CN108546287A - A kind of antitumor fungal immunomodulatory protein Fip-bbo and its application - Google Patents
A kind of antitumor fungal immunomodulatory protein Fip-bbo and its application Download PDFInfo
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- CN108546287A CN108546287A CN201810337961.XA CN201810337961A CN108546287A CN 108546287 A CN108546287 A CN 108546287A CN 201810337961 A CN201810337961 A CN 201810337961A CN 108546287 A CN108546287 A CN 108546287A
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- bbo
- fip
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- immunomodulatory protein
- fungal immunomodulatory
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- 201000010881 cervical cancer Diseases 0.000 abstract description 7
- 208000019065 cervical carcinoma Diseases 0.000 abstract description 4
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
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- 230000004957 immunoregulator effect Effects 0.000 description 1
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- 210000005265 lung cell Anatomy 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000003726 plant lectin Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/195—Proteins from microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The present invention relates to a kind of antitumor fungal immunomodulatory protein Fip bbo and its applications, and the amino acid sequence of the immune modulator is as shown in SEQ ID NO.1.It is used to prepare health food.The present invention can obviously induce cervical cancer Hela cells apoptosis；Therefore, antitumor fungal immunomodulatory protein Fip bbo have application potential in terms of the health food for cervical carcinoma.
Description
Technical field
The invention belongs to fungal immunomodulatory protein field, more particularly to a kind of antitumor fungal immunomodulatory protein Fip-
Bbo and its application.
Background technology
Fungal immunomodulatory protein (Fungal immunomodulatory protein, FIP) is detached from fungi
The one kind arrived has the small protein of immunoregulatory activity.Their structure function and phytolectin and immunoglobulin
Superfamily has a very high similitude, and with promoting lymphopoiesis (Hsieh KY et al., 2003), regulating cell
Period (Liao CH et al., 2008), the table for inducing Apoptosis (Hsiao YM et al., 2008), influencing cell factor
Up to (Li QZ et al., 2011), activating cell adhesion molecule (Wu CT et al., 2011) and antianaphylaxis (Ko JL
et al.,1995；Lin YL et al.,2009；Sun X et al., 2014) immunoregulation effects such as, possess good face
Bed application prospect and medicinal health value.
Although all having centainly on primary structure, secondary structure and higher structure between various fungal immunomodulatory proteins
Similitude, and have similar biological function.However, different FIPs is not showed also not in terms of certain bioactivity
Together.Such as in terms of inhibiting tumor cell proliferation, LZ-8 has lung cell A549 and human leukemia cell HL60 very strong
Antitumor activity, but FIP-fve but there is no this activity (Huang L et al., 2009；Li SY et al.,
2016), although their protein sequence similarity reaches 60% or more.Different fungal immunomodulatory proteins has different lifes
Therefore object activity excavates novel fungitype immune modulator in different fungies, will be explore with more biological functions or
The pharmaceutical protein of more high-immunity regulation activity provides abundant living resources, and is a kind of novel medicine or function food for its exploitation
Product provide valuable theoretical foundation and apply foundation.
Invention content
Technical problem to be solved by the invention is to provide a kind of antitumor fungal immunomodulatory protein Fip-bbo and its answer
With the immune modulator can obviously induce cervical cancer Hela cells apoptosis；Therefore, antitumor fungal immunomodulatory protein
Fip-bbo has application potential in terms of the health food for cervical carcinoma.
The present invention provides a kind of antitumor fungal immunomodulatory protein Fip-bbo, the amino of the immune modulator
Acid sequence is as shown in SEQ ID NO.1.
The present invention also provides a kind of antitumor fungal immunomodulatory protein Fip-bbo of coding in expression in escherichia coli
Optimize codon, the nucleotide sequence of the optimization codon is as shown in SEQ ID NO.2.
The present invention also provides a kind of carriers optimizing codon containing antitumor fungal immunomodulatory protein Fip-bbo.
Preferably, the carrier is pCreat-S II.
Preferably, antitumor fungal immunomodulatory protein Fip-bbo optimization codons are inserted on pCreat-S II
Between BamHI and XhoI restriction enzyme sites, recombination large intestine expression plasmid pCreat-Fip-bbo is obtained.
The present invention also provides a kind of recombinant bacteriums optimizing codon containing antitumor fungal immunomodulatory protein Fip-bbo
Strain.
The recombinant bacterial strain is DE3-Fip-bbo.
