CN101993869B

CN101993869B - Relevant experimental technique of epiphyte secondary metabolism function reformation and application thereof

Info

Publication number: CN101993869B
Application number: CN 201010000035
Authority: CN
Inventors: 崔承彬; 吴长景; 田从魁; 柴云晶; 卜秀嫣; 房士明; 李长伟
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2010-01-04
Filing date: 2010-01-04
Publication date: 2013-04-24
Anticipated expiration: 2030-01-04
Also published as: CN101993869A

Abstract

The invention relates to relevant experimental technique of epiphyte secondary metabolism function reformation and application thereof, in particular to the experimental technical method of DMSO (dimethyl sulfoxide) mediated epiphyte secondary metabolism function reformation and application of the technical method in the obtaining of active mutant strains such as anti-tumor and antipathogen epiphyte and the like through conversion and sieving of inactive epiphyte wild strains and the study on medicinal herbs resource active products. The method of the invention can be effectively used for the reformation from the epiphyte such as the inactive epiphyte into industrial usable epiphyte.

Description

Relevant experimental technique of epiphyte secondary metabolism function reformation and uses thereof

Technical field:

The present invention relates to relevant experimental technique of epiphyte secondary metabolism function reformation, the fungal secondary metabolic function that is specifically related to the DMSO mediation is transformed the experimental technique method, and this technological method obtains the purposes that antitumor, disease-resistant fungal pathogens isoreactivity mutant strain also supplies medicine source activity Study on product for conversion and the screening of non-activity fungi wild strain.

Background technology:

Microbial product is an important sources of new drug and guide structure thereof, and separable culturing micro-organisms is main source and the main flow development resources of microbial medicine and guide structure thereof so far always.Usually, in the conventional R﹠D process of the medicine source of separable culturing micro-organisms strain resource, separate the most bacterial strains that obtain and often can not be directly used in medicine source activity Study on product because of non-activity, thereby left unused in a large number or selected the destruction, cause the significant wastage of early investment and strain resource development and use efficient seriously low.Therefore, thus the non-activity bacterial strain that how will leave unused useless in a large number effectively changes into active bacterial strain to be expanded medicine source Microbial resources and has become important subject.

The microbial genome result of study showed in recent years, the contained secondary metabolite intercrescence of microbial genome sequence becomes gene cluster or gene many (such as document S.D.Bentley more than the product of its known production, et al.Complete genome sequence of the modelactinomycete Streptomyces coelicolor A3 (2) .Nature, 2002,417:141-147; Document H.Ikeda, et al.Completegenome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis.NatBiotechnol, 2003,21 (5): 526-531; Document M.Oliynyk, et al.Complete genome sequence of theerythromycin-producing bacterium Sacchropolyspora erthraea NRRL23338.Nat Biotechnol, 2007 (4): 447-453; Document Y.Ohnishi, et al.Genome sequence of the streptomycin-producing microorganismStreptomyces griseus IFO 13350.J Bcteriol, 2008,190 (11): 4050-4060 etc. put down in writing), thereby the potential ability that possesses synthetic more times grade product is (referring to document S.Donadio, et al.Impact of the first Streptomyces genome sequence on thediscovery and production of bioactive substances.Appl Microbiol Biotechnol, 2002,60:377-380 and document H.B.Bode, et al.The impact of bacterial genomics on natural product research.Angew Chem Int Ed, 2005,44 (12): associated viscera that 6828-6846 records and narrates).Therefore, the non-activity bacterial strain has the genome basis that changes into active bacterial strain, as long as the method that adopts is proper, might realize effectively transforming and therefrom finding medicine source activity compound fully, thereby expand the new strain resource of medicine source microorganism.This also just following non-activity actinomycetes have the genome basis that sensitization transforms potential, the ribosome engineering technology then is a kind of simple and effective means that excite this potential of actinomycetes.

The microorganism rrna has the function of regulation and control secondary metabolism, by intervening and changing complete these functions of ribose, can transform the microbial secondary metabolism, thus the relevant useful potential of digging utilization microorganism.Ribosome engineering is (referring to document K.Ochi, et al.Ribosome engineeringand secondary metabolite production.Advan Appl Microbiol, 2004,56:155-184 and document Ochi K.From microbial differentiation to ribosome engineering.Biosci Biotechnol Biochem, 2007,71 (6): the 1373-1386 record) correlative study shows, non-activity actinomycetes wild strain can by antibiotics resistance screening be converted into active mutant strain (referring to document " Yu Zhibin; etc. utilize the research of ribosome engineering technology developing marine microorganism medicine resource. hi-tech communication; 2005; 15 (5): 87-90 " and document " Sun Yuwen; etc. the screening of the separation and Culture of marine microorganism and wild-type thereof and mutant anti-tumor activity bacterial strain. institute of Military Medical Science Institute prints; 2008,32 (6): 532-536 " associated viscera); can be used for mutant strain and newly produce medicine source activity Study on product and transform the active mutant strain obtain; thus the new strain resource of efficient extn microorganism medicine source activity is (referring to document " Yu Zhibin, Deng. with the research of ribosome engineering technology secondary development marine microorganism strain resource. the hi-tech communication, 2006,16 (11): 1190-1194 " and document " Han Xiao, Deng. the ribosome engineering transformation of actinomycetes wild strain metabolic function and the new anti-tumor activity Study on product of producing. Inpharm research magazine, 2009,36 (6): 435-442﹠amp; 446 " associated viscera in).

Fungi is that ribosome engineering is studied so far a still untouched quasi-microorganism, not only with rrna function Relative Fungi ribosome engineering research so far there are no bibliographical information, and conversion and screening that the sensitization of relevant non-activity fungi wild strain transforms the experimental technique method of the especially relevant transformation fungal secondary metabolic function that the present invention relates to of correlative study and is used for non-activity fungi wild strain are obtained antitumor, disease-resistant fungal pathogens isoreactivity mutant strain and be there is not yet bibliographical information for the research of screening medicine source activity product.

Summary of the invention:

The purpose of this invention is to provide a kind of effective relevant experimental technique of epiphyte secondary metabolism function reformation and/or method and uses thereof.The inventor once attempted adopting conventional ribosome engineering experimental technique method, selected to act on ribosomal various microbiotic, fungi is explored carried out the relevant resistance screening experiment of transformation secondary metabolism function.But because the special construction of cell walls, act on ribosomal microbiotic and can't enter into site of action through fungal cell wall, therefore utilize conventional ribosome engineering resistant screening methods, can't screen at all and obtain the fungi antibiotics resistance mutant strain that the secondary metabolism gain-of-function is transformed.

On this basis, the present invention is freezing by system's examination, high temperature, ultrasonic, microwave, pH, ethyl acetate, acetone, DMSO, methyl alcohol, ethanol, the multiple Physicochemical factor such as butanols is on impact and the combination chain mycin of fungal cell wall membrane permeability, paraxin, Liu Suanyan NEOMYCIN SULPHATE, Rifampin, gentamicin, Multiple Classes of Antibiotics and/or the nitrosoguanidines (NTG) such as kantlex, ethyl sulfate (DES), ultrasonic, microwave, the acting in conjunction of the multiple physical chemistry mutagen such as ultraviolet is on the impact of fungal secondary metabolism, but groped the combination experiment condition of artificial reforming fungal secondary metabolism, and set up the antibiotics resistance screening of DMSO mediation, the experimental technique method of fungal secondary metabolic function is transformed in the artificial chemistry mutagenesis of DMSO mediation etc.Simultaneously, utilize this experimental technique method, success transforms non-activity fungi wild strain, the bioactive active mutant strains such as that efficient screening has obtained to have is antitumor, disease-resistant fungal pathogens, research has been illustrated active mutant strain and has newly been produced active result, and in this process, study and set up by directly comparing with original bacterium tunning, follow the tracks of fast the separating-purifying preparation method that the isolating active mutant strain newly produces active result.Result of study of the present invention shows, the relevant experimental technique method of transforming the fungal secondary metabolic function of the present invention can be used for effectively transforming the secondary metabolism function of non-activity fungi wild strain, and therefrom Efficient Conversion obtains medicine source activity mutant strain, thereby the new strain resource of efficient extn fungi medicine source activity, illustrate mutant strain by system simultaneously and newly produce active result, can also deeply develop the new product of fungi medicine source activity.The present invention is based on above-mentioned discovery and be accomplished.

Put it briefly, the invention provides the following:

The method of the transformation fungal secondary metabolic function of project 1, a kind of DMSO mediation, it is characterized in that, in DMSO solution, processed original fungi and the microbiotic that can act on microorganism rrna performance anti-microbial effect were interacted 0.5～200 day (for example 0.5～200 day, for example 0.5～180 day, for example 0.5～150 day, for example 0.5～125 day, for example 0.5～100 day, for example 0.5～90 day, for example 1～90 day, for example 1～75 day, for example 1～50 day, for example 1～40 day, for example 1～30 day; For example 0.5～200 day, for example 1～180 day, for example 2～150 days, for example 5～120 days, for example 5～100 days, for example 5～75 days, for example 5～50 days, for example 5～30 days) during, and in during this period in good time sampling (for example, the sampling interval can be 1 hour～20 days or 1h～30 day, for example about 1h, about 5h, about 10h, approximately 15 hours, approximately 20 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 5 days, approximately 10 days, approximately 15 days, approximately 20 days, about 30 days interval), select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

The method of the transformation fungal secondary metabolic function of project 2, a kind of DMSO mediation is characterized in that, in DMSO solution, with original fungi and chemical mutagen interact 0.5～200 day (for example 0.5～200 day, for example 0.5～180 day, for example 0.5～150 day, for example 0.5～125 day, for example 0.5～100 day, for example 0.5～90 day, for example 1～90 day, for example 1～75 day, for example 1～50 day, for example 1～40 day, for example 1～30 day; For example 0.5～200 day, for example 1～180 day, for example 2～150 days, for example 5～120 days, for example 5～100 days, for example 5～75 days, for example 5～50 days, for example 5～30 days) during, and in during this period in good time sampling (for example, the sampling interval can be 1 hour～20 days or 1h～30 day, for example about 1h, about 5h, about 10h, approximately 15 hours, approximately 20 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 5 days, approximately 10 days, approximately 15 days, approximately 20 days, about 30 days interval), select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

Project 3, a kind of DMSO mediation by fungi (fungi wild strain for example, the fungi wild strain of non-activity for example) transforms to obtain the method for the active mutant strain of anti-tumor pathogenic fungi, it is characterized in that, in DMSO solution, with described fungi (fungi wild strain for example, the fungi wild strain of non-activity for example) interacted 0.5～200 day (for example 0.5～200 day with the microbiotic and/or the chemical mutagen that act on microorganism rrna performance anti-microbial effect, for example 0.5～180 day, for example 0.5～150 day, for example 0.5～125 day, for example 0.5～100 day, for example 0.5～90 day, for example 1～90 day, for example 1～75 day, for example 1～50 day, for example 1～40 day, for example 1～30 day; For example 0.5～200 day, for example 1～180 day, for example 2～150 days, for example 5～120 days, for example 5～100 days, for example 5～75 days, for example 5～50 days, for example 5～30 days) during, and in during this period in good time sampling (for example, the sampling interval can be 1 hour～20 days or 1h～30 day, about 1h for example, about 5h, about 10h, approximately 15 hours, approximately 20 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 5 days, approximately 10 days, approximately 15 days, approximately 20 days, about 30 days interval), select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain of secondary metabolism gain-of-function transformation, through the anti-tumor pathogenic fungi screening active ingredients of mutant strain, obtain the mutant strain with anti-tumor pathogenic fungi activity again.

