CN103739684B - The preparation method and applications of ganoderma atrum fungal immunomodulatory protein - Google Patents
The preparation method and applications of ganoderma atrum fungal immunomodulatory protein Download PDFInfo
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Abstract
The preparation method and applications of the ganoderma atrum fungal immunomodulatory protein of a kind of technical field of biological genetic engineering, this ganoderma atrum fungal immunomodulatory protein gene has nucleotide sequence shown in SEQ ID NO.1.The present invention is expressed in escherichia coli and obtains high expressed amount destination protein this recombinant fungus immune adjustment protein gene, and proves that it has good agglutination to breast cancer cell.This provides a kind of new direction for utilizing engineered method to produce recombinant fungus immune modulator in a large number with the application utilizing this albumen and developing into a kind of novel anti-tumor agent, can be widely used for health food and biomedicine field.
Description
Technical field
A kind of method that the present invention relates to molecular biology, gene engineering technology field, specifically a kind of Black Ganoderma fungus is exempted from
The preparation method and applications of epidemic disease regulation albumen.
Background technology
Fungal immunomodulatory protein (fugal immunomodulatory proteins, FIPs) isolated from Ganoderma
After polysaccharide and triterpenoid compound, another class active substance.In FIPs family, at present it has been reported that there are 7 kinds of albumen, point
Not Wei named LZ-8 (FIP-glu), FIP-gts, FIP-fve, FIP-vvo, FIP-gja (AY987805), FIP-gmi and FIP-gsi,
It is respectively derived from red ganoderma (Ganodermalucidum), Ganoderma tsugae (G.tsugae), Flammulina velutiper (Fr.) Sing
(Flammulinavelutipes), Volvariella volvacea (Bull.Ex Franch.) Singer． (Volvarielavolvacea), Ganoderma sinense Zhao Xu et Zhang (G.japoncium), little spore Ganoderma
And Ganoderma (G.sinensis) [Li QZ, Wang XF, Zhou XW.Recent status and prospects (G.microsporum)
of the fungal immunomodulatory protein family.Critical Reviews in Biotechnology.2011,31(4):
365-375].In this protein family, directly from natural mushroom fungus, the fungal immunomodulatory protein of isolated only has four kinds, and they divide
Not: LZ-8 (FIP-glu), FIP-gts, FIP-fve, FIP-vvo, remaining fungal immunomodulatory protein is then to pass through gene recombinaton
Technological expression, its aminoacid sequence obtains after being translated by gene order.Wang XF etc. find through comparison, FIPs's
Amino acid sequence homology is higher, typically up to about 60-70% [Wang XF, Su KQ, Bao TW, et,
al.Immunomodulation effects of fungal proteins.Current Topics in Nutraceutical Research.
2012,10(1):1-11.]。
FIPs has certain immunoloregulation function, divides at antiallergic, antitumor, the propagation of promotion lymphocyte, inducing cell
Secreting the aspect such as cytokine and resisting transplant rejection all to play an important role, wherein antitumor action especially causes everybody interest.Lin JW etc.
