CN105237544B - Xantholipin B and its production and use - Google Patents
Xantholipin B and its production and use Download PDFInfo
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Abstract
The invention provides a kind of xantholipin B and its production and use.The structural formula of the compound is as follows：The preparation method includes the step of structure mutant strain Δ stnR and the step of producing the liquid of B fermentation containing xantholipin of being fermented to the mutant strain Δ stnR, and a kind of new xantholipin analog xantholipin B is made first.The xantholipin B has good bioactivity, available for preparing antitumor and antibacterials, and the isolated xantholipin class compound from streptonigrin producing strains first of the invention, new material is provided for the preparation and medicinal study of xantholipin class compound, drug resource is provided for antitumor and selection of antibacterial.
Description
Technical field
The present invention relates to antibiotics preparation field, and in particular to a kind of xantholipin analog --- xantholipin B's
Preparation method and purposes.
Background technology
Xantholipin (Xanthol ipin) is isolated sallow strepto- from the sandy soil in Chinese Bohai Sea Gulf in 2003
Found in the zymotic fluid of bacterium (Streptomyces flavogriesus).Xantholipin and the like is a kind of Oxoxanthone
(xanthone) class antibiotic.Xanthones antibiotic is a kind of microbial secondary metabolite with specific structure, such
Antibiotic bioactivity includes suppressing bacterium, and fungi and cytotoxicity, its avtive spot mainly act on cell membrane, can be with conjunction
Precursor substance into cell membrane produces Reverse transcriptase, so as to the synthesis of interference cell wall.
Research confirms that xantholipin has wide inhibitory action to gram-positive bacteria, while experiment shows that it can press down
Liver fibrosis processed, glomerulonephritis, between kidney in the illness such as duty ephritis heat shock protein HSP47 great expression, so as to conduct
The potential drugs of such illness.
Fine hair streptomycete (Streptomyces flocculus) CGMCC4.1223 has been found that the black bacterium of chain can be used as before this
The producing strains of element.We utilize molecular biology philosophy and technique, and streptonigrin biosynthesis is obtained from this streptomycete
Gene cluster, and selectivity inactivation is carried out to the biological synthesis gene cluster of streptonigrin in gene level and obtains corresponding mutant bacteria
Strain, and the mutant strain is fermented, xantholipin and xantholipin B are separated to from its metabolite first.
The content of the invention
For in the prior art the defects of, it is an object of the invention to provide a kind of new xantholipin analog-xantholipin
B and preparation method thereof and the application in antitumor and antibacterials are prepared.Fine hair streptomycete (Streptomyces
Flocculus) have been found before this can be as the producing strains of streptonigrin by CGMCC4.1223.The present invention utilizes molecular biology
Philosophy and technique, streptonigrin biological synthesis gene cluster is obtained from this streptomycete, and in gene level to streptonigrin
Biological synthesis gene cluster carry out selectivity inactivation and obtain corresponding mutant strain, and the mutant strain is fermented, first
Isolated xantholipin and the like -- the xantholipin B from its metabolite.
The purpose of the present invention is achieved through the following technical solutions：
The invention provides a kind of xantholipin B, its structural formula such as formula (I) are shown：
Present invention also offers a kind of xantholipin B preparation method, the preparation method includes structure mutant strain Δ
The step of stnR and the step of producing the liquid of B fermentation containing xantholipin of being fermented to the mutant strain Δ stnR.
Preferably, the step of structure mutant strain Δ stnR is specific as follows：
Using fine hair streptomycete (Streptomycesflocculus) CGMCC4.1223 as starting strain, clone obtains chain
Black rhzomorph biological synthesis gene cluster；
For the stnR genes design inactivation primer in the streptonigrin biological synthesis gene cluster, carry out selective mistake
It is living, and obtain PCR primer；The inactivation primer is as shown in SEQ ID No.1, SEQ ID No.2；
PCR primer electricity is transferred in E. coli BW25113/pIJ790/pLS1153, extracted
Mutant plasmid pLS1154；
With in mutant plasmid pLS1154 conversion E. colis ET12567/pUZ8002, large intestine bar is built
Bacterium E.coli ET12567/pUZ8002/pLS1154；
Engagement transfer flat board co-cultures the E. coli ET12567/pUZ8002/pLS1154 and fine hair chain
Mould (Streptomycesflocculus) CGMCC4.1223, produces mutant strain Δ stnR.
