CN104817635A - Straw mushroom immunomodulatory protein FIP-vvo80 and preparation method thereof - Google Patents
Straw mushroom immunomodulatory protein FIP-vvo80 and preparation method thereof Download PDFInfo
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Abstract
The invention relates to straw mushroom immunomodulatory protein FIP-vvo80 and a preparation method thereof. An amino acid sequence of the protein is shown in SEQ ID NO.1. The preparation method comprises the following steps of (1) transforming host cells by using a recombinant vector to obtain a recombination strain; (2) cultivating the recombination strain and inducing expression of a recombined straw mushroom immunomodulatory protein FIP-vvo80; and (3) performing ultrasonication to obtain the expressed straw mushroom immunomodulatory protein FIP-vvo80. Health food capable of improving immunity of human bodies can be prepared according to the fungus immunomodulatory protein which has immunomodulatory effects on the human bodies and the gene sequence of the fungus immunomodulatory protein, and diseases caused by weakened immunity are prevented and cured.
Description
Technical field
The invention belongs to fungal immunomodulatory protein field, particularly a kind of straw mushroom immune modulator FIP-vvo80 and preparation method thereof.
Background technology
Macro fungi (mushroom) and active substance treatment human diseases thereof is utilized to have long history in Asian countries, B.C. 3000 can be traced back to, because it is in the outstanding performance of immunomodulatory and anticancer aspect, also come into one's own gradually in western countries nearly ten years.Past focuses mostly at polysaccharide and triterpene for macro fungi actives Quality Research, relatively less for albumen Quality Research, but along with the application of biotechnology in drug research and development, particularly to gene in herbal medicine, between albumen and pharmacological action relation research gradually deeply, mycoprotein progressively obtains attention, wherein fungal immunomodulatory protein (Fungalimmunomodulatory protein, FIP), because it has immunobiologic activity widely, and enjoy domestic and international concern.
Fungal immunomodulatory protein (Fungal immunomodulatory protein, FIP) be from Higher basidiomycetes, be separated the small protein that the class obtained has immunoregulatory activity, its structure and fuction is similar with immunoglobulin heavy chain variable region to phytohemagglutinin, have promote lymphopoiesis, cell cycle regulation, bring out apoptosis, affect the expression of cytokine, the immunoregulation effect such as active cells adhesion molecule and antianaphylaxis, have good potential applicability in clinical practice and medicinal health value.From 1989, Japanese Kino is separated to first fungal immunomodulatory protein from glossy ganoderma (Ganoderma lucidum) sporophore, called after Ling Zhi-8 (LZ-8), and to its gene order, amino-acid sequence and immune physiologically active measure.Up to the present, have that 8 kinds of FIPs are separated to be purified, they come from glossy ganoderma (Ganoderma lucidium), Ganoderma tsugae (G.tsugae), purple sesame (G.japoncium), sporule glossy ganoderma (G.microsporum), sweet glossy ganoderma (G.sinense) Ganoderma fornicatum (Fr.) Pat (G.fornicatum), needle mushroom (Flammulina velutipes) and straw mushroom (Volvariella volvacea), be named as LZ-8 (FIP-glu) respectively, FIP-gts, FIP-gja (AY987805), FIP-gmi, FIP-sin, FIP-gfo, FIP-fve and FIP-vvo, jointly constitute a new protein man Zu – FIPs.In FIPs family except gold needle mushroom immunomodulatory protein (FIP-fve) and straw mushroom immune modulator (FIP-vvo), be separated in other FIPs glossy ganoderma all never of the same race and obtain, although this illustrates and is all glossy ganoderma but due to the difference of kind, and there is different immune modulators, just there is panimmunity Function protein in glossy ganoderma even of the same race.Such as leaf popin etc. are separated to 3 protein from glossy ganoderma (G.microsporum): LZP-1, LZP-2 and LZP-3.Experiment in vitro shows that 3 kinds of glossy ganoderma albumen have 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 occurs in straw mushroom equally.Except Hsu in 1997, H.C. people is waited in the sub-sporophore of straw mushroom by separation and purification and checking, and be about beyond a kind of straw mushroom immune modulator (FIP-vvo) of 12.67KDa through the direct amino acid molecular weight obtaining being made up of 112 amino acid that checks order, also likely there is other kind of straw mushroom immune modulator.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of straw mushroom immune modulator FIP-vvo80 and preparation method thereof, screen one by bioinformatics methods such as Comparative genomic strategy and derive from straw mushroom equally, but gene order and aminoacid sequence are all different from the immune modulator FIP-vvo80 with immunoregulation effect of FIP-vvo, it is applicable to use in healthcare products and medicine.