Through MTT(tetrazolium salts) test and flow cytomery, the FIP-glu expressed in Pichia sp. can be withered by inducing cell
Die suppression human leukemia NB4 expression (32 μ g/mL FIP-glu can induce about 35% human leukemia NB4 cell
Apoptosis), there is certain active anticancer [Lin JW, Hao LX, Xu GX, et al.Molecular cloning and recombinant
expression of a gene encoding a fungal immunomodulatory protein from Ganodermalucidum in
Pichiapastoris.World Journal of Microbiology and Biotechnology,2008,25(3):383-390]。Liao
CH etc. show anti-tumor activity at the FIP-gts of expression in escherichia coli in mankind's Lung Adenocarcinoma A 549 Cell.Work as FIP-gts
Concentration when reaching 4-10 μ g/mL, can effectively control the transfer of cancerous cell, there is good antitumous effect [Liao CH, Hsiao
YM,Lin CH.Induction of premature senescence in human lung cancer by fungal
immunomodulatory protein from Ganodermatsugae.Food and Chemical Toxicology,2008,46(5):
1851-1859].Liao etc. (2006) research is thought, though FIP-gts can significantly inhibit the growth of mankind's adenocarcinoma of lung A549 cancerous cell,
But not interfering with the growth of normal MRC-5 fibroblast, it lowers the expression of telomerase catalytic subunit in mRNA level in-site,
Suppress the activity of telomerase and there is dose-dependent effect.Telomerase is not typically activated in normal somatic cell, and its activity has
Without often becoming the key factor determining cell infinite multiplication.Meanwhile, FIP-gts can promote NK cell (natural killer cell),
Macrophage and the generation of activated serum antibody, regulate immunologic function effectively, to a certain extent can also the life of anticancer
Long [Liao CH, Hsiao YM, Hsu CP, et al.Transcriptionally mediated inhibition of telomerase of
fungal immunomodulatory protein from Ganodermatsugae in A549human lung
adenocarcinoma cell line.Molecular Carcinogenesis,2006,45(4):220-229]。
The research of FIPs pharmacological experiment proves, the albumen of FIPs family is when different pharmacological experiment, and different FIP are to different experiments sample
Product work and its exercising result has dose dependent.Using [3H] as labelling, process Mouse spleen cells with LZ-8 (FIP-glu).
When LZ-8 concentration is 3.13 μ g/mL, [3H] absorption value reaches peak-peak [Kino K, Yamashita A, Yamaoka K, et
al.Isolated and characterization of a new immunomodulatoryprotein Ling Zhi-8(LZ-8),from
Ganodermalucidum, Journal of Biological Chemistry, 1989,264 (1): 472-478].FIP-fve and FIP-vvo
The optium concentration of human peripheral lymphocyte proliferation activity is respectively 100 μ g/mL and 5 μ g/mL(Ko JL, Hsu CI, Lin RH,
et al.A new fungal immunomodulatoryprotein FIP-fveisolated from the edible mushroom,
Flammulianvelutlpesand its complete amino acid sequence,European Journal of Biochemistry,
1995,228(2):244-249.Hsu HC,HsuCI,Lin RH,et al.Fip-vvo,a new
fungalimmunomodulatoryprotein isolated from Volvarielavolvacea,Journal of Biological
Chemistry, 1997,323 (2): 557-565).Wang etc. study discovery, and FIP-fve shows and promotes human peripheral lymphocyte
By the G1/G0 phase to the mitogenetic effect of S phase transition.100 μ g FIP-fve and human peripheral blood mononuclear cell's co-cultivation 72h
After, found that the DNA content of lymPhocytes of 61% is consistent with the DNA content of G0/G1 phase cell, and the lymph of 26% and 12%
Cell DNA content, is respectively equivalent to DNA content (Wang PH, Hsu CI, Tang SC, the et al.Fungal of S and G2/M phase cell
immunomodulatoryprotein from Flammulinavelutipesinduces interferon-γproduction through
P38mitogen-activated protein kinase signaling pathway, Agricultural and Food Chemistry,
2004,52 (9): 2721-2725).But in the clinical practice with medical applications, the dosage using albumen all has certain wanting
Ask, therefore, this albuminoid is applied to the primary problem of field of medicaments is how to carry out scale to produce in a large number？Due to fungal immune
Regulation albumen content in natural mushroom (or mushroom fungus) the lowest (20-80mg/Kg) (Lin Jingwei, Sun Fei, Zhang Ren, etc. fungus
The progress of immune modulator. China Journal of Immunology .2005,21 (6): 477-480), in addition true during protein extraction
The interference of granulose and other composition so that the acquisition of albumen relatively costly.Hence set up stable protokaryon (or eucaryon) to express
System, produces fungal immunomodulatory protein in a large number by heterogenous expression and fermentation industry process imperative.