The present invention also provides a kind of preparation methods of antitumor fungal immunomodulatory protein Fip-bbo, including：
(1) the optimization Codon sequences of antitumor fungal immunomodulatory protein are connected into carrier, obtain recombinant vector；
(2) above-mentioned recombinant vector is converted into host cell, obtains recombinant bacterial strain；
(3) above-mentioned recombinant bacterial strain is cultivated, induction recombinates the expression of antitumor fungal immunomodulatory protein Fip-bbo；
(4) expressed antitumor fungal immunomodulatory protein Fip-bbo is obtained through Ni-NTA purifying, dialysis separation.
Host cell in the step (2) is Bacillus coli cells DE3-Fip-bbo.
The present invention also provides a kind of application of antitumor fungal immunomodulatory protein Fip-bbo, the immunological regulation eggs
It is used to prepare health food in vain.
Amino acid sequence of the present invention is subjected to BLAST in the irredundant protein sequence databank (nr) of NCBI and compares hair
It is existing, the albumen with from Ganoderma microsporum, G.applanatum, G.lucidum, G.atrum and
The similitude of the immune modulator Gmi, Fip-gap, LZ-8, Fip-gat and Fip-gja of G.japonicum are below 62%.
Illustrate that Fip-bbo is a kind of novel fungal immunomodulatory protein.
The present invention demonstrates the function of fungal immunomodulatory protein Fip-bbo through the following experiment：
1.MTT methods measure the antitumor activity of above-mentioned fungal immunomodulatory protein Fip-bbo.
2. flow cytometry detects fungal immunomodulatory protein Fip-bbo induced Hcla cell apoptosis.
Advantageous effect
(1) present invention obtains a fungal immune tune for including 112 amino acid residues by the method for bioinformatics
Save albumen Fip-bbo protein sequences (SEQ ID NO:1) it, and by artificial synthesized method obtains in Escherichia coli preference password
CDNA sequence (the SEQ ID NO of son:2), then the sequence in expression in escherichia coli and is isolated and purified, through MTT and streaming
Cell art method proves that the fungal immunomodulatory protein Fip-bbo from basidiomycetes B.botryosum genomes has and inhibits palace
The function of neck cancer cell Hela growths；
(2) present invention firstly discovers that a kind of having cervical cancer cell Hela the fungal immunomodulatory protein for inhibiting growth
Fip-bbo, and Fip-bbo high efficient expression (expression quantity 78.8mg/L) in escherichia expression system is provided, and through Ni-
NTA isolates and purifies the method for obtaining pure Fip-bbo.It may be implemented to prepare with anti-using genetic engineering means according to the present invention
The active fungal immunomodulatory protein Fip-bbo of cervical carcinoma has application potential in terms of the health food for cervical carcinoma.
Description of the drawings
Fig. 1 is the SDS-PAGE analyses of fungal immunomodulatory protein Fip-bbo of the present invention expression；Wherein, swimming lane 1, Fip-
Bbo pre-induction samples；Swimming lane 2, Fip-bbo postinduction samples；Swimming lane M, molecular weight standard.
Fig. 2 analyzes for fungal immunomodulatory protein Fip-bbo solubilities SDS-PAGE of the present invention；Wherein, swimming lane 1, Fip-
Bbo pre-induction samples；Swimming lane 2, Fip-bbo postinduction samples；Swimming lane 3, sediment after induction；Swimming lane 4, supernatant after induction；
Swimming lane M, molecular weight standard.
Fig. 3 is the SDS-PAGE analyses that fungal immunomodulatory protein Fip-bbo of the present invention is isolated and purified；Wherein, swimming lane 1,
It is precipitated after broken；Swimming lane 2, supernatant after being crushed；Swimming lane 3, efflux after Ni-NTA is incubated；Swimming lane 4, Buffer B clean sample；Swimming
Road 5, Buffer C clean sample；Swimming lane 6, Buffer D elute sample；Swimming lane M, molecular weight standard.
Fig. 4 is the MTT detection knots that fungal immunomodulatory protein Fip-bbo of the present invention inhibits cervical cancer Hela cells proliferation
Fruit.
Fig. 5 is that fungal immunomodulatory protein Fip-bbo of the present invention induces cervical cancer cell Hela apoptosis results.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.Test material：
1. cell strain：BL21 (DE3) competence is purchased from general biosystem (Anhui) Co., Ltd (CP01010)；People
Cervical cancer cell Hela is bought in Shanghai Inst. of Life Science, CAS cell resource center.
