NCI-H460;

FIG. 6PBY15-8 shows the inhibition of proliferation of human hepatoma cell BEL-7402;

FIG. 7PBY15-8 inhibition of human lung adenocarcinoma cell A549 proliferation;

FIG. 8PBY15-8 shows the inhibition of proliferation of human large cell lung carcinoma cell NCI-H460.

Enteromorpha Y15-8, the Latin name is Bacillus sp.Y15-8, the strain is preserved in the China center for type culture Collection in 2016, 12 months and 12 days, and the preservation addresses are as follows: the preservation number of Wuhan university in Wuhan, China is CCTCC M2016744.

Detailed Description

The technical solutions of the present invention are further illustrated by the following examples, but the scope of the present invention is not limited by the examples in any way. And are not limited to the following examples:

example 1 screening and isolation procedure for Bacillus enteromorpha Y15-8

(1) Active preliminary screening

Fresh enteromorpha is fished from Qingdao sea area, ground and crushed into primary pulp, and different culture mediums such as a seawater basic culture medium 2216E, LB culture medium and a beef extract peptone culture medium NRG are respectively adopted to carry out selective coating, lineation and separation culture on enteromorpha samples for multiple times, so that 27 single bacterial colonies are obtained. The result of primary screening of the anti-tumor activity of the fermentation product of the 27 enteromorpha symbiotic microorganisms shows that 2 enteromorpha symbiotic microorganisms have the inhibition rate of more than 50 percent on liver cancer cell BEL-7402.

(2) Bacterial strain rescreening

The proliferation inhibition effect of the fermentation liquor of the 2 active strains on other tumor cells including human liver cancer cells HepG2, human lung adenocarcinoma cells A549 and human lung cancer cells NCI-H460 is determined. The results show that the fermentation liquor of one strain Y15-8 has better cell proliferation inhibition effect on the tumor cells, wherein the inhibition rates of human liver cancer cells HepG2, human lung adenocarcinoma cells A549 and human lung cancer cells NCI-H460 are 53.2%, 58.9% and 55.6%.

Example 2 morphological characteristics and physiological and biochemical characteristics of Bacillus enteromorpha Y15-8

(1) Morphological characteristics

Gram-negative bacteria were obtained by gram-staining the strains. The strain Y15-8 has two important characteristics of bacillus morphology and spore production capacity, and after being cultured on beef extract peptone medium NRG for 36h, the bacterial colony is circular, has dry and rough and sticky surface, is not easy to pick up, has uniform texture, has uniform colors on the front side, the back side, the edge and the central part, and is milky white (as shown in figure 1).

The strain is aerobic, and can grow well at a pH value of 7.5 and a culture temperature of 30 ℃.

(2) Physiological and biochemical reaction characteristics

According to the methods of 'handbook of methods for ordinary bacteriology' and 'handbook of identification of common bacteria system', API20E Enterobacter and other gram-negative bacteria identification systems, API 20N identification system for parenteral gram-negative bacteria and oxidase test paper are adopted to determine physiological and biochemical characteristics, specific operation is carried out according to the instructions to determine part of physiological and biochemical characteristics, and the result is negative, namely β -galactosidase, arginine double hydrolase, lysine decarboxylase, H₂S production, urease, tryptophan dehydrogenase, indole experiments, gelatinase and oxidase experiments; positive: ornithine decarboxylase, VP experiments. Negative fermentation utilization: mannitol, inositol, sucrose, amygdalin, arabinose; positive: glucose, sorbitol, rhamnose, melibiose. Negative assimilation experiment: and (4) citric acid.