Preferably, in step C, the separation of fermentative broth purifying is specially：Zymotic fluid extract and separate is obtained into xantholipin B
Crude extract, then purified obtained xantholipin B sterlings.
Preferably, the process of the fermentation specifically includes：First the mutant strain Δ stnR is inoculated in seed culture medium
Culture, is then transferred and is fermented into fermentation medium again, and macroreticular resin is added in most backward fermentation medium and continues to ferment, i.e.,
The zymotic fluid of the xantholipin B must be contained.
Preferably, it is described be inoculated in the condition cultivated in seed culture medium for 30 DEG C, 220rpm, 2 days；It is described to transfer into hair
The condition that ferment culture medium is fermented is：30 DEG C, 220rmp, 2 days；The macroreticular resin that added into fermentation medium continues to send out
The condition of ferment is：30 DEG C, 220rmp, 5 days.
Preferably, the seed culture medium (TSBY culture mediums) is：Oxide tryptone beans powder 30g, dusty yeast 5g,
10.3% sucrose, distilled water 1000ml.
Preferably, the fermentation medium is：Glucose 25g, soybean cake powder 15g, NaCl 5g, KCl 0.5g,
MgSO4·7H2O 0.25g, K2HPO43g, Na2HPO4·12H2O 3g, pH 7.2.
Preferably, also include the zymotic fluid extract and separate of acquisition obtaining xantholipin B crude extracts after the fermentation, then pass through
The step of xantholipin B sterlings is made in purifying.
Preferably, the concrete operations that the zymotic fluid extract and separate obtains xantholipin B crude extracts are：Will be big after sterilizing
Hole resin is added in zymotic fluid, after fermented and cultured, isolates macroreticular resin, macroreticular resin is washed through middle polarity organic solvent A
It is de-, obtain xantholipin B crude extracts；The middle polarity organic solvent A be ethyl acetate, ether, chloroform, dichloromethane or
One kind in acetone.
Preferably, described macroreticular resin chooses XAD-16 models.
Preferably, the purifying concretely comprises the following steps：
Xantholipin B crude extracts are subjected to column chromatography with purification on normal-phase silica gel, mixed with middle polarity organic solvent B with methanol
The mixed solution arrived elutes as eluent gradient, analyzes and detects through HPLC, collects evaporating containing xantholipin similar compound
Point, concentrate drying obtains crude separation thing A；
Crude separation thing A is chromatographed through reverse silica gel (YMC*GEL ODS-A Ball-type packings, 120A apertures, 50 μm of particle diameters), used
Volumetric concentration is eluted for 50%~100% aqueous methanol gradient, and cut is merged after HPLC is detected, and concentrate drying is separated
Thing B；
Isolate B is chosen methanol, merged after HPLC is detected through gel post separation, mobile phase, is concentrated and dried, produces pure
Xantholipin B after change；
The middle polarity organic solvent B and methanol are 100 by volume:1~10:1 mixing；The middle polarity is organic
Solvent B is one kind in ethyl acetate, chloroform, dichloromethane or acetone.