Project 4, project 1 to 3 each described method, wherein said DMSO solution is that (for example DMSO percent by volume (%, v/v) is 2～100% or is 5～100% or is 10～100% or is 15～100% or is 20～100% or is 25～100% or is 30～95% or is 30～90% or is 35～90% or is 40～90% or is 50～90% or is 60～90% or is 70～90% the DMSO aqueous solution DMSO aqueous solution; Perhaps for or be 5～90% or be 10～80% or be 15～75% or be 20～70% or be 25～60% or be 30～50% the DMSO aqueous solution), the described microbiotic that acts on microorganism rrna performance anti-microbial effect is to be selected from Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, gentamicin, paraxin, the Rifampin one or more, and described chemical mutagen is to be selected from ethyl sulfate, the nitrosoguanidine one or more.

Project 5, the fast separating and purifying preparation method that the fungi mutant strain of secondary metabolism function through transforming that project 1 to 4 each described method obtains newly produces active result, it is characterized in that, original bacterium and active mutant strain thereof ferment respectively, (for example the acetone percent by volume is 30～95% with aqueous acetone solution to the gained fermented product, or be 35～90%, or be 40～85%, or be 50～85%, or be 55～80%, or be 60%～80% aqueous acetone solution) extract, then to this aqueous acetone extracting solution reclaim under reduced pressure acetone, then with residual water layer ethyl acetate extraction, obtain respectively original bacterium acetic acid ethyl ester extract and contain the mutant strain acetic acid ethyl ester extract that mutant strain newly produces active result, then all adopt active the tracking with chemical detection to analyze combination to each step lock out operation of mutant strain acetic acid ethyl ester extract, the experiment model of trace prerun guide → amplification test preparation, by mutant strain tunning and original bacterium tunning are directly compared, determine fast that in micro-prerun mutant strain newly produces on the basis of the TLC spot of active result or HPLC elution peak, amplification test under TLC or HPLC guidance or detection, sharp separation prepares mutant strain and newly produces active result.

The fungi activity mutant strain of secondary metabolism function through transforming that project 6, project 1 to 4 each described method obtain newly produces medicine source activity compound for the preparation of mutant strain purposes.

Project 7, project 1 to 4 each described method be in the secondary metabolism function that is used for transforming non-activity fungi wild strain, thus with non-activity fungi wild strain as the purposes of original bacterium resource with the new strain resource of efficient extn medicine source activity.

The mutant strain of secondary metabolism function through transforming that project 8, project 1 to 4 each described method obtain.Perhaps, the secondary metabolism function of project 8 ', project 1 to 4 each described method acquisition is through the mutant strain of improvement.

Formula I compound or formula II compound that the mutant strain of each described method of whole embodiment of project 9, specification sheets, process, test conditions, detection method, testing conditions, bacterial strain uses therefor, acquisition, secondary metabolite and project 1 to 4 each described method obtain.In one embodiment, the formula I compound that obtains of the mutant strain of described method, process, test conditions, detection method, testing conditions, bacterial strain uses therefor, acquisition, secondary metabolite and project 1 to 4 each described method or formula II compound are as described in the specification sheets.

The below is further described with characteristics to various aspects of the present invention.

All documents that the present invention quotes from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this these terms and phrase to be described in more detail and to explain, the term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.

First aspect of the present invention relates to the experimental technique method of the transformation fungal secondary metabolic function of DMSO mediation, it is characterized in that, in DMSO solution, processed original fungi and the microbiotic that acts on microorganism rrna performance anti-microbial effect were interacted 1～90 day, and during in good time the sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

Second aspect of the present invention relates to the experimental technique method of transforming the fungal secondary metabolic function by the artificial chemistry mutagenesis of DMSO mediation, it is characterized in that, in DMSO solution, original fungi and chemical mutagen were interacted 1～90 day, and during in good time the sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

What the 3rd aspect of the present invention related to DMSO mediation transforms the experimental technique method of obtaining the active mutant strain of anti-tumor pathogenic fungi by non-activity fungi wild strain, it is characterized in that, in DMSO solution, non-activity fungi wild strain and the microbiotic or the chemical mutagen that act on microorganism rrna performance anti-microbial effect were interacted 1～90 day, and during in good time the sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed, through the anti-tumor pathogenic fungi screening active ingredients of mutant strain, obtain the related activity mutant strain again.

The 4th aspect of the present invention relates to be implemented the necessary key factor of the successful modification of fungal secondary metabolic function, wherein, DMSO solution described in the experimental technique method of above-mentioned transformation fungal secondary metabolic function is that DMSO content is the DMSO aqueous solution of 20%～100% (v/v), the described microbiotic that acts on microorganism rrna performance anti-microbial effect is Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, gentamicin, paraxin, Rifampin, and described chemical mutagen is ethyl sulfate, nitrosoguanidine.

The fast separating and purifying preparation method that the fungi mutant strain that the 5th aspect of the present invention relates to the transformation of secondary metabolism gain-of-function newly produces active result, it is characterized in that, original bacterium and active mutant strain thereof ferment respectively, through the acetone percent by volume to the gained fermented product be 60%～80% aqueous acetone extract and to aqueous acetone extracting solution reclaim under reduced pressure acetone after the ethyl acetate extraction of residual water layer, obtain respectively original bacterium acetic acid ethyl ester extract and contain the mutant strain acetic acid ethyl ester extract that mutant strain newly produces active result, after this all adopt active the tracking with chemical detection to analyze combination to each step lock out operation of mutant strain acetic acid ethyl ester extract, the experiment model of trace prerun guide → amplification test preparation, by mutant strain tunning and original bacterium tunning are directly compared, determine fast that in micro-prerun mutant strain newly produces on the basis of the TLC spot of active result or HPLC elution peak, use separation means, amplification test under TLC or HPLC guidance, sharp separation prepares mutant strain and newly produces active result.Described separation means comprises the known liquid-liquid extraction of the professional person in natural product chemistry field, column chromatography, thin-layer chromatography, high performance liquid chromatography and recrystallization etc.

The fungi activity mutant strain that the 6th aspect of the present invention relates to the transformation of described secondary metabolism gain-of-function newly produces medicine source activity compound for the preparation of mutant strain purposes.

The 7th aspect of the present invention relates to the secondary metabolism function that described relevant experimental technique of epiphyte secondary metabolism function reformation method is used for transforming non-activity fungi wild strain, thereby with the purposes of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The above-mentioned either side according to the present invention, the concentration of wherein said microbiotic in described DMSO solution is 0.01～20% (w/v), for example be 0.02～18% (w/v), for example be 0.02～15% (w/v), for example be 0.02～12% (w/v), for example being 0.05～12% (w/v), for example is 0.1～12% (w/v).In one embodiment, described microbiotic is Streptomycin sulphate; In one embodiment, described microbiotic is Streptomycin sulphate, and its concentration in described DMSO solution is 0.1～15% (w/v), for example is 0.15～10% (w/v).In one embodiment, described microbiotic is Liu Suanyan NEOMYCIN SULPHATE; In one embodiment, described microbiotic is Liu Suanyan NEOMYCIN SULPHATE, its concentration in described DMSO solution is 0.1～10% (w/v), for example be 0.1～5% (w/v), for example be 0.1～2% (w/v), for example being 0.1～1% (w/v), for example is 0.2～0.67% (w/v).In one embodiment, described microbiotic is kantlex; In one embodiment, described microbiotic is kantlex, and its concentration in described DMSO solution is 0.01～20% (w/v), for example is 0.01～15% (w/v), for example being 0.015～15% (w/v), for example is 0.02～12% (w/v).In one embodiment, described microbiotic is gentamicin; In one embodiment, described microbiotic is gentamicin, and its concentration in described DMSO solution is 0.01～20% (w/v), for example is 0.02～15% (w/v), for example being 0.03～12% (w/v), for example is 0.05～10% (w/v).

The above-mentioned either side according to the present invention, the concentration of wherein said chemical mutagen in described DMSO solution is 0.01～20% (w/v), for example be 0.05～15% (w/v), for example be 0.1～10% (w/v), for example be 0.2～5% (w/v), for example being 0.5～5% (w/v), for example is 0.5～4% (w/v), for example is 0.5～2% (w/v).In one embodiment, described microbiotic is ethyl sulfate; In one embodiment, described microbiotic is ethyl sulfate, and its concentration in described DMSO solution is 0.1～10% (w/v), for example is 0.2～5% (w/v), for example is 0.5～2% (w/v).

The present invention adopts Leukemia K562 cell, with the mtt assay test evaluation anti-tumor activity of original bacterium and mutant strain fermented extracted sample and gained compound, employing separates the pathogenic fungi clinical separation strain Q-1 that obtains from intractable fungi infestation person disease sites, W-1, Y-1 and Candida albicans type strain, with the test evaluation of 8mm paper disk method the anti-mycotic activity of original bacterium and mutant strain fermented extracted sample and gained compound, and through experiment confirm, transforming the active mutant strain sample obtain by non-activity fungi wild strain and newly produce active compound with experimental technique of the present invention has significantly antitumor or disease-resistant fungal pathogens active.Thereby experiment confirm, the fungal secondary metabolic function transformation experimental technique of DMSO mediation of the present invention can be used for conversion and the screening of non-activity fungi wild strain to be obtained antitumor, disease-resistant fungal pathogens isoreactivity mutant strain and supplies medicine source activity Study on product, thereby has the purposes of the new strain resource of efficient extn fungi medicine source activity.

As used herein, term " secondary metabolism " has implication well known in the art, for example refer to certain growth period (for example being in the stable growth phase), the vital movement to microorganism itself that microorganism is synthesized take primary metabolite as precursor does not have the process of the material of clear and definite function.As used herein, term " original fungi " has implication well known in the art, for example refers to, include but not limited to, fungi known, unknown fungi, from the fungi of natural origin, through the fungi of artificial mutation, activity fungal, non-activity fungi etc. are arranged; With respect to for the fungi mutant strain of the inventive method through transforming, described " original fungi " can be understood as is the precursor of this mutant strain.

Method of the present invention can be effectively be used for from a kind of fungi for example the non-activity fungi be transformed into the available fungi of industry.

Embodiment:

The following example will further specify the present invention, but the present invention will not be construed as limiting.

In following examples, hereinafter referred to as the bacterial strain of G59 be from pick up from tideland, Bohai Sea Gulf, donkey coltfoal river, Tanggu, Tianjin ooze sample (on September 29th, 2004 early morning 4～7 o'clock take advantage of to ebb tide to depth approximately gather the tideland of 5km) the marine source fungi wild strain that separates; The bacterial strain that is called H4 is the Lu Sheng fungi wild strain that separates from picking up from the inner kiln earth sample of remote mountain areas, Badong County, Hubei Province brickkiln (gathering in November, 2005).Described G59 bacterial strain is preserved in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 30th, 2009, China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number is CGMCC No.3560, the Classification And Nomenclature of suggestion is penicillium purpurogenum, Penicillium purpurogenum.Described H4 bacterial strain is preserved in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 30th, 2009, China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number is CGMCC No.3559, the Classification And Nomenclature of suggestion is golden grey mould, Penicillium aurantiogriseum.