(3)16S rRNA sequence analysis

1.5mL of Y15-8 bacterial liquid in the logarithmic phase was aspirated, centrifuged at 12000rpm at 4 ℃ for 10 min. Discarding the supernatant, adding 400 μ L of TE buffer solution, blowing the precipitate with a gun head, adding 10 μ L of lysozyme, mixing well, placing in a 37 ℃ water bath for 90min, at 4 ℃, 12000rpm, centrifuging for 10min, and retaining the supernatant as DNA solution. 16S r D N A universal primer, forward primer: 27F 5 '-AGAGTTTGATCCTGGTCAG-3', reverse primer 1492R5 '-CGGCTACCTTGTTACGACTT-3'; the PCR conditions for the 16S rRNA sequence were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 40s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 90s, circulation for 30 times, and extension at 72 ℃ for 10 min. The PCR reaction system is as follows: reaction system: 50 μ L, template DNA: 3 μ L, forward primer: 2 μ L, downstream primer: 2 μ L, mix: 25 μ L, Free water: 18 μ L.

The PCR amplification product was detected by 1.5% agarose electrophoresis using D2000Marker as the standard molecular weight control reagent. The 16S rRNA PCR product of strain Y15-8 was sent to Beijing Dacron for sequencing. The sequence was finally determined to be 1430bp in length for the 16S rRNA sequence of strain Y15-8. The specific sequence is shown in SEQ and NO.1

BLAST comparison is carried out on the 16SrRNA sequence at an NCBI website, homologous sequence retrieval is carried out, the Bacillus is determined to be Bacillus (Bacillus), possibly a new Bacillus, and the Bacillus is named as Enteromorpha Bacillus sp.Y15-8. The marine bacillus Y15-8 is preserved in the China Wuhan type culture Collection in 2016, 12 months and 12 days, and the preservation number is CCTCC M2016744. The 16S rRNA gene sequence has different mutation probability at certain sites, but shows structural and functional conservation at the species and genus level, has the reputation of bacterial fossils and is a timer for the biological evolution history. By adopting 16SrRNA as a molecular index, the microorganism can be classified and identified quickly, accurately, slightly and simply.

EXAMPLE 3 fermentation culture of Bacillus Enteromorpha Y15-8

The screened enteromorpha Bacillus Y15-8 (Latin, the name of which is Bacillus sp.Y15-8, 2016, and 12 days in 12 months) is preserved in a China center for type culture collection, the preservation address is China Wuhan university of Wuhan, the preservation number is CCTCC M2016744, the screened enteromorpha Bacillus Y15-8 is connected in a test tube containing 4mL of seed culture solution, the seed culture solution comprises 3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, the final concentration of the components, the pH value is 7.5, the selected enteromorpha Bacillus Y is placed in a constant temperature shaking incubator with the temperature of 30 ℃ and the temperature of 200rmp for overnight culture, a gradient dilution coating plate is made, a solid culture medium comprises 3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, 20g/L of agar, the final concentration of the components, the pH value is 7.5, the coated plate is placed in a constant temperature incubator with the temperature of 30 ℃ for inverted culture for 18-36 hours, a, culturing in a constant-temperature shaking incubator at 30 ℃ and 200 rmp; when the logarithmic phase is reached, inoculating the strain to a 250mL triangular flask containing 50mL of fermentation culture solution according to the inoculation amount of 4%, and culturing for 12h in a constant-temperature shaking incubator of 200rmp at 30 ℃; then inoculating the mixture into a 2L triangular flask containing 400mL of fermentation culture solution according to the inoculation amount of 4 percent, wherein the fermentation culture solution: 3g/L beef extract, 10g/L peptone and 5g/L NaCl, wherein the final concentration of each component is pH 7.5. The fermentation broth was centrifuged at 9800rmp for 25min at 4 ℃ and 8L of fermentation supernatant was collected.

EXAMPLE 4 preparation of active protein of Bacillus enteromorpha Y15-8

(1) Ultrafiltration

8L of the collected fermentation supernatant was concentrated by ultrafiltration. And then carrying out ultrafiltration by using ultrafiltration membranes with the molecular weights of 3kDa and 50kDa to obtain an active component with the molecular weight of 3-50 kDa.

