CN104817637B - A kind of straw mushroom immune modulator FIP-vvo78 and preparation method thereof - Google Patents
A kind of straw mushroom immune modulator FIP-vvo78 and preparation method thereof Download PDFInfo
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- CN104817637B CN104817637B CN201510217510.9A CN201510217510A CN104817637B CN 104817637 B CN104817637 B CN 104817637B CN 201510217510 A CN201510217510 A CN 201510217510A CN 104817637 B CN104817637 B CN 104817637B
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Abstract
The present invention relates to a kind of straw mushroom immune modulator FIP vvo78 and preparation method thereof, and the amino acid sequence of albumen is as shown in SEQ ID NO.1.Preparation method includes：(1) host cell is converted using recombinant vector, obtains recombinant bacterial strain；(2) recombinant bacterial strain, the expression of induction restructuring straw mushroom immune modulator FIP vvo78 are cultivated；(3) ultrasonication obtains expressed straw mushroom immune modulator FIP vvo78.The fungal immunomodulatory protein and its gene order to body with immunoregulation effect according to the present invention, can prepare the health food improved the immunity of the human body, prevent the harm of the disease caused by immunity degradation.
Description
Technical field
The invention belongs to fungal immunomodulatory protein field, more particularly to a kind of straw mushroom immune modulator FIP-vvo78
And preparation method thereof.
Background technology
There is long history in Asian countries using macro fungi (mushroom) and its active material treatment human diseases, it can
To trace back to B.C. 3000, since it is in immunological regulation and the outstanding performance of anticancer aspect, nearly ten years in western countries
Also gradually it is taken seriously.Past focuses mostly on for macro fungi active matter Quality Research in polysaccharide and triterpene, for protein
Relatively fewer, but the application with biotechnology in drug research and development is studied, particularly to gene, albumen in Chinese herbal medicine
The gradual of relation research gos deep between pharmacological action, and mycoprotein is progressively paid attention to, wherein fungal immunomodulatory protein
(Fungal immunomodulatory protein, FIP), since it is with extensive immunobiologic activity, and receives the country
Outer concern.
Fungal immunomodulatory protein (Fungal immunomodulatory protein, FIP) is from Higher basidiomycetes
Isolated one kind has the small protein of immunoregulatory activity, structure and function and phytolectin and immune ball
Ferritin heavy chain variable region is similar, have promote lymphopoiesis, cell cycle regulation, induce Apoptosis, influence cell because
The immunoregulation effects such as expression, activating cell adhesion molecule and antianaphylaxis of son, possess good potential applicability in clinical practice and
Medicinal health value.From 1989, Japanese Kino was separated to first from ganoderma lucidum (Ganoderma lucidum) fructification
Fungal immunomodulatory protein is named as Ling Zhi-8 (LZ-8), and lives to its gene order, amino acid sequence and immune physiology
Property is determined.Up to the present, share 8 kinds of FIPs to be isolated and purified out, they come from ganoderma lucidum (Ganoderma
Lucidium), Ganoderma tsugae (G.tsugae), purple sesame (G.japoncium), microspore ganoderma lucidum (G.microsporum), sweet tea spirit
Sesame (G.sinense) Ganoderma fornicatum (Fr.) Pat (G.fornicatum), needle mushroom (Flammulina velutipes) and straw mushroom
(Volvariella volvacea) is named as LZ-8 (FIP-glu), FIP-gts, FIP-gja (AY987785) respectively,
FIP-gmi, FIP-sin, FIP-gfo, FIP-fve and FIP-vvo have collectively constituted a new protein families-FIPs.