In following examples, form hereinafter referred to as the substratum of fungi PDA substratum and to be: glucose 2%, agar 2%, NaCl 1.5%, with the water cooking liquid preparation of 200g/L potato, transfer pH 6.0; Substratum composition hereinafter referred to as the fungi liquid substratum is: glucose 2%, maltose 1%, N.F,USP MANNITOL 2%, L-glutamic acid 1%, peptone 0.5%, yeast soak powder 0.3%, with the water cooking liquid preparation of 200g/L potato, transfer pH 6.0.

The streptomycin resistant mutation strain screening of embodiment 1.G59 and secondary species detect to be analyzed

The collecting cells of fungi original strain G59

Get original bacterium G59 an amount of, be inoculated on the PDA solid medium, cultivate activation 3～10 days for 28 ℃, treat sporulation, the scraping spore is an amount of, places the 50mL triangular flask that 10mL sterilized water and granulated glass sphere are housed, and dispersal spore is smashed in concussion, spore suspension.Get this suspension 100 μ L, place 96 orifice plates, the OD value with under the microplate reader monitoring 600nm wavelength is diluted to the OD value with sterilized water and reaches 0.35.Extension rate according under the microplate reader monitoring with the same doubling dilution of all spores suspension, obtains the bacteria suspension of original bacterium G59 with sterilized water.

The streptomycin resistance screening of DMSO mediation

Bacteria suspension with original bacterium G59, add Streptomycin sulphate and DMSO is an amount of, respectively preparation contain Streptomycin sulphate 2,5,10,50mg/mL 20% (v/v) DMSO the G59 bacteria suspension and contain respectively the G59 bacteria suspension of 50% (v/v) DMSO of Streptomycin sulphate 0.15,0.2,0.3,0.6,1,2,10,100mg/mL, make processed group of mixed solution.Other gets the bacteria suspension of an amount of original bacterium G59, adds DMSO an amount of, and preparation does not contain 20% (v/v) of Streptomycin sulphate and the G59 bacteria suspension of 50% (v/v) DMSO respectively, makes processed control group mixed solution.In addition, the bacteria suspension of an amount of original bacterium G59 is used respectively and the dilution of the sterilized water of used DMSO equal volume, preparation does not contain the bacteria suspension of Streptomycin sulphate and DMSO, makes blank group mixed solution.Place 4 ℃ of refrigerators to process 1～30 day these mixed solutions, during respectively get 90 μ L in good time, coat respectively on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Fermentation culture and extraction preparation

The fermentation culture of original bacterium G59 and mutant strain thereof all adopts the fungi liquid fermention medium.Each bacterial strain is inoculated in respectively in the 500mL Erlenmeyer flask that contains 200mL fungi liquid substratum in right amount, cultivated 10～13 days in 28 ℃, 200rpm shaker fermentation, stop cultivating according to the microorganism growth situation in good time, lower shaking table gets fermented liquid.The acetone that adds 2 times of fermentating liquid volumes in the fermented liquid, make the volumn concentration of acetone reach approximately 66%, the ultrasonication thalline, the centrifuging and taking supernatant liquor, be evaporated to and do not contain acetone, with equal volume EtOAc extraction, extraction liquid reclaims EtOAc through concentrating under reduced pressure, lyophilize gets the fermented extracted sample of G59 and mutant strain thereof.

Mutant strain and original bacterium form are relatively

Original bacterium G59 and mutant strain thereof are distinguished streak inoculation on the PDA plate culture medium, cultivated 4～8 days in 28 ℃ of incubators, form and the external appearance characteristics such as visual inspection colony shape, thalline color and luster, chromogenesis color and infiltration substratum situation thereof.

The thin-layer chromatography of secondary species detects to be analyzed

The sample solution preparation: the fermented extracted sample that takes by weighing G59 and mutant strain thereof is an amount of, is made into respectively the sample solution of 10mg/mL with methyl alcohol, detects for secondary species and analyzes.

The thin-layer chromatography condition: thin-layer chromatography adopts Qingdao Marine Chemical Co., Ltd. to produce silica gel G F ₂₅₄(20cm * 10cm * 0.25mm), spot employing 254 or 365nm uviolizing, cerous sulfate ammonium molybdate reagent spray heating or spray the iron trichloride reagent colour development and detect thin layer plate.The point sample above-mentioned 10mg/mL methanol solution of G59 and mutant strain fermented extracted sample thereof, developping agent adopts respectively hexanaphthene-acetone (3: 1,1: 1,1: 2), chloroform-methanol (20: 1,9: 1,8: 2,7: 3), 60～90 ℃ of sherwood oil-acetone of bp (5: 1,2: 1,1: 1,1: 3), 60～90 ℃ of sherwood oil-chloroforms of bp (4: 1,2: 1,1: 1,1: 2), chloroform-acetone (9: 1,7: 1,5: 1,1: 1) or chloroform-methanol-ammoniacal liquor (27: 6: 1,90: 10: 2) etc.

The chromatography experiment method: each thin layer plate all adopts same capillary point sample, the G59 of equal volume and mutant strain fermented extracted sample solution thereof on the point, the sample that each thin layer plate is all put original bacterium G59 in contrast, the solvent of waiting the sample solution spot of putting fully volatilizes, place with corresponding developping agent presaturation and the chromatography cylinder of an amount of developping agent is housed, adopt tiltedly vertical thin layer plate expansion mode to carry out thin layer chromatography and separate.Launch to finish at once to take out thin layer plate, treat that solvent volatilizes, adopt above-mentioned colour developing mode to detect the thin layer spot, whether the difference of secondary species thin layer spot between each mutant strain of comparative analysis and original bacterium G59 and each mutant strain determines whether the new parity level of mutant strain product spot and is the same blob that duplicate detection arrives between the different mutant strains.

Experimental result

The screening of G59 streptomycin resistant mutation strain is obtained: in processed control group and the experiment of blank group, the G59 growth is not suppressed, and grows vigorous and full dull and stereotyped in flakes growth, but does not form the bacterium colony of picking.In contrast, in processed group of experiment, processed bacteria growing is subject to obvious inhibition, forms several limited, bacterium colonies that form is different, can picking list bacterium colony.Therefore, streptomycin resistance screening experiment under different volumes percentage ratio DMSO mediation, screening has obtained streptomycin resistant mutation strain totally 112 strains of G59,46 strains that 66 strains, 20% (v/v) the DMSO mediation of wherein obtaining by 50% (v/v) DMSO mediation is obtained, concrete outcome sees table 1 for details.

The DMSO mediation streptomycin resistant mutation strain screening statistics of table 1G59

Mutant strain and original bacterium form, external appearance characteristic: no matter institute's mutant strain that obtains is still compared with original bacterium G59 between mutant strain, form, surface shape and the color of the bacterium colony that forms, the thalline color and luster, producing the mode of appearance features such as pigmentary colours and infiltration substratum situation thereof all has notable difference.

The thin-layer chromatography of fermented extracted sample detects: through the thin-layer chromatographic analysis to 112 plant mutant strains and original bacterium G59 fermented sample, from most mutant strain fermented sample, detect the secondary species spot that does not have in the original bacterium G59 fermented sample, especially, from 9 strain anti-mycotic activity mutant strain fermented samples described in the active anti-tumor activity mutant strain of 34 strain anti-tumor activity mutant strains described in the following embodiment 2 especially 17 plant heights and the embodiment 3, all detect secondary species spot that do not have and that between each mutant strain sample, present different chromatography behaviors in the original bacterium G59 fermented sample.

Above experimental result shows, the streptomycin resistance screening of the G59 of above-mentioned DMSO mediation has changed the secondary metabolism of most mutant strain that obtains, make secondary species that variation occur, thereby experiment confirm, streptomycin resistance screening experiment technology of the present invention can be used for the secondary metabolism function of artificial reforming fungi.

The screening of the streptomycin resistance anti-tumor activity mutant strain of embodiment 2.G59 is obtained

Experimental strain

The streptomycin resistant mutation strain of the 112 strain G59 that original bacterium G59 and the streptomycin resistance screening experiment that mediates by DMSO in above-described embodiment 1 obtain.

Fermentation culture and sample preparation

Fermentation culture and sample extraction preparation: original bacterium G59 and its mutant strain thalline are inoculated in respectively in the 500mL Erlenmeyer flask that contains 200mL fungi liquid substratum in right amount, in 28 ℃, 200rpm shaker fermentation 10～13 days, stop cultivating according to the microorganism growth situation, lower shaking table gets fermented liquid in good time.The acetone (volumn concentration of acetone reach approximately 66%) that adds 2 times of fermentating liquid volumes in the fermented liquid, the ultrasonication thalline, the centrifuging and taking supernatant liquor, be evaporated to and do not contain acetone, extract with equal volume EtOAc, extraction liquid reclaims EtOAc through concentrating under reduced pressure, and lyophilize gets the fermented extracted sample of G59 and mutant strain thereof.

The active testing preparation of sample solution: the fermented extracted sample of precision weighing G59 and mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL and 10mg/mL with methyl alcohol, for the screening anti-tumor activity.

The antitumor activity screening experimental technique

Cell cultures and the maintenance of going down to posterity: the human leukaemia K562 cell passes into 5%CO with the RPMI-1640 substratum that contains each 100 μ g/mL of 10% foetal calf serum and penicillin and Streptomycin sulphate in 37 ℃ ₂With cellar culture in the incubator of 95% air and the maintenance of going down to posterity.

Activity test method: collect the K562 cell of logarithmic phase, with fresh RPMI-1640 substratum, being mixed with cell density is 2 * 10 ⁵The cell suspension of individual/mL is inoculated in 96 orifice plates, every hole 200 μ L.With postvaccinal cell in 37 ℃, pass into 5% CO ₂After cultivating 4h in the cell culture incubator of 95% air, every hole adds 2 μ L sample solutions, and each sample solution is established three parallel holes, establishes simultaneously three hole negative controls.Continue to cultivate 24h after the dosing, cultivating the cellular form that at first causes after optical microphotograph Microscopic observation drug treating after finishing changes, judge and have or not the morphological specificitys such as apoptosis, necrocytosis or cell abnormity, take pictures in case of necessity, then every hole adds 20 μ LMTT solution (the MTT solution of the 5mg/mL of the PBS liquid preparation of usefulness 0.01M), continues to place 37 ℃ of incubators to hatch 4h.The centrifugal 10min of 2000r/min sucks supernatant liquor in the hole.Every hole adds 150 μ L DMSO, vibrates 10 minutes, and crystallisate is fully dissolved, and utilizes microplate reader to measure the OD value of every hole solution at the 570nm place.

Blank and sample sets are got the average OD value in three holes respectively, are calculated as follows the inhibiting rate of sample on cell proliferation: inhibiting rate %=(OD _Blank-OD _Sample)/OD _Blank* 100%.