Present invention also offers it is a kind of can fermenting and producing Huang aspergin B mutant strain Δ stnR, the mutant strain Δ stnR
Construction step it is specific as follows：
Using fine hair streptomycete (Streptomycesflocculus) CGMCC4.1223 as starting strain, clone obtains chain
Black rhzomorph biological synthesis gene cluster；
For the stnR genes design inactivation primer in the streptonigrin biological synthesis gene cluster, carry out selective mistake
It is living, and obtain PCR primer；The inactivation primer is as shown in SEQ ID No.1, SEQ ID No.2；
PCR primer electricity is transferred in E. coli BW25113/pIJ790/pLS1153, extracted
Mutant plasmid pLS1154；
With in mutant plasmid pLS1154 conversion E. colis ET12567/pUZ8002, large intestine bar is built
Bacterium E.coli ET12567/pUZ8002/pLS1154；
Engagement transfer flat board co-cultures the E. coli ET12567/pUZ8002/pLS1154 and fine hair chain
Mould (Streptomycesflocculus) CGMCC4.1223, produces mutant strain Δ stnR.
Present invention also offers a kind of purposes of xantholipin B in antitumor and antibacterials are prepared.
The xantholipin B reaches to the minimum bacteriostatic activity of the staphylococcus aureus Mu50 bacterial strains of methicillin-resistant
0.025 mcg/ml is higher than vancomycin 40 times；0.037 microgram/milli is reached to human oral cavity epithelial JEG-3 KB IC50
Rise, 0.009 mcg/ml is reached to people's promyelocytic leukemia cell HL-60 IC50, to human gastric adenocarcinoma (low differentiation)
BGC-803 IC50 reaches 0.03 mcg/ml, and Non-small cell lung carcinoma cell A549 IC50 reaches 0.08 mcg/ml,
The IC50 of human breast cancer cell line Bcap-37 reaches 0.09 mcg/ml.
Compared with prior art, the present invention has following beneficial effect：
1st, the present invention from fine hair streptomycete (Streptomycesflocculus) CGMCC4.1223 mutant strain first
Isolated xantholipin with and the like -- xantholipin B.The structure diversity of xantholipin class compound is enriched,
Also new material and new technology are provided for xantholipin and the like xantholipin B preparation and application study.
2nd, xantholipin B of the present invention has good antitumor activity and excellent antibacterial activity particularly multiple medicine resistance to
Medicine positive bacteria, therefore xantholipin B can be developed into new antineoplastic or antibacterials.
Brief description of the drawings
The detailed description made by reading with reference to the following drawings to non-limiting example, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the xantholipin B of present invention chemical constitution schematic diagram；
Fig. 2 is stnR gene knockout mutant strain Δs stnR structure schematic diagram；
Fig. 3 is stnR gene knockout mutant strain Δs stnR genotype proof diagram；Wherein swimming lane 1 is 1kb DNAmarker；
Swimming lane 2 is the PCR primer of positive control (positive plasmid pLS1154)；Swimming lane 3 is negative control (Streptomyces
Flocculus CGMCC4.1223 STb gene) PCR primer；Swimming lane 4 is that single-swap mutant strain occurs in stnR gene knockouts
PCR primer；Swimming lane 5-8 is that double crossing over mutant strain PCR primer occurs in stnR gene knockouts；
Fig. 4 is mutant strain Δ stnR extractive from fermentative and wild type fine hair streptomycete (Streptomycesflocculus)
The HPLC detection collection of illustrative plates of CGMCC4.1223 extractive from fermentative；Wherein, line a is that the HPLC of mutant strain Δ stnR extractive from fermentative is examined
Mapping is composed, and line b is that the HPLC of wild-type strain extractive from fermentative detects collection of illustrative plates, and peak 1 is xantholipin, and peak 2 is xantholipin B.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
The structure of embodiment 1, transamination enzyme coding gene stnR Inactivating mutations strain Δs stnR
StnR gene knockout mutant strain Δ stnR, mutant strain Δ stnR structure are built using PCR-targeting method
It is as shown in Figure 2 to build schematic diagram.According to stnR sequences in the corresponding gene cluster of acquisition, the PCR-targeting of reference literature report
The requirement of technology, design the knockout inactivation primer of a pair of stnR genes
SftarstnR-For(SEQ ID No.1):
5’–CCCTACGGTGCCAGCCCCGCGGCGGTGGCCGCGGTGACGattccggggatccgtcgacc-3’
SftarstnR-Rev(SEQ ID No.2)：
5’-AATGCCGTGGTCGCGGGCCAGCACGTCCTGCGCCTCGGCtgtaggctggagctgcttc-3’
5 ' end 39bp capitalizations partial sequences of wherein primer match with stnR genes, and the lowercase portion at 3 ' ends
Sub-sequence includes the fragment both sides for shifting homing sequence and apramycin resistance gene with one in plasmid pIJ773 respectively
Consensus dna sequence or complementation.Plasmid is knocked out outside referring next to PCR-targeting method construct, and the plasmid is transferred to
Into the donor bacterium of Conjugative tiansfer.Comprise the following steps that：
(1) cosmid plasmids pLS1153 is transferred in E. coli BW25113/pIJ790 and obtains Escherichia coli
E.coli BW25113/pIJ790/pLS1153, with 10mmol/L L-arabinose induction λ/red restructuring in LB culture mediums
Expression system, and be prepared into stand-by for the competent cell of electricity conversion.