In FIPs families in addition to gold needle mushroom immunomodulatory protein (FIP-fve) and straw mushroom immune modulator (FIP-vvo), other
FIPs be all isolated in ganoderma lucidum never of the same race, although this explanation is all difference of the ganoderma lucidum due to species, and have
Panimmunity regulatory protein is there is in different immune modulators or even ganoderma lucidum of the same race.Such as leaf popin etc. is from ganoderma lucidum
(G.microsporum) 3 protein are separated in：LZP-1, LZP-2 and LZP-3.Experiment in vitro shows 3 kinds of ganoderma lucidum albumen tools
There is stronger promotion lymphocyte proliferation activity, and SDS-PAGE shows that its molecular weight is different from LZ-8, so they obtain
3 kinds of new Ganoderma lucidum immunoregulation proteins.Such situation is equally happened in straw mushroom.Hsu, H.C. et al. existed except 1997
By isolating and purifying and verifying in the sub- fructification of straw mushroom, and it is sequenced to obtain what is be made of 112 amino acid by direct amino acid
Beyond a kind of straw mushroom immune modulator (FIP-vvo) that molecular weight is about 12.67KDa, it is also possible to which there are other kinds of straw mushrooms
Immune modulator.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of straw mushroom immune modulator FIP-vvo78 and its preparation side
Method screens one kind by comparing bioinformatics methods such as genomes and equally derives from straw mushroom, but gene order and amino acid
Sequence is all different from the immune modulator FIP-vvo78 with immunoregulation effect of FIP-vvo, and it is suitable in health products
It is used in medicine.
The present invention provides a kind of straw mushroom immune modulator FIP-vvo78, the amino acid sequence such as SEQ of the albumen
Shown in ID NO.1.Straw mushroom immune modulator FIP-vvo78 113 amino acid of overall length, theoretical molecular weight 12.74kDa.
The present invention also provides and synthesized the gene of the above-mentioned straw mushroom immune modulator FIP-vvo78 of coding.The gene
CDNA sequence is as shown in SEQ ID NO.2.
The present invention by pair of primers FIP-vvo78-F (5 '-CAT ATG TCT ACC GAC TTG A-3 ' of NdeI) and
FIP-vvo78-R(EcoRI 5’-CTT AAG TTA TTC CAC TGG GCA-3’).This has been obtained with the method for gene cloning
The gene order of one straw mushroom immune modulator FIP-vvo78, DNA complete sequence analysis the result shows that, FIP-vvo78 full length genes
338bp。
The albumen belongs to a kind of straw mushroom immune modulator.Its cDNA sequence is subjected to BLAST comparisons in GenBank,
Do not find the gene order similar to its, and by its amino acid sequence in the irredundant protein sequence databank (nr) of NCBI
It carries out BLAST and compares discovery, the albumen and the similitude from microspore ganoderma lucidum (G.microsporum) immune modulator
Reach 64%, the similitude with coming from ganoderma applanatum (G.applanatum) immune modulator is 62%, red with coming from
The similitude of sesame (G.lucidum) immune modulator LZ-8 is 63%, with needle mushroom (F.velutipes) immune modulator
FIP-fve is 54%.It is a kind of new fungal immunomodulatory protein to illustrate FIP-vvo78, and the gene of coding is one new
Gene.And the genetic modification of this albumen and the excellent base of high efficient expression offer in various heterologous gene expression systems are encoded therefore
Because of resource.
It is preferential to select the present invention also provides the recombinant vector for including above-mentioned straw mushroom immune modulator FIP-vvo78 genes
For pET-28b (+).The straw mushroom immune modulator FIP-vvo78 genes of the present invention are inserted into expression vector suitably to limit
Between property restriction enzyme site, make its nucleotide sequence is operable to be connected with expression regulation sequence.One as the present invention is most
Preferred scheme, preferably by straw mushroom immune modulator FIP-vvo78 genes be inserted into EcoRI on pET-28b (+) and
Between NdeI restriction enzyme sites, restructuring large intestine expression plasmid pET-FIP-vvo78 is obtained.
The present invention also provides the recombinant bacterial strain for containing above-mentioned straw mushroom immune modulator FIP-vvo78 genes, preferably
For recombinant bacterial strain BL21-FIP-vvo78.
The present invention also provides a kind of methods for preparing straw mushroom immune modulator FIP-vvo78, comprise the following steps：
1. above-mentioned recombinant vector converts host cell, recombinant bacterial strain is obtained；
2. recombinant bacterial strain is cultivated, the expression of induction restructuring straw mushroom immune modulator FIP-vvo78；
3. ultrasonication obtains expressed straw mushroom immune modulator FIP-vvo78.