In case of necessity, identical test is independently carried out three times respectively, asks the sample of calculation different concns to the average inhibiting rate of K562 cell proliferation, and recycling Bliss method is calculated by the half-inhibition concentration IC of test agent to the K562 cell ₅₀

Experimental result

The active testing result of original bacterium sample: even original bacterium G59 sample does not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL, 1000 μ g/mL and 100 μ g/mL samples are respectively 6.9% and 4.8% in the experimental error scope to the inhibiting rate of K562 cell.Through repeatedly going down to posterity for several times, the screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, G59 sample 1000 μ g/mL or 100 μ g/mL all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell.

The screening active ingredients statistics of mutant strain sample: the 112 plant mutant strains of obtaining have been carried out repeatedly going down to posterity repeatedly with fungi PDA substratum respectively, and each mutant strain is adopted respectively the bacterial strain of different passage numbers, from fermentation culture and sample extraction preparation, the whole mutant strains of 112 strains that obtain have all independently been carried out respectively antitumor activity screening repeatedly.Through to the mutant strain of different passage numbers through the repeatedly anti-tumor activity repeated screening of 112 plant mutant strain samples of independent fermented extracted preparation, the stable active mutant strain that reappears of anti-tumor activity has 34 strains, wherein 100 μ g/mL samples to active mutant strain 17 strains between 30%～40% of the inhibiting rate of K562 cell, inhibiting rate greater than 40% high reactivity mutant strain 17 strains.In the active mutant strain of 34 strains, the active mutant strain that the streptomycin resistance screening experiment that mediates by 50% (v/v) DMSO obtains has 14 strains, the active mutant strain that the streptomycin resistance screening experiment that mediates by 20% (v/v) DMSO obtains has 20 strains, and concrete statistics sees table 2 for details.

Table 2 streptomycin resistant mutation strain sample is to the antitumor activity screening statistics of K562 cell

Microscopically cellular form detected result: microscopically observes, the K562 cell after original bacterium G59 sample 1000 μ g/mL and 100 μ g/mL or non-activity mutant strain sample 100 μ g/mL process 24 hours cellular form without noticeable change, be the morphological specificity identical with the blank cell, show these samples on the K562 cell without impact.

And after active mutant strain sample especially high reactivity mutant strain sample 100 μ g/mL process 24 hours, the part cell of some sample preparation is compared form generation noticeable change with the blank cell, the typical gangrenosum acne cell morphological characteristics such as some is that cell space expands, cytolemma dimly expands, tenuigenin is condensing, the part after birth breaks, some is the typical apoptotic cell morphological specificitys such as cell debris that fragmentary after birth wrapping up in or snowflake flap, some then is bar-shaped or the special-shaped shape such as 23 cell paste, coincide with following mtt assay active testing result.

The mtt assay active testing result of active mutant strain: through antitumor activity screening repeatedly, anti-tumor activity reappears stable active mutant strain and has 34 strains in 112 plant mutant strains, wherein 100 μ g/mL samples to the inhibiting rate of K562 cell greater than 40% 17 strains of high reactivity mutant strain, active mutant strain 17 strains of inhibiting rate between 30%～40%.In the 34 strain anti-tumor activity mutant strains that obtain, inhibiting rate greater than the active mutant strain sample of 40% 17 plant heights 100 μ g/mL to the inhibition active testing of K562 cell the results detailed in Table 3, the active mutant strain sample of 17 strains of inhibiting rate between 30%～40% 100 μ g/mL to the inhibition active testing of K562 cell the results detailed in Table 4.

Table 3 G59 and mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

Table 4 G59 and mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

Thin-layer chromatography detection analytical results to secondary species in above experimental result and above-described embodiment 1 shows, the active mutant strain of above-mentioned 34 strains metabolism produced non-activity the original fungi wild strain of marine source G59 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, streptomycin resistance screening experiment technological method of the present invention can successful modification marine source non-activity fungi wild strain metabolic function, and therefrom efficient screening obtains the anti-tumor activity mutant strain, thereby have the actual utility value of non-activity thalassiomycetes wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The screening of the active mutant strain of the disease-resistant fungal pathogens of the streptomycin resistance of embodiment 3.G59 is obtained

Experimental strain

The streptomycin resistant mutation strain of the 83 strain G59 that original bacterium G59 and the streptomycin resistance screening experiment that mediates by DMSO in above-described embodiment 1 obtain.

Fermentation culture and active testing sample preparation

Fermentation culture and sample extraction preparation: adopt experimental technique and experiment condition described in above-described embodiment 2, original bacterium G59 and 83 strain streptomycin resistant mutation strains thereof were fermented 10～13 days, get the fermented liquid of each bacterial strain, extract and the ethyl acetate extraction operation through aqueous acetone again, get the fermented extracted sample of original bacterium G59 and mutant strain thereof.

The preparation of active testing sample solution: the fermented extracted sample of precision weighing G59 and mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL, 50mg/mL, 10mg/mL and 5mg/mL with methyl alcohol, and is active for the disease-resistant fungal pathogens of screening.

Anti-mycotic activity screening experiment method

Tested pathogenic fungi: from pathogenic fungi clinical separation strain Q-1, W-1, Y-1 and the Candida albicans type strain ATCC10231 of intractable fungi infestation person disease sites separation.

The preparation of tested pathogenic fungi bacteria suspension: 1. under sterile state, the pathogenic fungi clinical separation strain of getting 28 ℃ of activation culture 3 days (W-1 and Y-1) or 7 days (Q-1) on the PDA substratum is an amount of, is made in right amount original suspension with sterilized water.Get original suspension 100 μ L, place 96 orifice plates, under the monitoring of the optical density(OD) (OD) at microplate reader 600nm place, with sterilized water be diluted to that Q-1 bacteria suspension OD value reaches 0.4, W-1 and Y-1 bacteria suspension OD value reach 0.2.According to the identical extension rate under the microplate reader monitoring, respectively the original suspension of remainder is all diluted with sterilized water again, prepare the bacteria suspension of tested pathogenic fungi clinical separation strain, for active testing.2. with Candida albicans type strain ATCC10231 28 ℃ of activation culture 3 days on the PDA substratum, get this bacterium thalline of an amount of activation culture, be mixed with bacteria suspension with an amount of sterilized water, for active testing.

Activity test method: the active 8mm paper disk method that adopts of disease-resistant fungal pathogens, test with the PDA solid medium.At first, get each 90 μ L of tested pathogenic fungi bacteria suspension, evenly coat on the PDA culture medium flat plate respectively, the PDA substratum of making tested pathogenic fungi detects dull and stereotyped.Then under aseptic condition, draw each 15 μ L of tested sample solution, inhale respectively and be stated from the 8mm filter paper, treat that solvent fully volatilizes, place the PDA substratum to detect on the flat board on the 8mm scraps of paper of adsorption sample, cultivate and observe at any time tested bacteria growing situation in 28 ℃.Q-1 cultivated 6 days, and W-1, Y-1 and Candida albicans were cultivated respectively 3 days, had or not the size of inhibition zone or inhibition zone according to visual inspection, and judgement sample has or not disease-resistant fungal pathogens active or active strong and weak.

Experimental result

The anti-mycotic activity test result of original bacterium sample: in active testing repeatedly, original bacterium G59 sample in the concentration range of trial 1500 μ g/ sheets～75 μ g/ sheets to all tested pathogenic fungies all without any anti-microbial activity, inhibition zone is zero.

The anti-mycotic activity screening statistics of mutant strain sample: through the disease-resistant fungal pathogens screening active ingredients of above-mentioned 83 plant mutant strain samples, 1500 μ g/ sheets are 7 strains that have that suppress active to Q-1, and W-1 and Y-1 are 3 strains that have that suppress active.Wherein, mutant strain G592DS20Z and G598DS20Z sample have remarkable inhibiting activity to Q-1, the 750 μ g/ scraps of paper are suitable to the effect of the restraining effect of Q-1 and the positive control nystatin 75 μ g/ scraps of paper, but when concentration is down to the 75 μ g/ scraps of paper, bacteriostatic action obviously die down (table 5).

Table 5 G59 and streptomycin resistant mutation bacterial strain sample thereof are to the inhibition active testing result of Q-1

The anti-mycotic activity test result of active mutant strain sample: through to the anti-mycotic activity screening of above-mentioned 83 plant mutant strain samples to pathogenic fungi clinical separation strain Q-1,7 plant mutant strain samples have in various degree anti-microbial effect to Q-1, the results are shown in Table 5.

Through the anti-mycotic activity of above-mentioned 83 plant mutant strain samples to pathogenic fungi clinical separation strain Y-1 and W-1 screened, 3 strains (wherein 1 strain also has restraining effect to Q-1) mutant strain sample has in various degree anti-microbial effect, result to see Table respectively 6 and table 7 to Q-1 and W-1.

Table 6 G59 and 3 strain streptomycin resistant mutation strain samples thereof are to the inhibition active testing result of Y-1

Table 7 G59 and 3 strain streptomycin resistant mutation strain samples thereof are to the inhibition active testing result of W-1

Thin-layer chromatography detected result to secondary species in above experimental result and above-described embodiment 1 shows, the active mutant strain of above-mentioned 9 strains of the present invention metabolism produced original bacterium G59 unproductive secondary metabolite with disease-resistant fungal pathogens activity, thereby experiment confirm, streptomycin resistance screening experiment technological method of the present invention can be transformed the metabolic function of non-activity fungi wild strain, and therefrom the active mutant strain of disease-resistant fungal pathogens is obtained in conversion, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The streptomycin resistance of embodiment 4.H4 and antitumor activity screening and secondary species detect to be analyzed

The collecting cells of fungi original strain H4

Get original bacterium H4 an amount of, be inoculated on the PDA solid medium, cultivate activation for 28 ℃, treat sporulation, according to same procedure described in above-described embodiment 1, preparation is monitored the bacteria suspension that the OD value is 0.43 original bacterium H4 with microplate reader 600nm, for following experiment.

The streptomycin resistance screening of DMSO mediation or acetone mediation

Bacteria suspension with original bacterium H4, add Streptomycin sulphate and DMSO is an amount of, preparation contains respectively the H4 bacteria suspension of 50% (v/v) DMSO of Streptomycin sulphate 0.3,0.6,1mg/mL, an amount of with the bacteria suspension of original bacterium H4 in addition, add Streptomycin sulphate and acetone is an amount of, preparation contains the H4 bacteria suspension of 50% (v/v) acetone of Streptomycin sulphate 0.6mg/mL, respectively as processed group of mixed solution.In addition, preparation does not contain 50% (v/v) DMSO of Streptomycin sulphate and the H4 bacteria suspension of 50% (v/v) acetone respectively, as processed control group mixed solution, the bacteria suspension with original bacterium H4 dilutes 1 times with the equal volume sterilized water in addition, as the blank mixed solution respectively.Place 4 ℃ of refrigerators to process 1～30 day these mixed solutions, during respectively get 90 μ L in good time, coat respectively on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Mutant strain and original bacterium form are relatively

Original bacterium H4 and mutant strain thereof are distinguished streak inoculation on the PDA plate culture medium, cultivated 4～8 days in 28 ℃ of incubators, form and the external appearance characteristics such as visual inspection colony shape, thalline color and luster, chromogenesis color and infiltration substratum situation thereof.