(2) Ammonium sulfate precipitation

Slowly adding ammonium sulfate powder into the 3-50 kDa component, slightly stirring to enable the concentration of the ammonium sulfate powder to reach 60% (mass percentage concentration), controlling the temperature to be 4 ℃ during the stirring, and standing for 10-12 hours in a refrigerator after the ammonium sulfate powder is completely dissolved; centrifuging at 9800rpm for 25min, collecting supernatant, continuously adding ammonium sulfate powder to make the concentration reach 75% (mass percentage concentration), controlling the temperature at 4 ℃ during the period, and standing in a refrigerator for 10-12 h after all the ammonium sulfate powder is dissolved; centrifuging at 9800rpm for 25min, collecting the precipitate, dissolving with 50mM Tris-HCl (pH8.0) twice the volume of the precipitate, dialyzing with dialysis bag having a molecular weight cut-off of 3.5kDa for 22h, and filtering the sample with 0.22 μm microporous membrane.

(3) Passing Q anion exchange chromatography column

Dissolving 60-75% (mass percent concentration) of the antitumor active component obtained by the ammonium sulfate precipitation in a certain amount of loading buffer solution, wherein the loading buffer solution is 50mmol/L Tris-HCl buffer solution with the pH value of 8.0, centrifuging the solution for 15min at 12000rmp, and filtering the sample by a 0.22 mu m microporous filter membrane; passing through a Q anion exchange chromatographic column, wherein the sample loading amount is 5mL each time, an eluent is 50mmol/LTris-HCl buffer solution containing 1mol/L NaCl and pH7, the eluent is eluted at the concentration of 0-100% (v/v), the flow rate is 2mL/min, the detection wavelength is 280nm, each penetration peak (Q1, Q2) and each elution peak (R1, R2, R3, R4, R5, R6, R7) are collected (as shown in figure 2), the antitumor activity of each peak component is detected, and the activity of the penetration peak Q1 is optimal. And desalting the sample collected by the penetration peak Q1, and freeze-drying to obtain the prepared enteromorpha bacillus Y15-8 active protein PBY 15-8.

Example 5 effects of 5PBY15-8 on morphological changes in human hepatoma cell BEL-7402

PBY15-8 acting human liver cancer cell BEL-7402 with the concentration of 10 mug/ml and the cell shape change conditions corresponding to different acting time (0h, 12h, 24h and 48h) are acted by polypeptide for 12h, most BEL-7402 cells become round, and a small amount of cells are disintegrated; after 24 hours of action, almost all BEL-7402 cells become round and a large amount of cells are disintegrated; when the reaction is carried out for 48 hours, only a small number of living cells are stored and are in a round state, and a large number of cells are dead (as shown in figure 3).

Example 6 effects of 6PBY15-8 on morphological changes in human Lung adenocarcinoma cells A549

PBY15-8 acting human lung adenocarcinoma cells A549 with the concentration of 10 mu g/ml and the cell morphology change conditions corresponding to different acting time (0h, 12h, 24h and 48h) are acted by polypeptide for 12h, most A549 cells become round, and a small amount of cells are disintegrated; after 24 hours of action, almost all A549 cells become round and a large number of cells are disintegrated; when the reaction is carried out for 48 hours, only a small number of living cells are stored and are in a round state, and a large number of cells are dead (as shown in figure 4).

Example 7 Effect of 7PBY15-8 on morphological changes in human Large cell Lung cancer cell NCI-H460

PBY15-8 acts on human large cell lung cancer cell NCI-H460, the concentration is 20 mug/ml, the cell shape change situation corresponding to different acting time (0H, 12H, 24H, 48H) is acted by polypeptide for 12H, most cells grow normally, and a small amount of cells are inhibited; after 24 hours of action, a large number of cells become round, and a small number of cells disintegrate; when the reaction is carried out for 48 hours, only a small number of living cells are stored and are in a round state, and a large number of cells are dead (as shown in figure 5).