Wherein, preferably described host cell is e. coli bl21 (DE3) cell, is preferably converted recombinant expression plasmid big
Coli cell BL21 (DE3), obtains recombinant bacterial strain BL21-FIP-vvo78.Carry out industrialization production grass with genetic engineering means
Mushroom immune modulator FIP-vvo78 products have not been reported.The present invention provides a fungal immunomodulatory protein for the first time
FIP-vvo78 can make industrial applied to health products and medicine etc..Technique according to the invention scheme, which can be realized, utilizes gene
Engineering means production straw mushroom immune modulator FIP-vvo78.
The method that the present invention utilizes bioinformatics is found that a kind of and known straw mushroom immunological regulation in straw mushroom genome
Albumen (FIP-vvo) otherness reaches the new straw mushroom immune modulator FIP-vvo78 and its volume that 7% i.e. similitude is 93%
Code gene order.And molecular biology and biotechnology are utilized, structure this straw mushroom immune modulator of high efficient expression
Engineered strain obtains restructuring straw mushroom immune modulator FIP-vvo78 using ultrasonication, immunologic function mirror has been carried out to it
It is fixed.The present invention not only enriches the species of FIPs, but also exploitation and application to carry out brand-new straw mushroom immune modulator in a deep going way
It lays the foundation.
Advantageous effect
The fungal immunomodulatory protein and its gene order to body with immunoregulation effect according to the present invention, can be with
The health food improved the immunity of the human body is prepared, prevents the harm of the disease caused by immunity degradation.
Description of the drawings
The straw mushroom immune modulator FIP-vvo78 that Fig. 1 is the present invention is analyzed in the SDS-PAGE of expression in escherichia coli；
Wherein, 1 is expression vector albumen；2 be molecular weight standard；3 be restructuring strain protein BL21-FIP-vvo78.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It is to 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
Scope.
1. cell line：Jurkat cell.
2. kit, enzyme, biochemical reagents and instrument：
Kit：ELISA KIT 96T Human IL-2 kits are purchased from four Zheng Bai bio tech ltd of Beijing.
96 well culture plates, blood counting chamber.
PCR primer synthesizes and gene sequencing is completed by Shanghai Sheng Gong biotech firms.
Enzyme：Restriction endonuclease is purchased from TaKaRa companies, and ligase is purchased from Invitrogen companies.
Instrument：Centrifuge；Vibrate shaking table；Microscope；Microplate reader.
Biochemical reagents：Ampicillin, kanamycins, penicillin, streptomysin, hyclone (FBS).
3. culture medium：RPMI-1640 culture solutions (contain 10% FBS, penicillin 100U/mL, streptomysin 100U/mL,
0.056%NaHCO3, pH value is adjusted to 7.4), is preserved for 4 DEG C after the sterilizing of 0.22 μ n membrane filtrations.
Explanation：Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiment《Molecular Cloning: A Laboratory
Guide》Listed specific method carries out or according to kit and product description in one book of (third edition) J. Pehanorm Brookers
It carries out.
Embodiment 1
The screening of straw mushroom immune modulator FIP-vvo78 encoding genes fip-vvo78 and clone
All gene orders of straw mushroom in Pfam protein structure regional data bases are searched for, 9084 is obtained and is divided into 2970 kinds
Then structural domain data message does not contain immune modulator with Coprinus cinereus, agaricus bisporus, schizophyllum commune and oyster cap fungus etc.
The structure numeric field data of edible fungus genome finds straw mushroom immune modulator FIP-vvo and an agnoprotein tool compared to
There are identical structural domain, and their GO annotations (GO:0030246 and GO:0002682) identical, GO annotation informations are shown
Showing the albumen of this 2 gene expressions all has immunoloregulation function.The phase of this albumen and straw mushroom immune modulator FIP-vvo
Reach 93% like property.Therefore, from straw mushroom genome, bioinformatics method is passed through, it was found that one kind has immunological regulation
The straw mushroom immune adjustment protein gene fip-vvo78 of effect.Fip-vvo78 gene orders according to obtaining devise pair of primers
FIP-vvo78-F(NdeI 5’-CAT ATGTCT ACC GAC TTG A-3 ') and FIP-vvo78-R (EcoRI 5 '-CTT AAGTTA TTC CAC TGG GCA-3 '), it has been cloned with the method for gene cloning from bacterial strain Volvariella
(its ACCC preserving number is volvacea V23：50897) straw mushroom immune modulator FIP-vvo78 genes, DNA complete sequences point
Analysis the result shows that, FIP-vvo78 overall length 338bp, cDNA sequence is as shown in SEQ ID NO.2.