In the heterogenous expression of fungal immunomodulatory protein, being used that carry out studying is prokaryotic expression system the earliest, this is also mesh
The expression system that front grasp is the most ripe.Conventional expression vector has pET-28a, pET-28b and pET-30 etc., the place of expression
Chief cell includes e. coli tg1 cell, escherichia coli M15 cell, e. coli bl21 cell etc..Li Qizhang etc. will originate
Proceed in pET-30a expression vector equally at e. coli bl21 cell in the FIP-gsi of Ganoderma (Ganodermasineses)
In expressed, it is thus achieved that [Li Qizhang, Wang Xuefei, Huang Lei, etc. Ganoderma immunomodulating egg for the Ganoderma fungal immunomodulatory protein of restructuring
The prokaryotic expression of white gene and functional analysis. northwest Botany Gazette, 2010,30 (1): 35-40].Escherichia coli M15 cell is also
Being presently expressed by one of fungal immune adjustment protein gene main host, Li QZ etc. is the fungal immunomodulatory protein deriving from Red Ganoderma
Gene LZ-8 (FIP-glu) is cloned in pQE-30 expression vector, is transformed in escherichia coli M15 cell, obtains restructuring equally
Fungal immunomodulatory protein FIP-glu [Li QZ, Wang XF, Bao TW, et al.In vitro synthesis of a recombinant
Fungal immunomodulatory protein from Lingzhi or Reishi medicinal mushroom, Ganoderma
lucidum(W.Curt.:Fr.)P.Karst.(Aphyllophoromycetideae)and analysis of its
Immunomodulatory activity.International Journal of Medicinal Mushrooms, 2010,12 (4):
347-358] for further scientific research or research and development of products.Equally, Li QZ et al. is also deriving from Ganoderma
(Ganodermasinensis) fungal immune adjustment protein gene FIP-gsi, proceeds in pQE-30 expression vector, equally greatly
Enterobacteria M15 cell is expressed, also obtain Ganoderma fungal immunomodulatory protein FIP-gsi [Li QZ, the Wang XF, Chen of restructuring
YY, et al.Cytokines expression induced by Ganoderma sinensis fungal immunomodulatory
Proteins (FIP-gsi) in mouse spleen cells.Applied Biochemistry and Biotechnology, 2010,162 (5):
1403-1413].Simultaneously, can also be used as, at sometimes e. coli tg1 cell, the host cell that FIPs expresses,
Lin in 1997 et al. is by being connected to FIP-gts gene in Bacillus coli cells TG1, it is thus achieved that form with glutathione S-transferase
Fusion protein (GST), then it is up to 20mg/L [Lin W H, Hung C H, Hsu by FIP-gts yield after thrombin digestion
CI,et al.Dimerization of the N-terminal amphipathic α-helix domain of the fungal
immunomodulatory protein from Ganoderma tsugae(Fip-gts)defined by a yeast two-hybrid
System and site-directed mutagenesis.Journal of Biological Chemistry, 1997,272 (32):
20044-20048].To sum up stating institute, the expression in escherichia coli of the existing fungal immune adjustment protein gene, its expression is about
About 20～30mg/L [leaf popin, Wang Fan, Liang Yixin wait the expression in escherichia coli of the .LZ-8 gene. Pharmaceutical Biotechnology,
2002,9(1):21-23；Bai Jieying, Zeng Lin, Li Yanfang, etc. expression in escherichia coli of. fungal immunomodulatory protein and anti-
Body prepares practicality medical magazine, and 2006,23 (10): 1205-1207], the carrier of expression is different with host cell, expressing protein
Modify and be also not quite similar, thus also show different physiologically actives.