The invention provides a kind of straw mushroom immune modulator FIP-vvo80, the aminoacid sequence of described albumen is as shown in SEQ ID NO.1.This straw mushroom immune modulator FIP-vvo80 total length 113 amino acid, theoretical molecular is 12.79kDa.
The present invention also provides and has synthesized the gene of coding above-mentioned straw mushroom immune modulator FIP-vvo80.This gene cDNA sequence is as shown in SEQ ID NO.2.
The present invention is by pair of primers FIP-vvo80-F (NdeI5 '-CAT ATG TCT ACC GAC TTG A-3 ') and FIP-vvo80-R (EcoRI5 '-CTT AAG TTA TTC CAC TGG GCA-3 ').Obtain the gene order of this straw mushroom immune modulator FIP-vvo80 by the method for gene clone, DNA complete sequence analysis result shows, FIP-vvo80 full length gene 339bp.
This albumen belongs to a kind of straw mushroom immune modulator.Its cDNA sequence is carried out BLAST comparison in GenBank, do not have to find the gene order similar to it, and its aminoacid sequence is carried out in the irredundant protein sequence databank (nr) of NCBI BLAST comparison discovery, this albumen reaches 63% with the similarity deriving from Ganoderma applanatum (pers) pat (G.applanatum) and sporule glossy ganoderma (G.microsporum) immune modulator, be 62% with the similarity coming from red sesame (G.lucidum) immune modulator LZ-8, be 57% with needle mushroom (F.velutipes) immune modulator FIP-fve.Illustrate that FIP-vvo80 is a kind of new fungal immunomodulatory protein, its gene of encoding is a new gene.And the genetic modification of this albumen of for this reason encoding and in various heterologous gene expression system high expression excellent genetic resources is provided.
Present invention also offers the recombinant vectors comprising above-mentioned straw mushroom immune modulator FIP-vvo80 gene, preferentially elect pET-28b (+) as.Straw mushroom immune modulator FIP-vvo80 gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As a most preferred scheme of the present invention, preferably straw mushroom immune modulator FIP-vvo80 gene is inserted between EcoRI and the NdeI restriction enzyme site on pET-28b (+), obtains restructuring large intestine expression plasmid pET-FIP-vvo80.
Present invention also offers the recombinant bacterial strain containing above-mentioned straw mushroom immune modulator FIP-vvo80 gene, be preferably recombinant bacterial strain BL21-FIP-vvo80.
Present invention also offers a kind of method preparing straw mushroom immune modulator FIP-vvo80, comprise the following steps:
1. above-mentioned recombinant vectors transformed host cell, obtains recombinant bacterial strain;
2. cultivate recombinant bacterial strain, the expression of induction restructuring straw mushroom immune modulator FIP-vvo80;
3. the straw mushroom immune modulator FIP-vvo80 expressed by ultrasonication acquisition.
Wherein, preferred described host cell is e. coli bl21 (DE3) cell, preferably by recombinant expression plasmid transform E. coli cells BL21 (DE3), obtains recombinant bacterial strain BL21-FIP-vvo80.Use genetic engineering means to carry out industrialization production straw mushroom immune modulator FIP-vvo80 product have not been reported.The present invention provides a fungal immunomodulatory protein FIP-vvo80 first, can be applied to the industry such as healthcare products and medicine.Just can realize utilizing genetic engineering means to produce straw mushroom immune modulator FIP-vvo80 according to technical scheme of the present invention.