(2) the restriction endonuclease EcoR I and digested plasmid pIJ773 of Hind III, recovery about 1.4kb is used to contain transfer site and A Bo
Draw the DNA fragmentation of mycin resistant gene, in this, as pcr template, with sftarstnR-For (SEQ ID No.1) and
SftarstnR-Rev (SEQ ID No.2) primer amplifies 1.4kb PCR primer, PCR reaction systems (50 μ l) by PCR:
The 2.5 μ l of μ l, dNTPs 0.5mM, DMSO of Taq archaeal dna polymerases 1U, 10x Buffer 5, each 0.5 μM of primer, DNA profiling is about
1ng, add water to 50 μ l.PCR reaction conditions are：94 DEG C of 5min of pre-degeneration；Amplification cycles are 94 DEG C of denaturation 45s, and 55 DEG C are annealed
30s, 72 DEG C of extension 90s, 35 circulations；Last 72 DEG C of extensions 5min.PCR primer (1.4kb) recovery purifying is stand-by.
(3) PCR primer is transferred to the competent cell prepared in step (1) by electricity and brings it about restructuring, in LB
Screening flat board is incubated overnight on (containing 100 μ g/ml ampicillins, 50 μ g/ml apramycins) in 37 DEG C, is chosen from flat board
Positive monoclonal, extract recombination mutation plasmid pLS1154, the Partial Fragments of the stnR genes in the plasmid be transferred site and Ah
Pool draws mycin resistant gene substitution.
(4) the recombination mutation plasmid pLS1154 built is gone into E. coli by electricity
In ET12567/pUZ8002, E. coli ET12567/pUZ8002/pLS1154 is built into.
By E. coli ET12567/pUZ8002/pLS1154 in 50ml containing 25 μ g/ml kanamycins, 25 μ g/
Grown in the LB fluid nutrient mediums of ml chloramphenicol, 100 μ g/ml ampicillins and 50 μ g/ml apramycins in 37 DEG C
OD600During value about 0.6, thalline (4000r/min, 10min) is collected by centrifugation, cleans thalline 2 times with LB, is suspended in 500 μ l LB
In culture medium, the donor bacterium as Conjugative tiansfer.
(5) wild type fine hair streptomycete (Streptomyces flocculus) CGMCC4.1223 bacterial strains are trained in SFM solids
Support and cultivated 7 days in base, dip the white spore on sterilized water collection flat board with cotton swab, 8000r/min centrifugations 10min collects spore
Son, spore is resuspended with 0.05M TES solution (pH8.5), in 50 DEG C of heat shock 10min.Isometric 500 μ l spores are added afterwards to sprout in advance
Lotion cultivates 2-4 hours in 37 DEG C, and supernatant is removed in centrifugation afterwards, is resuspended with 500 μ l LB, the recipient bacterium as Conjugative tiansfer.