Fermentation culture and sample preparation

The fermentation culture of original bacterium H4 and mutant strain thereof all adopts the fungi liquid fermention medium.Described identical method under fermentation culture and the sample preparation item in employing and above-described embodiment 1 through fermentation culture and extraction preparation, gets the fermented extracted sample of H4 and mutant strain thereof.

The thin-layer chromatography of secondary species detects to be analyzed

The sample solution preparation: the fermented extracted sample that takes by weighing H4 and mutant strain thereof is an amount of, is made into respectively the sample solution of 10mg/mL with methyl alcohol, detects for secondary species and analyzes.

Thin-layer chromatography condition: identical with the thin-layer chromatography condition of putting down in writing in above-described embodiment 1.

Chromatography experiment method: adopt chromatography experiment method described in above-described embodiment 1, carry out Development of Thin-Layer Chromatography and thin layer spot color developing detection, whether the difference of secondary species thin layer spot between each mutant strain of comparative analysis and original bacterium H4 and each mutant strain determines whether the new parity level of mutant strain product spot and is the same blob that duplicate detection arrives between the different mutant strains.

The antitumor activity screening experimental technique

The preparation of anti-tumor activity test sample solution: the fermented extracted sample of precision weighing H4 and mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL and 10mg/mL with methyl alcohol, for the screening anti-tumor activity.

The anti-tumor activity testing method: adopt antitumor activity screening experimental technique described in above-described embodiment 2, use the human leukaemia K562 cell, specimen is active to the inhibition of K562 cell.

Disease-resistant fungal pathogens screening active ingredients experimental technique

The preparation of sample solution of disease-resistant fungal pathogens active testing: the fermented extracted sample of precision weighing H4 and mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL, 50mg/mL, 10mg/mL and 5mg/mL with methyl alcohol, and is active for the disease-resistant fungal pathogens of screening.

The anti-mycotic activity testing method: adopt anti-mycotic activity screening experiment method described in above-described embodiment 3, specimen is to the restraining effect of the 4 pathogen strain fungies of putting down in writing in above-described embodiment 3.

Experimental result

The screening of H4 streptomycin resistant mutation strain is obtained: in processed control group and the experiment of blank group, the G59 growth is not suppressed, and grows vigorous and full dull and stereotyped in flakes growth, but does not form the bacterium colony of picking.In contrast, in processed group of experiment, processed bacteria growing is subject to obvious inhibition, forms several limited, bacterium colonies that form is different, can picking list bacterium colony.Therefore, the streptomycin resistance screening through 50% (v/v) DMSO mediation obtains mutant strain totally 9 strains, and screens through the streptomycin resistance of 50% (v/v) acetone mediation, obtains mutant strain totally 6 strains, and concrete outcome sees table 8 for details.

The DMSO of table 8H4 or acetone mediation streptomycin resistant mutation strain the selection result

Form, the external appearance characteristic of mutant strain and original bacterium: institute's mutant strain that obtains is no matter between mutant strain or compare with original bacterium H4, form, surface shape and the color of the bacterium colony that forms, the thalline color and luster, producing the mode of appearance features such as pigmentary colours and infiltration substratum situation thereof all has notable difference.

The thin-layer chromatography of secondary species detects in the fermented extracted sample: through the thin-layer chromatographic analysis to 15 plant mutant strains and original bacterium H4 fermented sample, detect the secondary species spot that does not have in the original bacterium H4 fermented sample from a strain through the mutant strain CTH42DS600S fermented sample that the screening of 0.6mg/mL streptomycin resistance obtains under 50% (v/v) DMSO mediation.

Antitumor activity screening result: without antitumor action, 100 μ g/mL are 7.9% to the inhibiting rate of K562 cell to original bacterium H4 sample to trial K562 cell.In the 15 strain streptomycin resistant mutation strains of H4, the sample of mutant strain CTH42DS600S has obvious restraining effect to the K562 cell, 100 μ g/mL are 46.5% to the inhibiting rate of K562 cell, coincide with the thin-layer chromatography detected result of respective sample, and preparing the stable reproduction of result the repeatedly screening active ingredients that begins independently to carry out from fermentation culture and sample extraction.

The anti-mycotic activity the selection result: original bacterium H4 and 15 plant mutant strain samples thereof in the concentration range of trial 1500 μ g/ sheets～75 μ g/ sheets to all tested pathogenic fungies all without any anti-microbial activity, inhibition zone is zero.

Above experimental result shows, the streptomycin resistance screening of the Lu Sheng fungi wild strain H4 of above-mentioned DMSO mediation has changed the secondary metabolism of the active mutant strain that obtains, make the mutant strain metabolism produced original bacterium H4 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, streptomycin resistance screening experiment technological method of the present invention also can artificial reforming Lu Sheng fungi wild strain the secondary metabolism function, thereby non-activity Lu Sheng fungi wild strain had too with its actual utility value as the new strain resource of original bacterium resource efficient extn medicine source activity.

The neomycin resistance mutant strain screening of embodiment 5.G59 detects with secondary species to be analyzed

The collecting cells of fungi original strain G59

According to identical method described in above-described embodiment 1, preparation is monitored the bacteria suspension that the OD value is 0.35 original bacterium G59 with microplate reader 600nm, for following experiment.

The neomycin resistance screening of DMSO mediation

The DMSO solution of preparation 10mg/mL Liu Suanyan NEOMYCIN SULPHATE is got an amount of four parts of this solution, and 1: 4 by volume, 1: 2,1: 1,2: 1 respectively, mix with the bacteria suspension of original bacterium G59, preparation contains the processed mixed solution of the original bacterium G59 of different concns Liu Suanyan NEOMYCIN SULPHATE and DMSO.In each mixed solution of gained, contain respectively Liu Suanyan NEOMYCIN SULPHATE 2,3.3,5,6.7mg/mL, and the DMSO concentration expressed in percentage by volume is followed successively by 20%, 33%, 50%, 67%.Mix by volume ratio same as described above respectively with original bacterium G59 bacteria suspension with the DMSO that does not contain Liu Suanyan NEOMYCIN SULPHATE in addition, prepare the processed control group mixed solution that contains respective volume percentage concentration DMSO and do not contain Liu Suanyan NEOMYCIN SULPHATE, press the bacteria suspension mixed diluting of corresponding proportion and original bacterium G59 with sterilized water, preparation does not contain the blank group mixed solution that Liu Suanyan NEOMYCIN SULPHATE does not contain DMSO yet again.In addition, get 3 parts of an amount of equivalent of original bacterium G59 spore, 1 part of usefulness contains an amount of suspendible of DMSO solution of 10mg/mL Liu Suanyan NEOMYCIN SULPHATE, the processed group of mixed solution of 100% (v/v) DMSO that contains the 10mg/mL Liu Suanyan NEOMYCIN SULPHATE of preparation G59,1 part of DMSO suspendible that does not contain Liu Suanyan NEOMYCIN SULPHATE with equal volume in addition, preparation does not contain the processed control group mixed solution of 100% (v/v) DMSO of Liu Suanyan NEOMYCIN SULPHATE, last 1 part of sterilized water suspendible with equal volume makes and neither contains the blank mixed solution that Liu Suanyan NEOMYCIN SULPHATE does not contain again DMSO.Place 4 ℃ of refrigerators to process 1～90 day these mixed solutions, during at processed the 1st, 2,5,10,15,30,50,60,90 day each 90 μ L of sampling respectively, coat respectively on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Fermentation culture and extraction preparation

According to identical method described in above-described embodiment 1, through fermentation culture and the extraction preparation of original bacterium G59 and mutant strain thereof, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

Mutant strain and original bacterium form are relatively

The thin-layer chromatography of secondary species detects to be analyzed

Thin-layer chromatography condition: identical with condition described in above-described embodiment 1.

Chromatography experiment method: according to identical method described in above-described embodiment 1, thin-layer chromatography through original bacterium G59 and mutant strain sample thereof separates and thin layer spot color developing detection, whether the difference of secondary species thin layer spot between each mutant strain of comparative analysis and original bacterium G59 and each mutant strain determines whether the new parity level of mutant strain product spot and is the same blob that duplicate detection arrives between the different mutant strains.

Experimental result

The screening of G59 neomycin resistance mutant strain is obtained: only contain each processed control group of DMSO not containing Liu Suanyan NEOMYCIN SULPHATE and neither contain in each blank group experiment that Liu Suanyan NEOMYCIN SULPHATE do not contain DMSO yet, tested bacterium G59 is not suppressed in the growth of PDA plate culture medium, grow vigorous and full dull and stereotyped in flakes growth, but do not form the bacterium colony of picking.In contrast, in each the processed group experiment that contains Liu Suanyan NEOMYCIN SULPHATE and DMSO, processed bacteria growing is subject to obvious inhibition, in the isolated growth of PDA flat board, form several limited, bacterium colonies that form is different, select by single bacterium colony, can obtain the mutant strain to the growth of different concns neomycin resistance.Therefore, the neomycin resistance screening experiment under different volumes percentage ratio DMSO mediation, the neomycin resistance mutant strain that screening has obtained to resist the G59 of different concns Liu Suanyan NEOMYCIN SULPHATE growth amounts to 56 strains, and concrete outcome sees table 9 for details.

The DMSO mediation neomycin resistance mutant strain screening statistics of table 9 G59

The thin-layer chromatography of secondary species detects in the fermented extracted sample: through the thin-layer chromatographic analysis to 56 plant mutant strains and original bacterium G59 fermented sample, from most mutant strain fermented sample, detect the secondary species spot that does not have in the original bacterium G59 fermented sample, especially, from 8 strain anti-mycotic activity mutant strain fermented samples described in 41 strain anti-tumor activity mutant strains described in the following embodiment 6 and the embodiment 7, all detect secondary species spot that do not have and that between each mutant strain sample, present different chromatography behaviors in the original bacterium G59 fermented sample.

Above experimental result shows, the neomycin resistance screening of the G59 of above-mentioned DMSO mediation has changed the secondary metabolism of most mutant strain that obtains, make secondary species that variation occur, thereby experiment confirm, neomycin resistance screening experiment technological method of the present invention can be used for the secondary metabolism function of artificial reforming fungi.

The screening of the neomycin resistance anti-tumor activity mutant strain of embodiment 6.G59 is obtained

Experimental strain

The neomycin resistance mutant strain of the 56 strain G59 that original bacterium G59 and the neomycin resistance screening experiment that mediates by DMSO in above-described embodiment 5 obtain.

Fermentation culture and sample preparation

Fermentation culture and sample extraction preparation: according to identical method described in above-described embodiment 2, through fermentation culture and the extraction preparation of original bacterium G59 and mutant strain thereof, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

The antitumor activity screening experimental technique

Activity test method: according to identical method described in above-described embodiment 2, specimen is active to the inhibition of K562 cell.

Experimental result

The active testing result of original bacterium sample: described in above-mentioned embodiment 2, even original bacterium G59 sample does not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL, the screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, 1000 μ g/mL or 100 μ g/mL samples all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell.