The present invention utilizes the method for information biology, found in straw mushroom genome a kind of reach with known straw mushroom immune modulator (FIP-vvo) otherness 5% that is similarity be new straw mushroom immune modulator FIP-vvo80 and the coding gene sequence thereof of 95%.And utilize molecular biology and biotechnology, build the engineering strain of this straw mushroom immune modulator of high expression, utilize ultrasonication to obtain restructuring straw mushroom immune modulator FIP-vvo80, immunologic function qualification has been carried out to it.The present invention has not only enriched the kind of FIPs, and lays the foundation for the exploitation and application carrying out brand-new straw mushroom immune modulator in a deep going way.
beneficial effect
According to fungal immunomodulatory protein and the gene order thereof body to immunoregulation effect of the present invention, can prepare and improve the protective foods of body immunity, prevent and treat the harm of the disease caused due to immunity degradation.
Accompanying drawing explanation
Fig. 1 is that straw mushroom immune modulator FIP-vvo80 of the present invention analyzes at the SDS-PAGE of expression in escherichia coli; Wherein, 1 is expression vector albumen; 2 is molecular weight standard; 3 is restructuring strain protein BL21-FIP-vvo80.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
1. cell strain: Jurkat cell.
2. test kit, enzyme, biochemical reagents and instrument:
Test kit: ELISA KIT 96T Human IL-2 test kit is purchased from Zheng Bai bio tech ltd, Beijing four.96 well culture plates, blood counting chamber.
PCR primer synthesis and gene sequencing complete by Shanghai Sheng Gong biotech firm.
Enzyme: restriction endonuclease is purchased from TaKaRa company, and ligase enzyme is purchased from Invitrogen company.
Instrument: whizzer; Vibration shaking table; Microscope; Microplate reader.
Biochemical reagents: penbritin, kantlex, penicillin, Streptomycin sulphate, foetal calf serum (FBS).
3. substratum: RPMI-1640 nutrient solution (containing the FBS of 10%, penicillin 100U/mL, Streptomycin sulphate 100U/mL, 0.056%NaHCO
3, adjust ph to 7.4), 4 DEG C of preservations after 0.22 μ n membrane filtration sterilizing.
Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, concrete grammar listed in equal reference " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book carries out, or carries out according to test kit and product description.
Embodiment 1
The screening of straw mushroom immune modulator FIP-vvo80 encoding gene fip-vvo80 and clone
All for straw mushroom gene orders are searched in Pfam protein structure regional data base, obtain the structural domain data message that 9084 are divided into 2970 kinds, then with Coprinus cinereus, Twospore Mushroom, the genomic structural domain data of edible fungus not containing immune modulator such as Split-gill and oyster cap fungus compare, find that a straw mushroom immune modulator FIP-vvo and agnoprotein has identical structural domain, and their GO annotation (GO:0030246 with GO:0002682) is identical, the albumen that GO annotation information shows these 2 genetic expressions all has immunoloregulation function.The similarity of this albumen and straw mushroom immune modulator FIP-vvo reaches 95%.Therefore, from straw mushroom genome, by bioinformatics method, found a kind of straw mushroom immune adjustment protein gene fip-vvo80 with immunoregulation effect.Pair of primers FIP-vvo80-F (NdeI 5 '-CAT ATG TCT ACC GAC TTG A-3 ') and FIP-vvo80-R (EcoRI 5 '-CTT AAGTTA TTC CAC TGG GCA-3 ') is devised according to the fip-vvo80 gene order obtained, clone by the method for gene clone and derived from bacterial strain Volvariella volvaceaV23 (its ACCC preserving number is: straw mushroom immune modulator FIP-vvo80 gene 50897), DNA complete sequence analysis result shows, FIP-vvo80 total length 339bp, its cDNA sequence is as shown in SEQ ID NO.2.