In experiment previously, we use homologous clone method, with Black Ganoderma (Ganodermaatrum) genomic DNA
For template, fungal immunomodulatory protein (FIPs) gene being carried out PCR amplification, analysis result shows: Black Ganoderma fungal immune regulates
Protein gene sequence comprises 336bp, 111 amino acid residues of codified, named FIP-gas.FIP-gas molecular weight is 12.4kDa,
Theoretical isoelectric point, IP is 4.80, meets the characteristic of FIPs, is FIPs family one newcomer.FIP-gas is carried out sequence analysis and finds its ammonia
Base acid sequence has the conserved sequence pattern of FIPs, and Ganoderma sinense Zhao Xu et Zhang (G.japoncium) (AAX98241), Ganoderma (G.sinensis),
Little spore Ganoderma (G.microsporum), red ganoderma (G.lucidum) (AAA33350), Ganoderma tsugae (G.tsugae), gold
The homology of the FIP sequence of pin mushroom (Flammulinavelutipes) (ADB24832) and Volvariella volvacea (Bull.Ex Franch.) Singer． (Volvarielavolvacea)
Being respectively 99%, 98%, 87%, 86%, 86%, 61%, 55%(Su Kai fine jade, Wang Xuefei, weekly selection is enclosed. Black Ganoderma immunity
The clone of regulation protein gene and bioinformatic analysis. Shanghai Communications University's journal (agricultural sciences version) .2012,30 (1): 65-71.).
Clone and the analysis of above-mentioned Black Ganoderma FIP gene provide genetic resources for further prokaryotic expression.
Summary of the invention
The present invention is directed to deficiencies of the prior art, propose a kind of ganoderma atrum fungal immunomodulatory protein preparation method and
Its application, it is possible to breast cancer cell is had good agglutination.This is true for utilizing engineered method to produce restructuring in a large number
Bacterial immunity regulation albumen provides a kind of new direction with the application utilizing this albumen to develop into a kind of novel anti-tumor agent, can be wide
General for health food and biomedicine field.
The present invention is achieved by the following technical solutions:
The present invention relates to the isolation and purification method of a kind of FIP-gas albumen, described FIP-gas gene, its nucleotide sequence is such as
Shown in Seq ID No.1, cloned from Black Ganoderma (G.atrum) mycelium by Homology-based cloning and obtain, by Black Ganoderma fungus
Immune modulator FIP-gas gene constructed one-tenth prokaryotic expression carrier pET-30a-FIP-gas is transformed into e. coli bl21 competence
In cell, it is induced to express through IPTG；Prokaryotic expression product obtains after purification through metallic nickel ions chelate column.
Described isolation and purification method, specifically includes following steps:
1) construction of expression vector；
2) convert to escherichia coli；
3) amplification culture；
4) IPTG abduction delivering；
5) isolated and purified；
Described ganoderma atrum fungal immunomodulatory protein is the expression product of ganoderma atrum fungal immunomodulatory protein gene FIP-gas,
Its sequence is as shown in SEQ ID NO.1；
Described e. coli host cell is e. coli strain bl21.
Preferably, described step 1) construction of expression vector method particularly includes:
1.1) according to the sequence of ganoderma atrum fungal immunomodulatory protein gene FIP-gas, design primer, and draw at upstream and downstream
Restriction endonuclease sites Bam H I and Hind III is added respectively, to build prokaryotic expression carrier on thing;
1.2) with FIP-gas gene as template, after PCR expands, FIP-gas gene is cloned into intermediate carrier;
1.3) with double digestion, FIP-gas segment is cut from intermediate carrier, reclaim respective segments, with 16 DEG C of companies of T4 ligase
Take over and obtain expression vector night.
Preferably, the described intermediate in described step 1.2 is pMD18-T simple vector or pET-30a.
Preferably, described step 2) method particularly includes: described expression vector is added competent escherichia coli cell ice
Upper placement 30min, 42 DEG C of circulator bath thermal shock 90s afterwards, rapid ice bath 2min.Add 800 μ L LB culture fluid 37 DEG C to shake slowly
Recovery 1h.Taking 200 μ L bacterium solution bacterium solution and be coated on the LB flat board of the kanamycin containing 50mg/mL, screening transformant is also chosen
Take positive colony.