(6) respectively take 100 μ l are well mixed to be coated on the IWL-4 without any antibiotic and consolidate above-mentioned recipient bacterium and donor bacterium
On body culture medium, after drying, cultivated 16 hours in 30 DEG C.Then the water of thiostrepton and A Baila antibiotic is contained with 1ml
Flat board, its final concentration of 35 μ g/ml apramycin and 25 μ g/ml thiostreptons are covered, are dried up as in 30 DEG C of incubators
Culture is observed after 4 days.
After monoclonal petite is grown on engagement transfer flat board, choose and expand on 35 μ g/ml apramycin flat boards
Culture, is not false positive clones to confirm it, then picking clone of normal growth on 35 μ g/ml apramycin flat boards connects
In the TSBY culture mediums for kind containing 35 μ g/ml apramycins to 25ml, 30 DEG C of cultures carry out the culture that relaxes, 50 μ of switching after 2 days
L is cultivated 2 days into nonreactive TSBY culture mediums, after being repeated 2 times, by the μ l of cumulative volume 500 culture according to thalline and culture medium
Volume ratio is 10-3~10-5, dilution spread was containing 35 μ g/ml apramycin SFM solid plates culture 4 days.The single bacterium that will be grown
Fall and be divided into two, be seeded to consolidate containing 35 μ g/ml apramycin SFM solid plates and 25 μ g/ml thiostreptons SFM respectively
Body flat board simultaneously carries out mark culture 4 days.Observe flat board afterwards, choose on 35 μ g/ml apramycin resistance flat boards growth but
It is that long clone is not inoculated into 25ml containing 35 μ g/ml apramycins TSBY trainings in 25 μ g/ml thiostrepton resistant panels
The genomic DNA that each mutant strain is extracted after being cultivated 2 days in base is supported, the correct of its mutant strain genotype is detected using detection primer
Property.
SftarstnA-T-For(SEQ ID No.3):5’-Atcgaggtgcacctcgac-3’
SftarstnA-T-Rev(SEQ ID No.4):5’-Acagttccttcgcgccgaa-3’
The clone that can be expanded by above-mentioned PCR primer and be only capable of amplifying 1434bp size fragments is positive colony, i.e.,
Obtain the mutant strain Δ stnR that stnR genes are substituted by apramycin resistance gene.Mutant strain Δ stnR genotype proof diagram
As shown in figure 3, its result shows that mutant strain Δ stnR is successfully built.
Embodiment 2, mutant strain Δ stnR fermentation and the extraction of tunning
1st, culture medium：
(1) seed culture medium (TSBY culture mediums)：Oxide tryptones beans powder 30g, dusty yeast 5g, 10.3% sucrose, distills
Water 1000ml.
(2) fermentation medium：Glucose 25g, soybean cake powder 15g, NaCl 5g, KCl 0.5g, MgSO4·7H2O0.25g,
K2HPO43g, Na2HPO4·12H2O 3g, pH 7.2.
(3) SFM culture mediums：Agar powder 20g, mannitol 20g, analysis for soybean powder 20g, running water 1000ml, pH are adjusted to 7.2 left
It is right.
(4) the pre- germination medium of spore：Difco yeast extracts 1%, Difco casamino acids 1%, CaCl20.01M (is needed
5M stoste is prepared, is separately added to after sterilizing in yeast extract/casamino acid solution).
(5) IWL-4 culture mediums：ISP4 powder 34g, yeast extract 1g, peptone 4g, are settled to 1000ml.After sterilizing
Add 8ml 2.5M MgCl2。
2nd, ferment：
Mutant strain Δ stnR is inoculated into 25ml seed culture mediums, 20 bottles of 25ml x, 30 DEG C, 220rpm, cultivated 2 days,
Then it is inoculated into by 1% inoculum concentration in 500ml fermentation mediums, 500mlx100 bottles, 30 DEG C, 220rmp, in fermentation 48 hours
The macroreticular resin of sterilizing is added afterwards, then proceedes to fermentation 5 days.