Embodiment 2
The preparation of restructuring straw mushroom immune modulator FIP-vvo80
Expression vector pET-28b (+) is carried out double digestion (EcoRI and NdeI), simultaneously by the gene fragment double digestion (EcoRI and NdeI) of the coding straw mushroom immune modulator FIP-vvo80 of synthesis, the gene fragment cutting out encoding mature straw mushroom immune modulator FIP-vvo80 is connected with expression vector pET-28b (+), obtain the recombinant plasmid pET-FIP-vvo80 containing straw mushroom immune modulator FIP-vvo80 gene and transformation of E. coli BL21 (DE3), obtain recombinant bacterial strain BL21-FIP-vvo80.
Draw 1mL respectively to contain the spend the night fresh bacterium liquid that shakes of recombinant bacterial strain BL21-FIP-vvo80 bacterial strain and 37 DEG C of BL21 bacterial strain containing expression vector pET-28b (+) and be inoculated in 100mL and contain in (50 μ g/mL) kantlex LB nutrient solution, 37 DEG C, 250rpm/min, 2h, OD ≈ 0.3, adding 200 μ L concentration is the IPTG of 50mg/mL, 25 DEG C, 250rpm/min, after inducing culture 4h, collected by centrifugation thalline.Then for ultrasonication (work 3s, interval 5s, operating voltage 300W, omnidistance 20-30min), and collected by centrifugation supernatant.The expression amount of result of study display restructuring straw mushroom immune modulator FIP-vvo80 is 436mg/L.SDS-PAGE result shows, restructuring straw mushroom immune modulator FIP-vvo80 obtains expression in intestinal bacteria.Expressed straw mushroom immune modulator FIP-vvo80 content reaches electrophoresis pure (Fig. 1).
Embodiment 3
The short cytokine interleukin element IL-2 secretion activity of restructuring straw mushroom immune modulator FIP-vvo80 measures
The effect that recombinant immune Function protein promotes human leukemia cell's Jurkat cell secretion interleukin (IL-2) is detected, to measure its immunoregulatory activity by ELISA method.
Jurkat cell suspension is poured in 15mL centrifuge tube, 1000rpm/min, centrifugal 3min, remove supernatant.Add 2mL RPMI-1640 nutrient solution (containing the FBS of 10%, penicillin 100U/mL, Streptomycin sulphate 100U/mL, 0.056%NaHCO
3, adjust ph to 7.4) and Eddy diffusion cell.Cell suspending liquid is transferred in culturing bottle, is placed in 37 DEG C, 5%CO
2in incubator, cellar culture is to logarithmic phase.
The Jurkat cell suspension of logarithmic phase is diluted to 1 × 10
7cell/mL, is inoculated in 96 well culture plates by every hole 200 μ L, adds recombinant immune Function protein and expression vector albumen PBS solution respectively, makes total protein final concentration be 10 μ g/mL.Separately establish negative control group (only add cell and nutrient solution, PBS replaces sample) and positive controls (only add PMA, PHA, cell and nutrient solution, do not add sample).Each sample does 3 parallel holes.48h is cultivated in 37 DEG C of incubators.
Sucking-off cell culture fluid is in 1.5mL centrifuge tube, and 1000rpm/min, centrifugal 3min, collect supernatant.The detection of interleukin-22 is carried out according to the specification sheets of ELISA KIT96T Human IL-2 test kit.
Add 100 μ L samples, negative control group (only adds cell and nutrient solution, PBS replaces sample) and positive controls (only add PMA, PHA, cell and nutrient solution, do not add sample) supernatant, get standard substance human IL-2 simultaneously, be diluted to respectively final concentration be 31.52,125,250,500pg/mL is as positive control and production standard curve, do negative control with PBS, add each sample and do 2 parallel holes.