The antitumor activity screening statistics of mutant strain sample: the 56 plant mutant strains of obtaining have been carried out repeatedly going down to posterity repeatedly with fungi PDA substratum respectively, and each mutant strain is adopted respectively the bacterial strain of different passage numbers, from fermentation culture and sample extraction preparation, the whole mutant strains of 56 strains that obtain have all independently been carried out respectively antitumor activity screening repeatedly.Warp is to the not anti-tumor activity repeated screening of 56 plant mutant strain samples, the stable active mutant strain that reappears of anti-tumor activity has 41 strains, and wherein 100 μ g/mL samples occupy active mutant strain 29 strains between 24%～50%, inhibiting rate greater than 50% high reactivity mutant strain 12 strains to the inhibiting rate of K562 cell.The screening of these active mutant strains is obtained statistics and is seen table 10 for details.

Table 10 neomycin resistance mutant strain sample is to the antitumor activity screening statistics of K562 cell

Microscopically cellular form detected result: microscopically observes, the K562 cell after original bacterium G59 sample 1000 μ g/mL and 100 μ g/mL or non-activity mutant strain sample 100 μ g/mL process 24 hours cellular form without noticeable change, be the morphological specificity identical with the blank cell, show these samples on the K562 cell without impact.And after active mutant strain sample especially high reactivity mutant strain sample 100 μ g/mL process 24 hours, the form of the part cell of some sample preparation is compared the generation noticeable change with the blank cell, the typical gangrenosum acne cell morphological characteristics such as some is that cell space expands, cytolemma dimly expands, tenuigenin is condensing, the part after birth breaks, some is the typical apoptotic cell morphological specificitys such as cell debris that fragmentary after birth wrapping up in or snowflake flap, some then is bar-shaped or the special-shaped shape such as 23 cell paste, coincide with following mtt assay active testing result.

The mtt assay active testing result of active mutant strain: 41 strains are for there being the active mutant strain of anti-tumor activity to the K562 cell in 56 plant mutant strains, wherein 100 μ g/mL sample inhibiting rates have 29 strains greater than 50% have 12 strains and inhibiting rate between 24%～50%, and 100 μ g/mL samples see Table respectively 11 and table 12 to the inhibition active testing result of K562 cell.

Table 11 G59 and neomycin resistance mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

Table 12 G59 and neomycin resistance mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

Secondary species thin-layer chromatography detected result shows among above experimental result and the embodiment 5, above-mentioned 41 active mutant strains metabolism produced the original fungi wild strain of non-activity marine source G59 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, neomycin resistance screening experiment technological method of the present invention can successful modification marine source non-activity fungi wild strain metabolic function, and therefrom efficient screening obtains the anti-tumor activity mutant strain, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity thalassiomycetes wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

Implementing the screening of the active mutant strain of the disease-resistant fungal pathogens of neomycin resistance of 7.G59 obtains

Experimental strain

Fermentation culture and active testing sample preparation

Fermentation culture and sample extraction preparation: adopt experimental technique and experiment condition described in above-described embodiment 1, with original bacterium G59 and 56 strain neomycin resistance mutant strain fermentation culture thereof 10～13 days, get the fermented liquid of each bacterial strain, extract and the ethyl acetate extraction operation through aqueous acetone again, get the fermented extracted sample of original bacterium G59 and mutant strain thereof, for following experiment.

The active testing preparation of sample solution: the fermented extracted sample of precision weighing G59 and mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL, 50mg/mL, 10mg/mL and 5mg/mL with methyl alcohol, and is active for the disease-resistant fungal pathogens of screening.

Anti-mycotic activity screening experiment method

Take the pathogenic fungi clinical separation strain Q-1, W-1, Y-1 and the Candida albicans type strain ATCC10231 that separate from intractable fungi infestation person disease sites as tested pathogenic fungi, adopt the method for anti-mycotic activity screening experiment described in above-described embodiment 3 fully, specimen is to the antifungic action of tested pathogenic fungi, and have or not the size of inhibition zone or inhibition zone, judgement sample to have or not disease-resistant fungal pathogens active or active strong and weak according to visual inspection.

Experimental result

The anti-mycotic activity the selection result of mutant strain sample: through the disease-resistant fungal pathogens screening active ingredients of above-mentioned 56 plant mutant strain samples, only 8 strain neomycin resistance mutant strain samples have in various degree bacteriostatic action to Q-1, but whole samples of trial 56 plant mutant strains all do not show anti-mycotic activity to all the other tested pathogenic fungies.There is the anti-Q-1 fungi activity test result of the active mutant strain sample of 8 strains of antifungic action to see Table 13 to tested pathogenic fungi Q-1.

Table 13 G59 and neomycin resistance mutant strain sample thereof are to the anti-mycotic activity test result of Q-1

Secondary species thin-layer chromatography detected result shows among above experimental result and the embodiment 5, the active mutant strain of above-mentioned 8 strains metabolism has produced the unproductive disease-resistant fungal pathogens active secondary metabolites of original bacterium G59, thereby experiment confirm, experimental technique method of the present invention can be by transforming the metabolic function of non-activity fungi wild strain, Efficient Conversion obtains the active mutant strain of disease-resistant fungal pathogens, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The ethyl sulfate mutagenic mutant screening of embodiment 8.G59 detects with secondary species to be analyzed

The collecting cells of fungi original strain G59

The ethyl sulfate mutagenic mutant screening of DMSO mediation

Prepare respectively the ethyl sulfate percent by volume and be 2.5%, 5% and 10% DMSO solution.Each is an amount of to get these three kinds of DMSO solution, mixes with the bacteria suspension of the original bacterium G59 of 4 times of volumes respectively, and preparation ethyl sulfate concentration expressed in percentage by volume is respectively the processed mixed solution of the original bacterium G59 of 20% (v/v) DMSO of 0.5%, 1% and 2%.It is an amount of that other gets DMSO, mixes with the bacteria suspension of the original bacterium G59 of 4 times of volumes, prepares not 20% (v/v) DMSO bacteria suspension of the original bacterium G59 of sulfur acid diethyl ester, makes processed control group mixed solution.In addition, mix with the bacteria suspension of an amount of sterilized water with the original bacterium G59 of 4 times of volumes, prepare the bacteria suspension diluent that sulfur acid diethyl ester does not contain the original bacterium G59 of DMSO yet, make blank group mixed solution.In addition, preparing respectively the ethyl sulfate percent by volume is 1%, 2% and 4% DMSO solution.Each is an amount of to get these three kinds of DMSO solution, mixes with the bacteria suspension of the original bacterium G59 of equal volume respectively, and preparation ethyl sulfate concentration expressed in percentage by volume is respectively the processed mixed solution of the original bacterium G59 of 50% (v/v) DMSO of 0.5%, 1% and 2%.It is an amount of that other gets DMSO, mixes with the bacteria suspension of the original bacterium G59 of equal volume, prepares not 50% (v/v) DMSO bacteria suspension of the original bacterium G59 of sulfur acid diethyl ester, makes processed control group mixed solution.In addition, mix with the bacteria suspension of an amount of sterilized water with the original bacterium G59 of 4 times of volumes, prepare the not bacteria suspension diluent of the original bacterium G59 of sulfur acid diethyl ester and DMSO, make blank group mixed solution.Place 4 ℃ of refrigerators to process 1～90 day these mixed solutions, during in good time each 90 μ L of sampling respectively, coat respectively on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Fermentation culture and extraction preparation

According to same procedure described in above-described embodiment 1, through fermentation culture and the extraction preparation of original bacterium and mutant strain thereof, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

Mutant strain and original bacterium form are relatively

The thin-layer chromatography of secondary species detects to be analyzed

Chromatography experiment method: adopt method described in above-described embodiment 1, thin-layer chromatography through original bacterium G59 and mutant strain sample thereof separates and thin layer spot color developing detection, whether the difference of secondary species thin layer spot between each mutant strain of comparative analysis and original bacterium G59 and each mutant strain determines whether the new parity level of mutant strain product spot and is the same blob that duplicate detection arrives between different mutant strains.

Experimental result

The screening of G59 neomycin resistance mutant strain is obtained: in processed control group and the experiment of blank group, tested bacteria growing is not suppressed, and grows vigorous and full dull and stereotyped in flakes growth, does not get bacterium colony but do not form picking.In contrast, in sulfur acid diethyl ester and DMSO each processed group experiment, processed bacteria growing is subject to obvious inhibition, forms several limited, bacterium colonies that form is different on the flat board, can carry out single bacterium colony and select.Therefore, the DES mutagenesis experiment under different volumes percentage ratio DMSO mediation, screening has obtained mutant strain totally 54 strains, and concrete outcome sees table 14 for details.

The DMSO mediation ethyl sulfate mutagenic mutant screening statistics of table 14G59

The thin-layer chromatography of secondary species detects in the fermented extracted sample: through the thin-layer chromatographic analysis to 54 plant mutant strains and original bacterium G59 fermented sample, from most mutant strain fermented sample, detect the secondary species spot that does not have in the original bacterium G59 fermented sample, especially, from 9 strain anti-mycotic activity mutant strain fermented samples described in 31 strain anti-tumor activity mutant strains described in the following embodiment 9 and the embodiment 10, all detect secondary species spot that do not have and that between each mutant strain sample, present different chromatography behaviors in the original bacterium G59 fermented sample.

Above experimental result shows, by the above-mentioned ethyl sulfate mutagenesis experiment that the DMSO of G59 is mediated, the secondary metabolism function of most mutant strain of the original bacterium G59 that obtains has obtained transformation, make secondary species that variation occur, thereby experiment confirm, the ethyl sulfate mutagenesis experimental technique method of DMSO mediation of the present invention can be used for the secondary metabolism function of artificial reforming fungi.

The screening of the ethyl sulfate mutagenesis anti-tumor activity mutant strain of embodiment 9.G59 is obtained

Experimental strain

Original bacterium G59 and the ethyl sulfate mutagenic mutant of in above-described embodiment 8, testing the 54 strain G59 that obtain by the induced mutations of DMSO mediation.

Fermentation culture and sample preparation

Fermentation culture and sample extraction preparation: according to identical method described in above-described embodiment 1, through fermentation culture and the extraction preparation of original bacterium G59 and mutant strain thereof, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

The antitumor activity screening experimental technique

Experimental result

The anti-tumor activity test result of original bacterium sample: described in above-mentioned embodiment 2, even original bacterium G59 sample does not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL, the screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, 1000 μ g/mL or 100 μ g/mL samples all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell.

The screening active ingredients statistics of mutant strain sample: repeatedly going down to posterity repeatedly carried out in the 54 plant mutant strains that obtain respectively, each mutant strain all adopts the bacterial strain of different passage numbers, from fermentation culture and sample extraction preparation, all independently carried out respectively antitumor activity screening repeatedly.Warp is to the anti-tumor activity repeated screening of 54 plant mutant strain samples of different passage numbers, the stable active mutant strain that reappears of anti-tumor activity has 31 strains, and wherein 100 μ g/mL samples occupy active mutant strain 9 strains between 30%～50%, inhibiting rate greater than 50% high reactivity mutant strain 22 strains to the inhibiting rate of K562 cell.The screening of these active mutant strains is obtained statistics and is seen table 15 for details.