3rd, the extract and separate of tunning：
The acquisition of mutant strain Δ stnR tunnings is by separating macroreticular resin, then elutes macropore with ethyl acetate
Resin, collect ethyl acetate and concentrate the crude extract for obtaining xantholipin B.Metabolite crude extract detects through HPLC, HPLC inspections
Survey condition is：C18 reversed-phase columns (4.6*150mm,Aperture, 50 μm of particle diameters), mobile phase is A (MilliQ-water)/B
(CH3CN) gradient mixed solution.Flow velocity is 0.6ml/min.Injection procedure：10%B to 70%B (linear gradient, 0-28min),
70%B to 100%B (linear gradient, 28-33min), 100%B (33-38min), 100%B to 10%B (38-40min).Its
As a result as shown in figure 4, line a, which is mutant strain Δ stnR fermentations HPLC, detects collection of illustrative plates, line b is that wild-type strain fermentation HPLC detections are schemed
Spectrum.As shown in Figure 4, by gene knockout means, the metabolic pathway of thalline is changed to the knockout of rear modifier in gene cluster,
So as to produce xantholipin and the like xantholipin B, this finds for the patent medicine improvement of xantholipin and Combinatorial biosynthesis
Antibiotics provide good example.
The purifying of embodiment 3, xantholipin B
The xantholipin B obtained after extract and separate crude extract is subjected to column chromatography with purification on normal-phase silica gel (200~300 mesh),
With middle polarity organic solvent B, (organic solvent B is ethyl acetate, chloroform or dichloromethane；Chloroform is selected in the present embodiment)
With methanol with volume ratio 100：1~10:1 elutes as mobile phase, is detected through HPLC, collects the cut containing target compound,
It is concentrated to give crude separation thing A；Crude separation thing A through reverse silica gel (YMC*GEL ODS-A Ball-type packings,Aperture, 50 μm of grains
Footpath) chromatography, eluted with volumetric concentration for 40~100% aqueous methanol gradients, cut is merged after HPLC is detected, obtains rough segmentation
From thing B；Crude separation thing B passes through gel column, is eluted using pure methanol system, merges after HPLC is detected, and is concentrated and dried, must purify
Xantholipin B afterwards.
Xantholipin B：Yellow crystals, by high resolution mass spectrum HR-ESI-MS (m/z536.0713, [M+H]+) obtain its point
Minor is C27H1809NCl.Push away xantholipin B structure is as shown in Figure 1.Its1H NMR (600MHz, DMSO-d6) and13C NMR
(150MHz, DMSO-d6) is shown in Table 1.Nuclear magnetic data listed by table 1 confirms xantholipin B chemical constitution.
The xantholipin B's of table 11H and3C NMR and necessary 2D NMR datas
From upper each embodiment, the present invention is to fine hair streptomycete (StreptomycesflocculusCGMCC4.122
3) the streptonigrin biological synthesis gene cluster that clone obtains in carries out selective knockout, obtains the base that aminopherase stnR is knocked out
Because of mutant strain Δ stnR.The mutant strain is fermented, and to its metabolite using purification on normal-phase silica gel, reverse silica gel, gel
Operated Deng chromatographic techniques such as column chromatography method and HPLC, isolated xantholipin and one are new from mutant strain Δ stnR
Xantholipin analog -- xantholipin B.Present invention finds one to have the natural of excellent antitumor and antibacterial activity
Compound, drug candidate resource is provided for antitumor and selection of antibacterial.