Embodiment 2
Recombinate the preparation of straw mushroom immune modulator FIP-vvo78
Expression vector pET-28b (+) is subjected to double digestion (EcoRI and NdeI), while the coding straw mushroom of synthesis is immunized
The genetic fragment double digestion (EcoRI and NdeI) of regulatory protein FIP-vvo78, cuts out encoding mature straw mushroom immune modulator
The genetic fragment of FIP-vvo78 is connected with expression vector pET-28b (+), is obtained and is contained straw mushroom immune modulator FIP-vvo78
The recombinant plasmid pET-FIP-vvo78 of gene simultaneously converts e. coli bl21 (DE3), obtains recombinant bacterial strain BL21-FIP-
vvo78。
1mL is drawn respectively contains recombinant bacterial strain BL21-FIP-vvo78 bacterial strains and containing expression vector pET-28b's (+)
The fresh bacterium solution that 37 DEG C of BL21 bacterial strains have shaken overnight is inoculated in 100mL and contains in (50 μ g/mL) kanamycins LB culture solutions, and 37
DEG C, 250rpm/min, 2h, OD ≈ 0.3 add in the IPTG of 200 μ L50mg/mL, after 25 DEG C, 250rpm/min, Fiber differentiation 4h,
Thalline were collected by centrifugation.Ultrasonication (work 3s, interval 5s, operating voltage 300W, whole 20-30min) is subsequently used for, and is centrifuged
Collect supernatant.The expression quantity of result of study display restructuring straw mushroom immune modulator FIP-vvo78 is 196mg/L.SDS-PAGE
The result shows that restructuring straw mushroom immune modulator FIP-vvo78 is expressed in Escherichia coli.Expressed straw mushroom is immunized
It is pure (Fig. 1) that regulatory protein FIP-vvo78 contents reach electrophoresis.
Embodiment 3
The rush cytokine interleukin element IL-2 secretion activities for recombinating straw mushroom immune modulator FIP-vvo78 measure
Detecting recombinant immune regulatory protein by ELISA method promotes human leukemia cell's Jurkat cell to secrete interleukin
(IL-2) effect, to measure its immunoregulatory activity.
Jurkat cell suspension is poured into 15mL centrifuge tubes, 1000rpm/min, centrifuge 3min, remove supernatant.It adds in
The RPMI-1640 culture solutions of 2mL (contain 10% FBS, penicillin 100U/mL, streptomysin 100U/mL, 0.056%NaHCO3, adjust
PH value is saved to 7.4) suspension cell again.Cell suspending liquid is transferred in blake bottle, is placed in 37 DEG C, 5%CO2In incubator often
Rule are cultivated to exponential phase.
The Jurkat cell suspension of exponential phase is diluted to 1 × 107Cell/mL is inoculated in 96 by every 200 μ L of hole
Well culture plate respectively plus recombinant immune regulatory protein and expression vector albumen PBS solution, makes the final concentration of 10 μ g/ of total protein
mL.It separately sets negative control group (refinement born of the same parents and culture solution, PBS substitutions sample) and positive controls (only adds PMA, PHA, cell
And culture solution, it is not added with sample).Each sample does 3 parallel holes.48h is cultivated in 37 DEG C of incubators.
Cell culture fluid is suctioned out in 1.5mL centrifuge tubes, 1000rpm/min centrifuges 3min, collects supernatant.According to ELISA
The specification of KIT96T Human IL-2 kits carries out the detection of interleukin (IL-2).
It adds in 100 μ L samples, negative control group (refinement born of the same parents and culture solution, PBS substitutions sample) and positive controls is (only
Add PMA, PHA, cell and culture solution, be not added with sample) supernatant, while standard items human IL-2 is taken, it is diluted to respectively final concentration of
31.52nd, 125,250,500pg/mL as positive control and makes standard curve, does negative control with PBS, each sample is added to do
2 parallel holes.