Example 8PBY15-8 inhibition of proliferation of human hepatoma cell BEL-7402

The human liver cancer cell BEL-7402 is used as an action cell, and the MTT method finds that PBY15-8 has better proliferation inhibition effect on BEL-7402, and the concentration gradient is 1 mu g/ml, 2 mu g/ml, 4 mu g/ml, 8 mu g/ml and 10 mu g/ml along with the change of the polypeptide concentration in a dose-dependent relationship, the action time is 48h, and the IC is IC₅₀It was 6.95. mu.g/mL (see FIG. 6).

Example 9PBY15-8 inhibition of proliferation of human Lung adenocarcinoma cells A549

Human lung adenocarcinoma cells A549 are taken as acting cells, and the MTT method finds that PBY15-8 has better proliferation inhibition effect on BEL-7402, the concentration gradient is 1 mu g/ml, 2 mu g/ml, 4 mu g/ml, 8 mu g/ml and 10 mu g/ml along with the change of polypeptide concentration, the acting time is 48h, and the IC is₅₀11.48. mu.g/mL (see FIG. 7).

Example 10PBY15-8 inhibition of proliferation of human Lung cancer cell NCI-H460

The human lung cancer cell NCI-H460 is taken as an action cell, and the MTT method finds that PBY15-8 has better proliferation inhibition effect on NCI-H460, and the concentration gradient is 2 mu g/ml, 4 mu g/ml, 8 mu g/ml, 10 mu g/ml, 20 mu g/ml and 40 mu g/ml along with the change of the polypeptide concentration in a dose-dependent manner, the action time is 48H, and the IC is₅₀It was 21.13. mu.g/mL (see FIG. 8).

The MTT method described in this example: collecting liver cancer cell BEL-7402 in logarithmic growth phase, discarding old culture solution, adding 0.25% pancreatin 1.5ml, digesting for 2-3 min, and microscopic examinationWhen the intercellular substance is retracted and the intercellular spaces are clear, pancreatin is discarded under the observation of a mirror. Adding 5mL of fresh DMEM culture solution, sufficiently blowing and beating to prepare single cell suspension, and carrying out centrifugation at 4X 10³Inoculating the solution into a 96-well plate at the density of 180 mu L per well, pre-culturing for 12h in a 5% CO2 incubator at 37 ℃ and adding 20 mu L of sample into each well, filtering and sterilizing the sample solution through a 0.22 mu m microporous filter membrane, setting a negative control group and a blank control group, setting 4 repeated wells, and continuously culturing for 48 h; then, 20 μ of LMTT solution (5mg/mL, i.e., 0.5% MTT) was added and incubated for 4h at 37 ℃ in a 5% CO2 incubator; after the culture was terminated, the culture medium was carefully aspirated from the wells, and then dimethyl sulfoxide DMSO (150 μ L) was added to each well, the wells were placed on a shaker at room temperature and low speed for 10min to sufficiently dissolve formazan crystals, and the absorbance (OD) of each well was measured at 570nm using an microplate reader, and the cell inhibition ratio (%) (control well OD value-administration well OD value)/control well OD value × 100% was calculated. Calculating the median inhibitory concentration IC by using Excel analysis software₅₀Data analysis was performed using SPSS18.0 software, expressed as mean. + -. standard deviation, and comparisons between groups were performed using t-test, P<0.05 means that the difference between the two groups is significant, P<0.0l indicated a very significant difference between the two groups.

Bacillus sp.Y15-816 s rRNA sequence:

TCTGACCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTGCAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTATGGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGT CTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTGCA

SEQUENCE LISTING

<110> research institute for aquatic products in yellow sea of China institute for aquatic science

<120> Enteromorpha bacillus Y15-8 and anti-tumor active protein thereof

<130> do not

<160>3

<170>PatentIn version 3.3

<210>1

<211>1430

<212>DNA

<213>Bacillus sp.