Embodiment 4, xantholipin B drug effect checking test
1) antibacterial activity is tested
Xantholipin B antibacterial activities are surveyed with 96 well plate methods.Xantholipin B is dissolved with DMSO, and configures 12.8mg/ml mother
Liquid.A small amount of mother liquor is taken, xantholipin B concentration dilutions to 6.4 μ g/ml are regard as initial concentration by the use of fermentation medium.Take 100 μ l yellow
The culture medium that fat rhzomorph B concentration is 6.4 μ g/ml is added in first hole, and gradient dilution is extremely in the form of dichotomy successively
0.003125 μ g/ml, and one group of blank test for being free of xantholipin B is set.The golden yellow of 5 μ l methicillin-resistants is added per hole
Staphylococcus Mu50 seed liquors, its optical activity is surveyed with ELIASA after being cultivated 16 hours in 37 DEG C of shaking tables, record result.
As a result show that xantholipin B reaches to the minimum bacteriostatic activity of the staphylococcus aureus Mu50 bacterial strains of methicillin-resistant
It is higher than vancomycin 40 times to 0.025 mcg/ml.
2) antitumor activity is tested
Xantholipin B is determined respectively with mtt assay to human oral cavity epithelial JEG-3 KB, people's promyelocytic leukemia cell
HL-60, human gastric adenocarcinoma (low differentiation) BGC-803, Non-small cell lung carcinoma cell A549, human breast cancer cell line Bcap-37
Antitumor activity.Comprise the following steps that：
1.0mg xantholipins B is dissolved in 1ml DMSO, obtains the mother liquor that concentration is 1mg/ml.Phosphoric acid buffer is used again
Liquid makees gradient dilution, and it is respectively 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, 0.01 μ g/ml, 0.001 μ to obtain concentration
G/ml dilute sample；The cell in exponential phase is taken, pancreatin digestion, collects, be resuspended after centrifugation, adjusting cell suspending liquid
Density is about 1 × 105 cell/ml.In 96 orifice plates, negative control hole, by reagent hole are set.90 μ l cells are added per hole, in
37 DEG C, 5%CO2Cultivated 6 hours in cell culture incubator.The xantholipin B diluted is added in corresponding flat 96 orifice plate, often
The μ l of hole 10, negative control hole are not added with, and continue at 37 DEG C, 5%CO2Cultivated in cell culture incubator.After 48h, 10 μ l are added in every hole
5mg/ml MTT solution, continue to be incubated 4 hours in incubator.100 μ l lysates are added per hole, continue to be incubated in incubator
Overnight, generation Jia Za crystal is made fully to dissolve.Determine 570nm absorbance values.Sample is calculated to each tumour finally by software
The IC of cell50。
The formula of wherein phosphate buffer is：NaCl 8g, KCl 0.2g, Na2HPO41.15g KH2PO40.2g, it is dissolved in 1L
Distilled water, 121 DEG C of autoclave sterilization 20min, 4 DEG C of preservations.MTT (AMRESCO) solution formula：5mg/ml is made into phosphate buffer
Solution.Dissolve formula of liquid：Per 100ml pure water 10g containing SDS, isobutanol 5ml, concentrated hydrochloric acid 0.1ml.
Test result is as shown in table 2.