It is put in 37 DEG C of incubators and cultivates 90min, carefully suction out supernatant, washed 4 times per 350 μ L cleaning solutions of hole, is inverted
On filter paper, empty dry liquids, pat dry for the last time as far as possible.
100 μ L biotinylated antibody working solutions are added in per hole, is put in 37 DEG C of incubators and cultivates 60min.Cleaning solution board-washing
Ibid.
100 μ L enzyme conjugates working solutions are added in per hole, is put in 37 DEG C of incubators and cultivates 30min.Cleaning solution board-washing is same as above.
100 μ L developing solutions are added in per hole, are put in dark reaction 10-20min in 37 DEG C of incubators.
100 μ L terminate liquids are added in per hole, are immediately placed in microplate reader, light absorption value is measured in 450nm and record reading, in 5min
Complete this step operation.It makes standard curve and calculates IL-2 contents in sample.
Result of study shows that 1 μ g restructuring straw mushroom immune modulators FIP-vvo78 promotes human leukemia cell Jurkat thin
Intracrine interleukin-22 (IL-2) content is in 62.945pg.
Claims (8)
1. a kind of straw mushroom immune modulator FIP-vvo78, it is characterised in that：The amino acid sequence of the albumen such as SEQ ID
Shown in NO.1.
2. a kind of recombinant vector containing straw mushroom immune modulator FIP-vvo78 as described in claim 1.
3. a kind of recombinant vector of straw mushroom immune modulator FIP-vvo78 according to claim 2, it is characterised in that：
The recombinant vector is pET-28b (+).
4. a kind of recombinant vector of straw mushroom immune modulator FIP-vvo78 according to claim 3, it is characterised in that：
EcoRI and NdeI restriction enzyme sites straw mushroom immune modulator FIP-vvo78 genes being inserted on pET-28b (+)
Between, obtain recombinant vector pET-FIP-vvo78.
5. a kind of recombinant bacterial strain containing straw mushroom immune modulator FIP-vvo78 as described in claim 1.
6. a kind of recombinant bacterial strain of straw mushroom immune modulator FIP-vvo78 according to claim 5, it is characterised in that：
The recombinant bacterial strain is BL21-FIP-vvo78.
7. a kind of preparation method of straw mushroom immune modulator FIP-vvo78, including：
(1) using the recombinant vector conversion host cell of Claims 2 or 3, recombinant bacterial strain is obtained；
(2) recombinant bacterial strain, the expression of induction restructuring straw mushroom immune modulator FIP-vvo78 are cultivated；
(3) ultrasonication obtains expressed straw mushroom immune modulator FIP-vvo78.
8. a kind of preparation method of straw mushroom immune modulator FIP-vvo78 according to claim 7, it is characterised in that：
Host cell in the step (1) is e. coli bl21 (DE3) cell.
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| CN106243204A (en) * | 2016-10-12 | 2016-12-21 | 上海市农业科学院 | A kind of poison fly goose cream immune modulator Fip amu1 and its preparation method and application |
| CN106432441B (en) * | 2016-10-12 | 2019-07-05 | 上海市农业科学院 | A kind of Lentinus tigrinus immune modulator Fip-lti2 and its preparation method and application |
| CN106432442B (en) * | 2016-10-12 | 2019-07-05 | 上海市农业科学院 | A kind of Lentinus tigrinus immune modulator Fip-lti1 and its preparation method and application |
| CN107118263B (en) * | 2017-04-08 | 2020-07-03 | 张喜田 | Recombinant ganoderma lucidum immunomodulatory protein mutant and application thereof |
| TWI702291B (en) * | 2018-12-28 | 2020-08-21 | 薩摩亞商康多富國際有限公司 | Method for determining a set of personalized cancer healthy foods and non-transitory computer readable storage medium |
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| CN102925403A (en) * | 2012-11-21 | 2013-02-13 | 黑龙江省科学院微生物研究所 | Recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), construction method, protein expression and purification method, and protein application |
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