Preferably, described step 3) method particularly includes: the positive colony of acquisition is inoculated in the card containing 50mg/mL
In the LB fluid medium of that mycin, 37 DEG C of shaking overnight incubation, inoculate with 2% inoculum concentration next day, 37 DEG C are continued fermentation.
Preferably, described step 4) method particularly includes: as fermentation liquid OD600When reaching 0.5～0.7, add IPTG
Induce, the final concentration of 1mM of IPTG.
Preferably, described step 5) method particularly includes:
5.1) the bacterium solution centrifugal collecting cell will induced through IPTG, is suspended in buffer；Mixture is placed in liquid nitrogen cold
Freeze, melt in frozen water afterwards；
5.2) the sample ultrasonic disruption in mixture of ice and water after melting；
5.3) the bacterium solution centrifuging and taking supernatant that supersound process is crossed, upper Ni-NTA post, after washing post with buffer 1, will with buffer 2
Destination protein is eluted out；
Described buffer 1 is 50mM NaH2PO4, 300mMNaCl, 20mM imidazole, pH are the water of 8.0
Solution；
Described buffer 2 is 50mM NaH2PO4, 300mMNaCl, 250mM imidazole, pH8.0's is water-soluble
Liquid.
The present invention relates to the application of the FIP-gas albumen that said method prepares, use it for preparing antitumor drug.
Described FIP-gas albumen, measures it to mankind mastopathy cell strain MDA-MB-231(Breast through cell agglutination test
Adenocarcinoma) agglutination, result shows the fungal immunomodulatory protein FIP-gas of recombinant gene expression, in vitro
Human breast cancer cell MDA-MB-231(Breast adenocarcinoma can be suppressed) breeding, illustrate that this albumen is to this tumor
Cell strain has inhibitory action.
Biomaterial involved in the present invention and reagent, in addition to indicating especially, all can obtain from commercially available channel.
Heretofore described ganoderma atrum fungal immunomodulatory protein FIP-gas gene order, by the open report of this laboratory,
See Shanghai Communications University's journal (agricultural sciences version), volume 30 the 1st phase the 65-71 page in 2012, its nucleotide sequence such as SEQ
Shown in ID NO.1.
E. coli bl21 involved in the present invention (Li Qizhang, Wang Xuefei, Huang Lei, weekly selection is enclosed. Ganoderma immunity is adjusted
The prokaryotic expression of joint protein gene and functional analysis, northwest Botany Gazette, 2010,30 (1): 0,035 0040) disclosed in；BL21
Coli strain can obtain by disclosing commercially available commercial channel, steps Deco skill Development Co., Ltd as Beijing is rich, CompanyAddress:
Road, West 4th Ring Road, Haidian District, Beijing City first 59.
Heretofore described metallic nickel ions chelating (Ni-NTA) post usually contains continuous 6 groups be applicable to extracting from antibacterial
The soluble recombinant protein matter (Native Protein) with natural structure of propylhomoserin tail (His Tag Protein), can be from open canal
Road is bought, Bioisystech Co., Ltd as great in Shanghai, CompanyAddress: SHILONG road, Xuhui District of Shanghai Lane 345 7 commercial affairs in spring
Room, building 401.
Heretofore described cell agglutination test is carried out according to the operating procedure of conventional cell agglutination reaction.The people wherein selected
Breast cancer cell MDA-MB-231(Breast adenocarcinoma) purchased from Chinese Academy of Sciences's cell bank, address: Shanghai City Xu
Remittance Yueyang road, district 320.The cultivation of tumor cell is conventionally carried out.
In the present invention, it may include the carrier containing recombinant fungus immune adjustment protein gene FIP-gas, described vector
Host cell.
The present invention utilizes the fungal immunomodulatory protein FIP-gas of recombinant gene expression, can suppress people and breast cancer cell in vitro
MDA-MB-231(Breast adenocarcinoma) breeding, illustrate that this albumen has inhibitory action to this tumor cell line.