Table 15 ethyl sulfate mutagenic mutant sample is to the antitumor activity screening statistics of K562 cell

The mtt assay active testing result of active mutant strain: 31 have anti-tumor activity to the K562 cell in 54 plant mutant strain samples, and the anti-tumor activity test result that 100 μ g/mL sample inhibiting rates occupy 9 plant mutant strain samples between 30%～50% greater than 50% 22 strains and inhibiting rate sees Table respectively 16 and table 17.

Table 16 G59 and DES mutagenesis mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

Table 17 G59 and DES mutagenesis mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

The thin layer detected result of secondary species shows among above experimental result and the embodiment 8, the 31 strain neomycin resistance anti-tumor activity mutant strains of above-mentioned G59 metabolism produced G59 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, the induced mutations experimental technique method of DMSO of the present invention mediation can successful modification marine source non-activity fungi wild strain metabolic function, and therefrom efficient screening obtains the anti-tumor activity mutant strain, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The screening of the ethyl sulfate mutagenesis anti-mycotic activity mutant strain of embodiment 10.G59 is obtained

Experimental strain

Fermentation culture and sample preparation

Fermentation culture and sample extraction preparation: according to identical method described in above-described embodiment 1, fermentation culture and extraction preparation through original bacterium G59 and mutant strain thereof, make the fermented extracted sample of G59 and 54 strain ethyl sulfate mutagenic mutants thereof, for following experiment.

Anti-mycotic activity screening experiment method

Experimental result

The anti-mycotic activity the selection result of mutant strain sample: through the disease-resistant fungal pathogens screening active ingredients of above-mentioned 54 plant mutant strain samples, only 9 strain ethyl sulfate (DES) mutagenic mutant samples have in various degree bacteriostatic action to Q-1, but whole samples of trial 54 plant mutant strains all do not show anti-mycotic activity to all the other tested pathogenic fungies.There is the anti-Q-1 fungi activity test result of the active mutant strain sample of 9 strains of antifungic action to see Table 18 to tested pathogenic fungi Q-1.

Table 18G59 and DES mutagenesis mutant strain sample thereof are to the inhibition active testing result of Q-1

The thin layer detected result of secondary species shows among above experimental result and the embodiment 8, above-mentioned 9 strain ethyl sulfate mutagenic activity mutant strains of the present invention metabolism produced original bacterium G59 unproductive secondary metabolite with disease-resistant fungal pathogens activity, thereby experiment confirm, the induced mutations experimental technique method of DMSO mediation of the present invention can be by transforming the metabolic function of non-activity fungi wild strain, Efficient Conversion obtains the active mutant strain of disease-resistant fungal pathogens, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The gentamicin resistant mutant strain screening of embodiment 11.G59 detects with secondary species to be analyzed

The collecting cells of fungi original strain G59

The gentamicin resistant mutant strain screening of DMSO mediation

Get the bacteria suspension of original bacterium G59, add an amount of gentamicin and DMSO, preparation contain respectively gentamicin 1,2,5,10,20mg/mL 50% (v/v) DMSO the G59 bacteria suspension and contain respectively the G59 bacteria suspension of 20% (v/v) DMSO of gentamicin 0.5,1,2,5,10,20,50,100mg/mL, make respectively processed group of mixed solution.Other gets the bacteria suspension of an amount of original bacterium G59, adds an amount of DMSO, and preparation does not contain 20% (v/v) of gentamicin and the G59 bacteria suspension of 50% (v/v) DMSO, makes respectively processed control group mixed solution.In addition, get the bacteria suspension of an amount of original bacterium G59, use respectively and the dilution of the sterilized water of used DMSO equal volume, preparation does not contain the bacteria suspension of gentamicin and DMSO, makes blank group mixed solution.Place 4 ℃ of refrigerators to process 1～20 day these mixed solutions, during respectively get 90 μ L in good time, coat respectively on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Fermentation culture and sample extraction preparation

The fermentation culture of original bacterium G59 and mutant strain thereof all adopts the fungi liquid fermention medium.Each bacterial strain is inoculated in respectively in the 500mL Erlenmeyer flask that contains 200mL fungi liquid substratum in right amount, cultivated 10～13 days in 28 ℃, 200rpm shaker fermentation, stop cultivating according to the microorganism growth situation in good time, lower shaking table gets fermented liquid.The acetone (volumn concentration of acetone reach approximately 66%) that adds 2 times of fermentating liquid volumes in the fermented liquid, the ultrasonication thalline, cross the leaching supernatant liquor, be evaporated to and do not contain acetone, with extracting with volume EtOAc, extraction liquid reclaims EtOAc through concentrating under reduced pressure, lyophilize, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

Mutant strain and original bacterium form are relatively

The thin-layer chromatography of secondary species detects to be analyzed

Experimental result

The screening of G59 gentamicin resistant mutant strain is obtained: do not containing gentamicin but contain the processed control group of DMSO and neither contain in the blank group experiment that gentamicin do not contain DMSO yet, tested bacteria growing is not subjected to any inhibition, grow vigorous and full dull and stereotyped in flakes growth, but do not form the bacterium colony of picking.In contrast, in containing gentamicin and DMSO each processed group experiment, processed bacteria growing is subject to obvious inhibition, forms several limited, bacterium colonies that form is different on the flat board, can carry out single bacterium colony and select.Therefore, gentamicin resistance screening experiment under different volumes percentage ratio DMSO mediation, screening has obtained the gentamicin resistant mutant strain and has amounted to 181 strains, mutant strain 45 strains of wherein, obtaining through the resistance screening experiment of 50% (v/v) DMSO mediation, mutant strain 136 strains of obtaining through the resistance screening experiment of 20% (v/v) DMSO mediation.The concrete statistics that this 181 strain gentamicin resistant mutant strain is obtained in screening sees table 19 for details.

The DMSO mediation gentamicin resistant mutant strain screening statistics of table 19G59

Annotate: " " expression do not carry out coated plate and cultivate screening; "-" expression is not chosen poor resistant mutant strain of leading; After cultivating, "～" expression coated plate do not have colony growth.

The thin-layer chromatography of secondary species detects in the fermented extracted sample: through the thin-layer chromatographic analysis to above-mentioned 181 plant mutant strains and original bacterium G59 fermented sample, from many mutant strain fermented samples, especially through being shown with the mutant strain sample of anti-tumor activity, active primary dcreening operation detects the secondary species spot that does not have in the original bacterium G59 fermented sample from 20 strains described in the following embodiment 12, especially, all detect that do not have in the original bacterium G59 fermented sample and secondary species spot that between each mutant strain sample, present different chromatography behaviors from sieving again active the reproduction the 9 stable strain anti-tumor activity mutant strain fermented samples through anti-tumor activity described in the following embodiment 12.

Above experimental result shows, gentamicin resistance screening experiment by above-mentioned DMSO mediation, the secondary metabolism function of many mutant strains of the original bacterium G59 that obtains has obtained transformation, make secondary species that variation occur, thereby experiment confirm, the gentamicin resistance screening experimental technique method of DMSO mediation of the present invention can be used for the secondary metabolism function of artificial reforming fungi.

The screening of the gentamicin resistance anti-tumor activity mutant strain of embodiment 12.G59 is obtained

Experimental strain

Original bacterium G59 and the gentamicin resistant mutant strain of in above-described embodiment 11, testing the 181 strain G59 that obtain by the gentamicin resistance screening of DMSO mediation.

Fermentation culture and sample preparation

The antitumor activity screening experimental technique

Experimental result

The screening active ingredients statistics of mutant strain sample: through the anti-tumor activity primary dcreening operation to first batch of fermented sample of above-mentioned 181 plant mutant strains, test in the 181 plant mutant strain samples 20 plant mutant strain samples preliminary show the K562 cell had suppress in various degree active, wherein 100 μ g/mL samples have 10 strains, inhibiting rate greater than 40% high reactivity mutant strain 10 strains to be arranged to the active mutant strain that the inhibiting rate of K562 cell occupy between 30%～40%.Be shown with in the mutant strain of anti-tumor activity at this 20 strain primary dcreening operation, through what 50% (v/v) DMSO mediation was obtained 9 strains that have that 11 strains, 20% (v/v) DMSO mediation obtains arranged.Concrete statistics sees Table 20, the primary dcreening operation active testing the results are shown in Table 21.

Table 20 gentamicin resistant mutant strain sample is to the anti-tumor activity primary dcreening operation statistics of K562 cell

Table 21 G59 and 20 plant mutant bacterial strain samples, 100 μ g/mL thereof are to the active primary dcreening operation test result of the inhibition of K562 cell

The activated mutant strain of above-mentioned 20 strain primary dcreening operations has been carried out 3 times gone down to posterity, and prepared through again fermentation and the extraction of the 3rd bacterial strain that goes down to posterity, made new fermented sample, then carried out anti-tumor activity with new fermented sample and sieved again.The result shows, 11 strain inactivations in 20 strains, 9 plant mutant strain sample anti-tumor activities are stable to be reappeared but still have, wherein 100 μ g/mL samples occupy active mutant strain 5 strains between 30%～40%, inhibiting rate greater than 40% active mutant strain 4 strains to the inhibiting rate of K562 cell, wherein through what 50% (v/v) DMSO mediation was obtained 2 strains that have that 7 strains, 20% (v/v) DMSO mediation obtains are arranged.The concrete statistics of the activated mutant strain fresh sample of 20 strain primary dcreening operations being sieved again anti-tumor activity sees Table 22, and 9 plant mutant strain sample activity are sieved again test result and seen Table 23.

Table 22 20 mutant strain samples 100 μ g/mL sieve statistics again to the anti-tumor activity of K562 cell

Table 23 G59 and gentamicin resistant mutant strain sample 100 μ g/mL thereof sieve test result again to the inhibition activity of K562 cell

In addition, from 181 strain gentamicin resistant mutant strains, through mutant strain and original bacterium G59 fermented sample 100 μ g/mL thereof are sieved again to the anti-tumor activity of K562 cell, the statistics that the stable active mutant strain that reappears of 9 strain anti-tumor activities is obtained in screening sees Table 24.

Table 24 screens the statistics of obtaining 9 strain anti-tumor activity mutant strains from 181 plant mutant strains

The thin-layer chromatography of secondary species detection analytical results shows among above experimental result and the embodiment 11, above-mentioned active mutant strain metabolism produced original fungi wild strain G59 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, the gentamicin resistance screening experimental technique method of DMSO of the present invention mediation can successful modification marine source non-activity fungi wild strain metabolic function, and therefrom screening obtains the anti-tumor activity mutant strain, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The kalamycin resistance mutant strain screening of embodiment 13.G59 detects with secondary species to be analyzed

The collecting cells of fungi original strain G59

The kalamycin resistance mutant strain screening of DMSO mediation

Get the bacteria suspension of original bacterium G59, add an amount of kantlex and DMSO, preparation contains respectively the G59 bacteria suspension of 20% (v/v) DMSO of kantlex 0.2,0.5,1,2,5,10,20,50,100,120mg/mL, as processed group of mixed solution.Other gets the bacteria suspension of an amount of original bacterium G59, adds an amount of DMSO, and preparation does not contain the G59 bacteria suspension of 20% (v/v) DMSO of gentamicin, as processed control group mixed solution.In addition, get the bacteria suspension of an amount of original bacterium G59, use and the dilution of the sterilized water of used DMSO equal volume, preparation does not contain the bacteria suspension of kantlex and DMSO, as blank group mixed solution.Place 4 ℃ of refrigerators to process 1～30 day these mixed solutions, during at processed the 1st, 3,5,10,20,30 day each 90 μ L of sampling respectively, coat on the PDA solid medium, cultivated 4～8 days in 28 ℃ of incubators.Observe the growing state of tested bacterium every day in culturing process, treats that bacterium colony forms, and in good time picking form, single bacterium colony that color is different with repeatedly streak culture, the separation and purification of PDA plate culture medium, obtain pure bacterial strain until separate.The gained mutant strain is inoculated on the PDA test tube slant substratum, preserves also in 4 ℃ of refrigerators and goes down to posterity in good time.