The sample of table 2 is in vitro to the IC of various tumor cell strains50(μg/ml)
As can be known from the results of Table 2, xantholipin B has good antitumor activity.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (5)
1. a kind of xantholipin B preparation method, it is characterised in that shown in the structural formula such as formula (I) of the xantholipin B：
Described xantholipin B preparation method includes the step of structure mutant strain Δ stnR and the mutant strain Δ stnR is sent out
Ferment produces the step of liquid of B fermentation containing xantholipin；
The step of structure mutant strain Δ stnR, is specific as follows：
Using fine hair streptomycete (Streptomyces flocculus) CGMCC4.1223 as starting strain, it is black that clone obtains chain
Rhzomorph biological synthesis gene cluster；
It is used to carry out selective inactivation for the stnR genes design inactivation primer in the streptonigrin biological synthesis gene cluster,
And obtain PCR primer；The inactivation primer is as shown in SEQ ID No.1, SEQ ID No.2；
PCR primer electricity is transferred in E. coli BW25113/pIJ790/pLS1153, extracting to be mutated
Plasmid pLS1154；
With in mutant plasmid pLS1154 conversion E. colis ET12567/pUZ8002, structure engagement transfer supplies
Body E. coli ET12567/pUZ8002/pLS1154；
Engagement transfer flat board co-cultures the E. coli ET12567/pUZ8002/pLS1154 and fine hair streptomycete
(Streptomyces flocculus) CGMCC4.1223, produces mutant strain Δ stnR；
The process of the fermentation specifically includes：First the mutant strain Δ stnR is inoculated in seed culture medium and cultivated, Ran Houzai
Transfer and fermented into fermentation medium, macroreticular resin is added in most backward fermentation medium and continues to ferment, is produced containing the Huang
Fat rhzomorph B zymotic fluid；
It is described be inoculated in the condition cultivated in seed culture medium for 30 DEG C, 220rpm, 2 days；Described transfer is entered into fermentation medium
Row fermentation condition be：30 DEG C, 220rmp, 2 days；It is described that the condition that macroreticular resin continues fermentation is added into fermentation medium
For：30 DEG C, 220rmp, 5 days.
2. xantholipin B as claimed in claim 1 preparation method, it is characterised in that also include obtaining after the fermentation
Zymotic fluid extract and separate obtain xantholipin B crude extracts, then the step of purified obtained xantholipin B sterlings.
3. xantholipin B as claimed in claim 2 preparation method, it is characterised in that the zymotic fluid extract and separate obtains
The concrete operations of xantholipin B crude extracts are：Macroreticular resin after sterilizing is added in zymotic fluid, after fermented and cultured, separation
Go out macroreticular resin, macroreticular resin elutes through middle polarity organic solvent A, obtains xantholipin B crude extracts；The middle polarity has
Solvent A is one kind in ethyl acetate, ether, chloroform, dichloromethane or acetone.
4. xantholipin B as claimed in claim 2 preparation method, it is characterised in that the purifying concretely comprises the following steps：
Xantholipin B crude extracts are subjected to column chromatography with purification on normal-phase silica gel, are mixed to get with middle polarity organic solvent B and methanol
Mixed solution elutes as mobile phase, and concentrate drying obtains crude separation thing A；
By crude separation thing A through reverse silica gel column chromatography, eluted with methanol aqueous solution, concentrate drying obtains crude separation thing B；
Crude separation thing B is chosen methanol, be concentrated and dried and produce xantholipin B sterlings through gel post separation, mobile phase；
Described middle polarity organic solvent B and methanol are 100 by volume:1~10:1 mixing；The middle polarity is organic molten
Agent B is one kind in ethyl acetate, chloroform, dichloromethane or acetone.
5. it is a kind of can fermenting and producing yellow aspergin B as claimed in claim 1 mutant strain Δ stnR, it is characterised in that it is described
Mutant strain Δ stnR construction step is specific as follows：
Using fine hair streptomycete (Streptomyces flocculus) CGMCC4.1223 as starting strain, it is black that clone obtains chain
Rhzomorph biological synthesis gene cluster；
It is used to carry out selective inactivation for the stnR genes design inactivation primer in the streptonigrin biological synthesis gene cluster,
And obtain PCR primer；The inactivation primer is as shown in SEQ ID No.1, SEQ ID No.2；
PCR primer electricity is transferred in E. coli BW25113/pIJ790/pLS1153, extracting to be mutated
Plasmid pLS1154；
With in mutant plasmid pLS1154 conversion E. colis ET12567/pUZ8002, Escherichia coli are built
E.coli ET12567/pUZ8002/pLS1154；
Engagement transfer flat board co-cultures the E. coli ET12567/pUZ8002/pLS1154 and fine hair streptomycete
(Streptomyces flocculus) CGMCC4.1223, produces mutant strain Δ stnR.
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