2. main agents consumptive material and instrument：
Reagent：Skimmed milk power is purchased from Erie, and BSA is purchased from Shanghai life work (A0903), 6 × His Tag Antibody, HRP
Conjugate is purchased from Invitrogen (MA1-21315-HRP), and Pre-stained Protein Marker are purchased from Thermo public affairs
It takes charge of (26617), IPTG is purchased from Amresco (0487), and Ni NTA beads 6FF are purchased from Changzhou people from world and biotechnology is limited
Company (SA005100), BeyoECL Plus are purchased from Beyotime (P0018), and SUMO Protease are purchased from general biosystem
(Anhui) Co., Ltd, Annexin V-FITC/PI cell apoptosis detection kits have purchased from Nanjing Keygen Biotech's development in science and technology
Limit company, glacial acetic acid, glycerine (glycerine), absolute ethyl alcohol, methanol, sodium hydroxide, sodium chloride, potassium chloride, disodium hydrogen phosphate, ten
Two hypophosphite monohydrate sodium dihydrogens, potassium dihydrogen phosphate, imidazoles etc. are purchased from Sinopharm Chemical Reagent Co., Ltd..
Instrument：Sai Mo flies generation that centrifuge, ELISA instrument, Vertial electrophorestic tank, constant-temperature shaking incubator, clasmatosis
Instrument, U.S.'s Thermo scientific cell incubators, U.S.'s Becton Dickinson flow cytometers.Shanghai Cai Kang falls
Set microscope；
3. culture medium：Raw work LB culture mediums；U.S.'s GIBCORPMI DMEM culture mediums, fetal calf serum.
Embodiment 1
The clone of fungal immunomodulatory protein Fip-bbo protein sequences and expression vector establishment
Using Ganoderma lucidum immunoregulation protein LZ-8 as template, BLAST ratios are carried out in basidiomycetes B.botryosum full-length genomes
To finding Fip-bbo protein sequences (the SEQ ID NO for being 62% with LZ-8 similitudes:1) it, and according to the optimization of this protein sequence sets
Count Codon sequences (the SEQ ID NO in Escherichia coli:2).General biosystem (Anhui) Co., Ltd is entrusted to complete base
The base sequence of cause synthesizes, and the sequence construct of synthesis extracts plasmid, with double to cloning vector pUC57, transformed clone bacterial strain DH5 α
After digestion (BamHI and XhoI) digestion, agarose gel electrophoresis recycles Fip-bbo genetic fragments, with ligase (T4ligase)
It is connected to so that on II carriers of pCreat-S of BamHI and XhoI digestion, expression vector pCreat-Fip-bbo is transformed into BL21
(DE3) in competent bacteria, 30min on ice is set, 42 DEG C of heat shock 90s set rapidly 5min in ice；500 μ L LB culture solutions are added,
37 DEG C, 220rpm shakes 1h, and resistant LB tablets, 37 DEG C of inversion overnight incubations are all coated on after centrifugation.5 tablets of picking
Upper monoclonal is inoculated in the test tube of the 4mL LB culture solutions containing appropriate resistance, and 37 DEG C of 220rpm, which shake to thalline OD600, is
0.6-0.8 takes out 1mL cultures, and 12000g room temperatures centrifuge 5min, abandon supernatant, and it is heavy that thalline is resuspended with 80 μ 1 × PBS buffer solution of L
IPTG is added into remaining culture to final concentration of 0.5mM, 37 by the 5 × Loading Buffer for forming sediment and being added 20 μ L
DEG C 220rpm shakes 4h, and induced fusion protein expression takes out 0.5mL cultures, and 12000g room temperatures centrifuge 5min, abandon supernatant, use
80 μ L1 × PBS buffer solution is resuspended bacterial sediment and 5 × Loading Buffer of 20 μ L is added.Finally carry out SDS-PAGE points
It analyses (see Fig. 1), as shown in Figure 1, correct plasmid is converted into BL21 (DE3) Escherichia coli, and the fusion protein of Fip-bbo
Expression is normal.