Be put in 37 DEG C of incubators and cultivate 90min, careful sucking-off supernatant liquor, every hole 350 μ L washings washs 4 times, and be inverted on filter paper, empty dry liquids, pats dry for the last time as far as possible.
Every hole adds 100 μ L biotinylated antibody working fluids, is put in 37 DEG C of incubators and cultivates 60min.It is the same that plate washed by washings.
Every hole adds 100 μ L enzyme conjugates working fluids, is put in 37 DEG C of incubators and cultivates 30min.It is the same that plate washed by washings.
Every hole adds 100 μ L nitrite ions, is put in dark reaction 10-20min in 37 DEG C of incubators.
Every hole adds 100 μ L stop buffers, puts into microplate reader immediately, measures light absorption value and record reading in 450nm, completes this step operation in 5min.Production standard curve IL-2 content in calculation sample.
Result of study shows that 1 μ g recombinant immune Function protein FIP-vvo80 promotes that human leukemia cell's Jurkat cell secretion interleukin-22 (IL-2) content is at 68.404pg.
Claims (9)
1. a straw mushroom immune modulator FIP-vvo80, is characterized in that: the aminoacid sequence of described albumen is as shown in SEQ ID NO.1.
2. a gene of coding straw mushroom immune modulator FIP-vvo80 as claimed in claim 1, is characterized in that: the cDNA sequence of described gene is as shown in SEQ ID NO.2.
3. the recombinant vectors containing straw mushroom immune modulator FIP-vvo80 as claimed in claim 1.
4. the recombinant vectors of a kind of straw mushroom immune modulator FIP-vvo80 according to claim 3, is characterized in that: described recombinant vectors is pET-28b (+).
5. the recombinant vectors of a kind of straw mushroom immune modulator FIP-vvo80 according to claim 4, it is characterized in that: straw mushroom immune modulator FIP-vvo80 gene is inserted between EcoRI and the NdeI restriction enzyme site on pET-28b (+), obtains recombinant vectors pET-FIP-vvo80.
6. the recombinant bacterial strain containing straw mushroom immune modulator FIP-vvo80 as claimed in claim 1.
7. the recombinant bacterial strain of a kind of straw mushroom immune modulator FIP-vvo80 according to claim 6, is characterized in that: described recombinant bacterial strain is BL21-FIP-vvo80.
8. a preparation method of straw mushroom immune modulator FIP-vvo80, comprising:
(1) adopt the recombinant vectors transformed host cell of claim 3 or 4, obtain recombinant bacterial strain;
(2) recombinant bacterial strain is cultivated, the expression of induction restructuring straw mushroom immune modulator FIP-vvo80;
(3) the straw mushroom immune modulator FIP-vvo80 expressed by ultrasonication acquisition.
9. the preparation method of a kind of straw mushroom immune modulator FIP-vvo80 according to claim 8, is characterized in that: the host cell in described step (1) is e. coli bl21 (DE3) cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106432442A (en) * | 2016-10-12 | 2017-02-22 | 上海市农业科学院 | Lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as preparation method and application thereof |
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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 |
| CN103980354A (en) * | 2014-05-30 | 2014-08-13 | 中国农业科学院农产品加工研究所 | Immunomodulatory protein FIP-ppl of fungus and gene of protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN103980354A (en) * | 2014-05-30 | 2014-08-13 | 中国农业科学院农产品加工研究所 | Immunomodulatory protein FIP-ppl of fungus and gene of protein |
Non-Patent Citations (2)
| Title |
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| HAO-CHI HSU ET AL.: "Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea", 《BIOCHEM. J.》 * |
| JING-WEI LIN ET AL.: "Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from Ganoderma lucidum in Pichia pastoris", 《WORLD J MICROBIOL BIOTECHNOL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432442A (en) * | 2016-10-12 | 2017-02-22 | 上海市农业科学院 | Lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as preparation method and application thereof |
| 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 |
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