This gene will proceed to escherichia coli by transgenic technology, after inducing culture, by Protein expression and purification, at anti-breast cancer medicines
Exploitation in have huge application potential.
Accompanying drawing explanation
Fig. 1 is restructuring ganoderma atrum fungal immunomodulatory protein electrophoretogram (A) and Western Blot detection figure (B).
Fig. 2 is that restructuring ganoderma atrum fungal immunomodulatory protein is to human breast cancer cell MDA-MB-231(Breast
Adenocarcinoma) the agglutination schematic diagram of cell；
Wherein: Fig. 2-1 is breast cancer cell coagulation situation after reFIP-gas albumen addition 6h, and Fig. 2-2 is reFIP-gas egg
Breast cancer cell coagulation situation after white addition 12h；Fig. 2-3 is breast cancer cell coagulation situation after reFIP-gas albumen addition 24h.
Detailed description of the invention
Elaborating embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention,
Give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The structure of ganoderma atrum fungal immunomodulatory protein gene FIP-gas prokaryotic expression carrier:
1) according to the sequence of ganoderma atrum fungal immunomodulatory protein gene FIP-gas, design can amplify the primer of its full length gene,
And on upstream and downstream primer, add restriction endonuclease sites Bam H I and Hind III respectively, to build prokaryotic expression carrier.
2) with ganoderma atrum fungal immunomodulatory protein gene FIP-gas as template, after PCR expands, by FIP-gas gene gram
Grand to intermediate carrier (such as pMD18-T simple vector), sequencing result and the ganoderma atrum fungal immunomodulatory protein base reported
Because of FIP-gas gene comparision, concordance is 100%, shows to expand and mispairing does not occur.
3) according to the restriction enzyme site of primer two ends design, with double digestion (pET-30a carrier can use Hind III and Bam H I)
FIP-gas segment is cut from pMD18-T simple vector, reclaims respective segments, connect overnight with T4 ligase 16 DEG C.
4) connect product and convert e. coli bl21 host cell, with the LB flat screen of the kanamycin containing 50mg/mL
Select transformant picking positive colony, order-checking.
5) extract cloned plasmids, i.e. obtain fungal immunomodulatory protein expression vector pET30a-FIP-gas in recombinant Ganoderma lucidum belongs to.
Embodiment 2
The abduction delivering of ganoderma atrum fungal immunomodulatory protein FIP-gas and purification
1. in recombinant Ganoderma lucidum belongs to, abduction delivering and the Western blot of fungal immunomodulatory protein FIP-gas detect
1) monoclonal obtained in embodiment 1 is inoculated in the LB fluid medium of the kanamycin containing 50mg/mL,
37 DEG C of shaking overnight incubation.
2) it is inoculated in larger volume culture fluid with 2% inoculum concentration next day.37 DEG C are continued to cultivate, to OD600When reaching 0.5～0.7,
Add IPTG, make the final concentration of IPTG reach 1mM.
3) every 0,0.5,1,2,3,4,5h takes out 1mL to-20 DEG C and saves backup.By acquired bacterium solution sample
Product 5,000rpm is centrifuged 20min to collect thalline, removes supernatant, thalline 100 μ L2 × SDS-PAGE Sample buffer (100
MM pH6.8Tris-Cl, 4% (W/V) SDS, 0.2% (W/V) BPB, 20% (W/V) glycerine, 2% (W/V) β-ME)
Suspend, and in 95 DEG C of water-baths, be incubated 5min.The most respectively with two pieces of 15%SDS-PAGE glue detections.One piece is used for examining
Maas light blue dyes, and another block detects (Fig. 1) for Western blot.
Shown in Fig. 1, top (A) is that the e. coli bl21 cell containing recombinant expression carrier pET30-FIP-gas is through IPTG
Induced protein expresses electrophoretogram, and lower section (B) is the Western Blot inspection of Ganoderma ganoderma atrum fungal immunomodulatory protein FIP-gas
Survey.Swimming lane M is marker, and it is thin that swimming lane 1-7 is respectively the induction time escherichia coli of 0,0.5,1,2,3,4,5 hours
Born of the same parents, are ganoderma atrum fungal immunomodulatory protein FIP-gas shown in arrow.Induce 5 little constantly, expression of recombinant proteins amount is the highest.