Embodiment 2
The soluble analysis of recombinant fungus immune modulator Fip-bbo
BL21 (DE3) bacterium solution 2mL, 12000g room temperatures centrifuge 5min after taking 37 DEG C of inductions, supernatant are abandoned, with the BufferA of 1mL
(20mM Tris, 300mM NaCl, 10%Glycerol, pH8.0) resuspension centrifuged deposit, ultrasonic disruption (Φ 3,15%,
3s/6s, 5min), the sample preparation respectively of supernatant precipitation carries out SDS-PAGE analyses, and under 37 DEG C of inductions, fusion protein is solvable (see figure
2)。
Embodiment 3
The amplification expression of recombinant fungus immune modulator Fip-bbo and affinity purification
Optimal clone conservation bacterium is taken to be seeded in antibiotic 1L LB culture mediums, 37 DEG C of 220rpm shake to OD600=
0.6~0.8, after final concentration of 1.0mM IPTG are added, 37 DEG C of 220rpm shake 4h.10min is centrifuged using 8000rpm, is discarded
Supernatant collects all thalline.Bacterium is resuspended using Buffer A (20mM Tris, 300mM NaCl, 10%Glycerol, pH8.0)
Body, ultrasonication (Φ 10,15%, 3s/6s, 20min).16000rpm centrifuges 10min, collects supernatant.3mL is added in supernatant
Ni-NTA, 4 DEG C of incubation 1h after mixing.Object will be incubated, void column pipe is added, and collect efflux.Respectively with Buffer B (20mM
Tris, 300mM NaCl, 10%Glycerol, 20mM Imidazole, pH8.0) and Buffer C (20mM Tris, 300mM
NaCl, 10%Glycerol, 40mM Imidazole, pH8.0) cleaning filler, collect cleaning solution.Utilize Buffer D (20mM
Tris, 300mM NaCl, 10%Glycerol, 250mM Imidazole, pH8.0) elution, collect eluent.SDS-PAGE electricity
Swimming detection, FIP-bbo is in E. coli, expression quantity 78.8mg/L；After column purification, the content of protein reaches
It is pure (Fig. 3) to electrophoresis.
Conclusion：FIP-bbo realizes high efficient expression in the escherichia expression system, therefore can quickly provide and quite count
The protein sterling of amount, to meet the application demand of exploitation medicine and health food.
Embodiment 4
Mtt assay detects recombinant fungus immune modulator Fip-bbo antitumor activities
Experimental method：The secondary culture Hela cells in DEME culture solutions count tumour cell, are diluted to final concentration of 5-
10×104A/ml.100 μ L tumor cell suspensions are added per hole in 96 well culture plates, are put in 5%CO2, in 37 DEG C of incubators
Cultivate a night.Second day, the culture solution in hole is absorbed, 100 μ L recombinant protein samples, the μ of final concentration of 0,1,2,4,8 and 16 is added
g/ml.Addition is diluted to 50mmol/ml adriamycins and does positive control simultaneously.Each 3, sample is parallel.It is put in 5%CO2, 37 DEG C
It is cultivated 24 hours in incubator.After reaching action time, into above-mentioned cell plates, each medicine feeding hole enters 10 μ L MTT reagents (MTT need
It is protected from light, is put into incubator, liquid in hole is discarded after 4h, 150 μ L DMSO are added, concussion makes crystal fully dissolve, in more work(
Energy microplate reader A570nm and A630nm measure absorbance, calculate cell survival rate.
Experimental result：FIP-bbo to cervical cancer Hela cells have toxic effect (Fig. 4), FIP-bbo pairs of 16 μ g/ml
The toxic effect of Hela is close with positive control.
Conclusion：FIP-bbo has high toxic effect to Hela cells.
Embodiment 5
Flow cytometry recombinant fungus immune modulator Fip-bbo inducing apoptosis of tumour cell
Experimental method：In 5%CO2, DMEM culture solution culture Hela cells, cell count is used in 37 DEG C of incubators to make it
Final concentration of 5-10 × 105A/ml.1ml tumor cell suspensions are added in 6 well culture plates, after cultivating 24 hours, are added eventually
The FIP-bbo of a concentration of 16 μ g/ml, negative control is done with the tumour cell of normal growth；In 5%CO2, train in 37 DEG C of incubators
It supports 24 hours.
Experimental result：FIP-bbo has the function of Hela cells to induce its apoptosis, the FIP-bbo processing of 16 μ g/ml
After Hela cells 36h, apoptosis rate is 35% (Fig. 5).
Conclusion：FIP-bbo has the function of Hela tumour cells to induce its apoptosis, and its apoptotic activity is higher,
It is the antitumor drug of great potential.