<400>1

tctgaccacc ttcggcggct ggctccataa aggttacctc accgacttcg ggtgttgcaa 60

actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 120

tgatccgcga ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa 180

ctgagaacag atttatggga ttggctaaac cttgcggtct cgcagccctt tgttctgtcc 240

attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc 300

ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actaaatgct ggcaactaag 360

atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420

accatgcacc acctgtcact ctgtccccga agggaaagcc ctatctctag ggttgtcaga 480

ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540

cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg 600

gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc 660

atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc 720

ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta 780

cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag tttcccagtt 840

tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc 900

gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc 960

tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtgcgag cagttactct 1020

cgcacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg 1080

gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140

agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200

gtcgccttgg tgagccatta ccccaccaac tagctaatgc gccgcgggtc catctgtaag 1260

tgacagccga aaccgtcttt catccttgaa ccatgcggtt caaggaacta tccggtatta 1320

gctccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc 1380

gtccgccgct aacatccggg agcaagctcc cttctgtccg ctcgactgca 1430

<210>2

<211>19

<212>DNA

<213>Artificial

<220>

<223> Forward primer 27F

<400>2

agagtttgat cctggtcag 19

<210>3

<211>19

<212>DNA

<213>Artificial

<220>

<223> reverse primer 1492R

<400>3

cggctacctt gttacgact 19

Claims

1. Enteromorpha bacillus Y15-8, which are classified and namedBacillussp.Y15-8, characterized in that the Enteromorpha prolifera Bacillus Y15-8 is preserved in the Wuhan type culture Collection in China in 2016, 12 months and 12 days, with the preservation number of CCTCC M2016744.

2. The enteromorpha bacillus protein produced by fermenting the enteromorpha bacillus Y15-8 according to claim 1, wherein the enteromorpha bacillus protein has anti-tumor activity; the preparation method of the enteromorpha bacillus protein comprises the following steps:

(1) fermentation culture

Fermenting enteromorpha bacillus Y15-8 under the fermentation conditions: the final concentration of each component in the fermentation liquor is 2-6 g/L of beef extract, 7-12 g/L of peptone, 2-6 g/L of sodium chloride, pH6.0-9.0, 1.05kg/cm²Sterilizing at 115-121 ℃ for 15-35 min, and culturing in a constant-temperature shaking table at 150-250 r/min and 25-37 ℃ for 48-80 h;

(2) ultrafiltration interception

Centrifuging fermentation liquor obtained by fermenting and culturing enteromorpha bacillus Y15-8 at 8000-10000 rpm for 10-25 min, collecting supernatant, and performing ultrafiltration by using ultrafiltration membranes with molecular weights of 3kDa and 50kDa to obtain components with molecular weights of 3-50 kDa;

(3) ammonium sulfate precipitation

(4) anion exchange chromatography, passing the fraction with anti-tumor activity obtained in step (3) through 0.22 μm microporous membrane; passing through a Q or DEAE anion exchange chromatography column, wherein the sample loading amount is 5-50 mL each time, the eluent is Tris-HCl with the concentration of 20-50 mmol/L and the pH value of 7.5-8.5, the eluent contains 1-2 mol/L NaCl, the concentration of 0-100% v/v is eluted, the flow rate is 3-6mL/min, the detection wavelength is 280nm, a penetration peak Q1 is collected, and the Q1 component is desalted and then is subjected to vacuum freeze drying to obtain the enteromorpha symbiotic microbial active protein which is named as enteromorpha bacillus protein.

3. The Bacillus enteromorpha protein of claim 2, wherein the concentration of NaCl in the eluate obtained in the step (4) is 1 mol/L.

4. The Bacillus enteromorpha protein of claim 2, wherein the eluent in the step (4) has a flow rate of 2.0 mL/min.

5. The enteromorpha bacillus protein of claim 2, wherein the sample amount in step (4) is 5 mL.

6. A medicament for treating or preventing human liver cancer cell HepG2, human lung adenocarcinoma cell a549, or human lung cancer cell NCI-H460, said medicament comprising the enteromorpha bacillus protein of claim 2.

7. The use of the enteromorpha bacillus protein of claim 2 in the preparation of medicaments for treating or preventing human liver cancer cells HepG2, human lung adenocarcinoma cells A549 or human lung cancer cells NCI-H460.