Through Escherichia coli fermentation, recombinant Ganoderma lucidum belongs to the yield of interior fungal immunomodulatory protein and is up to 29.76mg/L.
2. recombinant fungus immune modulator is isolated and purified
1) bacterium solution 5 will induced through IPTG, 000rpm is centrifuged 20min to collect cell, with the Lysis of 1/10 volume
buffer(50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH8.0) suspend；Mixture is placed in liquid nitrogen
Freezing, melts afterwards in frozen water；
2) the sample ultrasonic disruption in mixture of ice and water after melting, 200～300W ultrasonic 6 times, each 10s, the phase
Between be spaced 10s；
3) bacterium solution that supersound process is crossed 12,000rpm at 4 DEG C is centrifuged 20～30min；Take supernatant, use Ni-NTA post
Purification (balances with the Lysis buffer of 10 times of column volumes) in advance；With the Wash buffer (50mM of 20 times of column volumes
NaH2PO4, 300mMNaCl, 20mM imidazole, pH8.0) and wash post；Destination protein is by Elution buffer (50mM
NaH2PO4, 300mMNaCl, 250mM imidazole, pH8.0) it is eluted out.
Embodiment 3
Recombinant Ganoderma lucidum belongs to the interior fungal immunomodulatory protein FIP-gas cell agglutination test to human breast cancer cell MDA-MB-231.
1. the cultivation of cancerous cell
The breast cancer cell line MDA-MB-231 DMEM culture medium containing 10% hyclone, at 5%CO2, 37 DEG C full
Cultivating with under damp condition, tumor cell monolayer adherence is cultivated.After cell is paved with bottle, with 0.25% trypsin and 0.04%EDTA
PBS liquid digestion, be prepared as single-cell suspension in the DMEM solution for standby of 0.5% fetal bovine serum albumin (BSA), people
Breast cancer cell MDA-MB-231 density is adjusted to 5-7 × 103/100μL。
2. cell agglutination experiment
1) cell concentration in embodiment 3.1 is adjusted to the breast cancer cell of 5-7 × 103/100 μ L and adds 96 orifice plates, and
CO237 DEG C of overnight incubation in incubator.
2) recombiant protein FIP-gas bag filter carrying out dialysis and remove the imidazoles composition in solution, dialysis solution is PBS(10mM
Na2HPO4, 137mM NaCl, 2.7mM KCl, 2mM KH2PO4, pH7.4).It is diluted to work with PBS and cell culture fluid
Make concentration .(20 μ g/mL, 15 μ g/mL, 10 μ g/mL, 5 μ g/mL, 200ng/mL).
3) in 96 orifice plates add test recombiant protein mixture, mixture have 4 Concentraton gradient 5 μ g/mL, 10 μ g/mL,
15 μ g/mL and 20 μ g/mL, each gradient 3 repetition, add PBS solution as negative control.And at CO2In incubator
37 DEG C of cultivations, observe at 6h, 12h and 24h respectively.
4) result will be examined under a microscope and take pictures (Fig. 2-1,2-2,2-3).
Shown in Fig. 2, wherein A is PBS negative control；It is 5 μ g/ that the reFIP-gas protein concentration that B-E adds is respectively as follows: B
ML, C are 10 μ g/mL, and D is 15 μ g/mL, and E is 20 μ g/mL；Fig. 2-1reFIP-gas albumen adds breast carcinoma after 6h
Cell agglutination situation, Fig. 2-2reFIP-gas albumen adds breast cancer cell coagulation situation after 12h；Fig. 2-3reFIP-gas albumen
Add breast cancer cell coagulation situation after 24h.