SEQUENCE LISTING
<110>Academy of Agricultural Sciences, Shanghai City
<120>A kind of antitumor fungal immunomodulatory protein Fip-bbo and its application
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 112
<212> PRT
<213>Artificial sequence
<400> 1
Met Ser Thr Asp Thr Thr Ala Gln Ile Phe His Leu Val Ala Ser Leu
1 5 10 15
Pro Lys Leu Ser Tyr Asp Tyr Thr Pro Asn Trp Val Lys Asp Arg Gln
20 25 30
Tyr Ile Asn Ser Val Thr Phe Ala Lys Val Leu Ala Asp Lys Ala Tyr
35 40 45
Thr Tyr Arg Val Met Ala Gly Asp Lys Asp Leu Gly Val Lys Pro Ser
50 55 60
Tyr Ala Val Gly Ser Asp Gly Ser Gln Lys Ile Asn Phe Leu Glu Tyr
65 70 75 80
Asn Gly Gly Tyr Gly Ile Asp Gly Asn Ala Asn Thr Val Tyr Val Tyr
85 90 95
Val Val Asp Pro Glu Thr Gly Asn Gln Tyr Lys Ile Ala Gln Ser Lys
100 105 110
<210> 2
<211> 336
<212> DNA
<213>Artificial sequence
<400> 2
atgagcaccg ataccaccgc acagattttt catctggtgg ccagtctgcc gaaactgagc 60
tatgattata ccccgaattg ggttaaagat cgtcagtata ttaacagtgt tacctttgca 120
aaagtgctgg ccgataaagc atatacctat cgcgttatgg caggcgataa agatctgggc 180
gttaaaccga gttatgccgt gggtagcgat ggcagccaga aaattaattt tctggaatat 240
aacggcggct atggcattga tggcaatgcc aataccgtgt atgtttatgt tgtggaccct 300
gaaaccggta atcagtataa aattgcccag agtaaa 336
Claims (10)
1. a kind of antitumor fungal immunomodulatory protein Fip-bbo, it is characterised in that：The amino acid sequence of the immune modulator
Row are as shown in SEQ ID NO.1.
2. a kind of coding antitumor fungal immunomodulatory protein Fip-bbo as shown in claim 1 is in expression in escherichia coli
Optimization codon, it is characterised in that：The nucleotide sequence of the optimization codon is as shown in SEQ ID NO.2.
3. a kind of carrier optimizing codon containing antitumor fungal immunomodulatory protein Fip-bbo as claimed in claim 2.
4. a kind of carrier of antitumor fungal immunomodulatory protein Fip-bbo optimizations codon according to claim 3,
It is characterized in that：The carrier is pCreat-S II.
5. a kind of carrier of antitumor fungal immunomodulatory protein Fip-bbo optimizations codon according to claim 4,
It is characterized in that：By antitumor fungal immunomodulatory protein Fip-bbo optimization codons be inserted into BamHI on pCreat-S II and
Between XhoI restriction enzyme sites, recombination large intestine expression plasmid pCreat-Fip-bbo is obtained.
6. a kind of recombinant bacterium optimizing codon containing antitumor fungal immunomodulatory protein Fip-bbo as claimed in claim 2
Strain.
7. a kind of recombinant bacterium of antitumor fungal immunomodulatory protein Fip-bbo optimizations codon according to claim 6
Strain, it is characterised in that：The recombinant bacterial strain is DE3-Fip-bbo.
8. a kind of preparation method of antitumor fungal immunomodulatory protein Fip-bbo, including：
(1) the optimization Codon sequences of antitumor fungal immunomodulatory protein are connected into the carrier such as claim 3 or 4, are obtained
To recombinant vector；
(2) above-mentioned recombinant vector is converted into host cell, obtains recombinant bacterial strain；
(3) above-mentioned recombinant bacterial strain is cultivated, induction recombinates the expression of antitumor fungal immunomodulatory protein Fip-bbo；
(4) expressed antitumor fungal immunomodulatory protein Fip-bbo is obtained through Ni-NTA purifying, dialysis separation.
9. the preparation method of antitumor fungal immunomodulatory protein Fip-bbo according to claim 8 a kind of, feature exist
In：Host cell in the step (2) is Bacillus coli cells DE3-Fip-bbo.
10. a kind of application of antitumor fungal immunomodulatory protein Fip-bbo as described in claim 1, it is characterised in that：Institute
It states immune modulator and is used to prepare health food.
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