Observe display ganoderma atrum fungal immunomodulatory protein FIP-gas to the suppression result of human breast cancer cell as shown in Figure 2.When
When reFIP-gas concentration is 5 μ g/mL, MDA-MB-231 cell occurs as soon as agglutination, cell shrinkage or elongation, and part is thin
Born of the same parents' gathering is agglomerating, and some cell takes off wall, and cell surface reflective is deteriorated.Cell membrane shrinkage, evagination or adhesion, the little follicular rupture having,
Or the formation apoptotic body that comes off.MDA-MB-231 cells blocks in the Gl/S phase, is disturbed the normal increasing of tumor cell by FIP-gas
Grow activity.Along with incubation time increases and the increase of FIP-gas concentration, cell agglutination increased activity, but reinforced effects is inconspicuous.
Claims (3)
1. the application of a FIP-gas albumen, it is characterised in that use it for preparing anti-human breast cancer cell MDA-MB-231
The medicine of tumor；
The encoding gene of described FIP-gas albumen, its nucleotide sequence as shown in Seq ID No.1, by Homology-based cloning from
Black Ganoderma (Ganoderma atrum) mycelium is cloned and obtains, ganoderma atrum fungal immunomodulatory protein FIP-gas is gene constructed
Become prokaryotic expression carrier pET-30a-FIP-gas to be transformed in e. coli bl21 competent cell, induce it to express through IPTG；
Prokaryotic expression product obtains after purification through metallic nickel ions chelate column.
Application the most according to claim 1, is characterized in that, described FIP-gas albumen, obtains as follows:
1) construction of expression vector, method particularly includes:
1.1) according to the sequence of ganoderma atrum fungal immunomodulatory protein gene FIP-gas, primer is designed, and at upstream and downstream primer
Upper interpolation restriction endonuclease sites Bam H I and Hind III respectively, to build prokaryotic expression carrier；
1.2) with FIP-gas gene as template, after PCR expands, FIP-gas gene is cloned into intermediate carrier；
1.3) with double digestion, FIP-gas segment is cut from intermediate carrier, reclaim respective segments, with T4 ligase 16 DEG C connection
Overnight obtain expression vector；
2) convert to escherichia coli, method particularly includes: described expression vector is added competent escherichia coli cell and places on ice
30min, afterwards 42 DEG C of circulator bath thermal shock 90s, rapid ice bath 2min；Add 800 μ L LB culture fluid 37 DEG C and shake recovery 1h slowly；
Take 200 μ L bacterium solution bacterium solution and be coated on the LB flat board of the kanamycin containing 50mg/mL, screening transformant picking positive colony；
Described intermediate is pMD 18-T simple vector or pET-30a；
3) amplification culture, method particularly includes: the positive colony of acquisition is inoculated in the LB liquid of the kanamycin containing 50mg/mL
In body culture medium, 37 DEG C of shaking overnight incubation, inoculate with 2% inoculum concentration next day, 37 DEG C are continued fermentation；
4) IPTG abduction delivering, method particularly includes: as fermentation liquid OD600When reaching 0.5～0.7, add IPTG and induce,
The final concentration of 1mM of IPTG；
It is 5) isolated and purified, method particularly includes:
5.1) the bacterium solution centrifugal collecting cell will induced through IPTG, is suspended in buffer；Mixture is placed in liquid nitrogen freezing,
Melt in frozen water afterwards；
5.2) the sample ultrasonic disruption in mixture of ice and water after melting；
5.3) the bacterium solution centrifuging and taking supernatant that supersound process is crossed, upper Ni-NTA post, after washing post with buffer 1, with buffer 2 by mesh
Albumen be eluted out.
Application the most according to claim 2, is characterized in that, described buffer 1 is 50mM NaH2PO4, 300mMNaCl,
20mM imidazole, pH are the aqueous solution of 8.0；Described buffer 2 is 50mM NaH2PO4, 300mMNaCl,
The aqueous solution of 250mM imidazole, pH 8.0.
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