Fermentation culture and sample extraction preparation

According to identical method described in above-described embodiment 1, through fermentation culture and the extraction preparation of original bacterium G59 and mutant strain thereof, make the fermented extracted sample of G59 and kalamycin resistance mutant strain thereof, for following experiment.

Mutant strain and original bacterium form are relatively

The thin-layer chromatography of secondary species detects to be analyzed

Experimental result

The screening of G59 kalamycin resistance mutant strain is obtained: do not containing kantlex but contain the processed control group of DMSO and namely do not contain in the blank group experiment that kantlex do not contain DMSO yet, tested bacteria growing is not subjected to any inhibition, grow vigorous and full dull and stereotyped in flakes growth, but do not form the bacterium colony of picking.In contrast, in containing each processed group of different concns kantlex and 20% (v/v) DMSO experiment, processed bacteria growing is subject to obvious inhibition, forms several limited, bacterium colonies that form is different on the flat board, can carry out single bacterium colony and select.Therefore, the kalamycin resistance screening experiment under 20% (v/v) DMSO mediation, screening has obtained totally 99 strains of kalamycin resistance mutant strain, and concrete statistics sees table 25 for details.

The DMSO mediation kalamycin resistance mutant strain screening statistics of table 25 G59

The thin-layer chromatography of secondary species detects in the fermented extracted sample: through the thin-layer chromatographic analysis to above-mentioned 99 plant mutant strains and original bacterium G59 fermented sample, from many mutant strain fermented samples, detect the secondary species spot that does not have in the original bacterium G59 fermented sample, especially, from all detecting the secondary species spot that does not have and between each mutant strain sample, present different chromatography behaviors in the original bacterium G59 fermented sample the 50 strain anti-tumor activity mutant strain fermented samples described in the following embodiment 14.

Above experimental result shows, by the above-mentioned kalamycin resistance screening experiment that the DMSO of G59 is mediated, the secondary metabolism function of many mutant strains of the original bacterium G59 that obtains has obtained to transform, make secondary species that variation occur, thereby experiment confirm, the kalamycin resistance screening experiment technological method of DMSO mediation of the present invention can be used for artificial spot and make the secondary metabolism function of fungi.

The screening of the kalamycin resistance anti-tumor activity mutant strain of embodiment 14.G59 is obtained

Experimental strain

The kalamycin resistance mutant strain of the 99 strain G59 that original bacterium G59 and the kalamycin resistance screening experiment that mediates by DMSO in above-described embodiment 13 obtain.

Fermentation culture and sample preparation

Fermentation culture and sample extraction preparation: according to identical method described in above-described embodiment 2, fermentation culture and extraction preparation through original bacterium G59 and kalamycin resistance mutant strain thereof, make the fermented extracted sample of G59 and mutant strain thereof, for following experiment.

The active testing preparation of sample solution: the fermented extracted sample of precision weighing G59 and kalamycin resistance mutant strain thereof is an amount of, is made into respectively the sample solution of 100mg/mL and 10mg/mL with methyl alcohol, for the screening anti-tumor activity.

The antitumor activity screening experimental technique

Experimental result

The screening active ingredients statistics of mutant strain sample: through the antitumor activity screening to the 99 strain kalamycin resistance mutant strain samples that obtain, acquisition has mutant strain totally 50 strains of anti-tumor activity to the K562 cell, wherein 100 μ g/mL samples to the inhibiting rate of K562 cell greater than 40% 28 strains of high reactivity mutant strain, active mutant strain 22 strains of inhibiting rate between 30%～40%.The concrete screening of these active mutant strains is obtained statistics and is seen table 26 for details.

Table 26 kalamycin resistance mutant strain sample is to the antitumor activity screening statistics of K562 cell

The mtt assay active testing result of active mutant strain: in 50 strain kalamycin resistance anti-tumor activity mutant strains, through the antitumor activity screening of 100 μ g/mL samples to the K562 cell, inhibiting rate sees table 27 for details greater than the anti-tumor activity test result of the active mutant strain sample of 40% 28 plant heights, and the anti-tumor activity test result of the active mutant strain sample of 22 strains of inhibiting rate between 30%～40% sees table 28 for details.

Table 27G59 and kalamycin resistance mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

This table has only been concluded 28 strain inhibiting rates (IR%) greater than the active testing data of 40% high reactivity mutant strain sample

Table 28 G59 and kalamycin resistance mutant strain sample 100 μ g/mL thereof are to the inhibition active testing result of K562 cell

This table has only been concluded the active testing data of the active mutant strain samples of 22 strain inhibiting rates (IR%) between 30%～40%

The thin-layer chromatography of secondary species detection analytical results shows among above experimental result and the embodiment 13, the active mutant strain of above-mentioned 50 strains metabolism produced the original fungi wild strain of marine source G59 unproductive secondary metabolite with anti-tumor activity, thereby experiment confirm, kalamycin resistance screening experiment technological method of the present invention can successful modification marine source non-activity fungi wild strain metabolic function, and therefrom efficient screening obtains the anti-tumor activity mutant strain, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity thalassiomycetes wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The neomycin resistance anti-tumor activity mutant strain 2-2-3 of embodiment 15.G59 newly produces the anti-tumor activity Study on product

Experimental strain

Original bacterium G59 and the neomycin resistance anti-tumor activity mutant strain 2-2-3 that in above-described embodiment 5, the DMSO of G59 is mediated neomycin resistance screening and the G59 that the antitumor activity screening experiment screening obtains in above-described embodiment 6.

The screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, product do not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL even the fermentation of original bacterium G59 slightly gets sample, and 1000 μ g/mL or 100 μ g/mL samples all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell; And the fermentation product that slightly get sample of the neomycin resistance anti-tumor activity mutant strain 2-2-3 of G59 are stablized the anti-tumor activity that reappears the K562 cell substantially, be 27.0% such as 100 μ g/mL samples in above-described embodiment 6 to the inhibiting rate of K562 cell, fluctuate in 20%～30% scope and the inhibiting rate of 100 μ g/mL samples is basicly stable in the repeatedly repeated screening afterwards.

Fermentation culture and sample preparation

The neomycin resistance mutant strain 2-2-3 that gets G59 from the PDA test tube slant is an amount of, and streak inoculation is on the PDA plate culture medium, and activation culture is 5 days in 28 ℃ of incubators, newborn spore on the scraping PDA flat board, and be mixed with spore suspension with an amount of sterilized water.An amount of respectively equivalent of this spore suspension is inoculated in 100 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium 28 ℃, 200rpm shaker fermentation 11 days.Fermented product (approximately 20L) is divided into supernatant liquor and thalline two portions through the centrifugal 20min of 3000rpm.Supernatant liquor is directly used the equal-volume ethyl acetate extraction 3 times, gets the acetic acid ethyl acetate extract of supernatant liquor.Thalline then adds an amount of 80% (v/v) acetone water, and the ultrasonication thalline extracts, the centrifuging and taking aqueous acetone, and concentrating under reduced pressure boils off acetone, and remaining water layer gets the acetic acid ethyl acetate extract of thallus extract with equal-volume ethyl acetate extraction 3 times.Merge supernatant liquor and thalline source acetic acid ethyl acetate extract, concentrating under reduced pressure gets the ethyl acetate extract (16.8g) of mutant strain 2-2-3, newly produces active result for follow the tracks of the separation mutant strain in following experiment.In addition, adopt identical method, through to the 200mL of G59 fermentation with extract operation, make the ethyl acetate extract (93mg) of G59, in following experiment in contrast.

The tracking that mutant strain 2-2-3 newly produces the anti-tumor activity product separates

In following experiment, antitumor activity screening all adopts identical experimental technique described in above-described embodiment 2, and specimen is active to the inhibition of K562 cell.Each step lock out operation of the following stated all adopts the experiment model that antitumor activity screening is combined with the thin-layer chromatography chemical monitoring, micro-prerun guide → amplification test prepares, by directly comparing with original bacterium G59 sample, follow the tracks of the mutant strain 2-2-3 that does not have in the original bacterium G59 sample of separation and newly produce the anti-tumor activity product.

With the ethyl acetate extract (16.8g) of mutant strain 2-2-3 with an amount of chloroform-methanol (v/v=1: 1) dissolving, add 30g silica gel (100～200 order) adsorption dry, add the glass decompression post that 90g silica gel (100-200 order) is housed to, use sherwood oil → chloroform → methyl alcohol volume system, gradient elution reduces pressure again.The every 300mL of elutriant is a stream part, accesses altogether 60 stream parts, and again according to the silica gel thin-layer chromatography detected result, merging obtains 11 component Fr-1～Fr-11.Wherein, be active ingredient by an only component Fr-2 (0.8g) of chloroform wash-out, 100 μ g/mL are 87.8% to the inhibiting rate of K562 cell.With component Fr-2 (0.8g) with an amount of chloroform-methanol (v/v=1: 1) dissolving, SephadexLH-20 post on the wet method, and with identical chloroform-methanol (v/v=1: 1) wash-out separation is divided into 3 components such as Fr-2-1, Fr-2-2 and Fr-2-3.Wherein active ingredient Fr-2-1 is through (v/v=1: recrystallization 1), the mutant strain 2-2-3 that obtains not having in the original bacterium G59 sample newly produces anti-tumor activity product formula I compound (40mg) at chloroform-methanol.

Formula I

Arabic numerals represent mark among the formula I

The spectral data of formula I compound and anti-tumor activity

Formula I compound: light yellow needle (CHCl ₃), positive ion ESI-MS m/z:393[M+H] ⁺, 415[M+Na] ⁺ ¹H-NMR(400MHz，CDCl ₃)δ：6.59(1H，d，J＝9.4Hz，H-7)，6.01(1H，d，J＝9.4Hz，H-6)，5.72(1H，s，H-4)，5.24(1H，dd，J＝7.6，15.2Hz，H-23)，5.18(1H，dd，J＝7.6，15.2Hz，H-22)，2.52(1H，m，H-1)，2.47(1H，m，H-15)，2.37(2H，m，H-2，15)，2.14(1H，m，H-20)，2.10(2H，m，H-9，12)，1.99(1H，m，H-1)，1.85(1H，m，H-25)，1.79(1H，m，H-2)，1.77(1H，m，H-16)，1.68(1H，m，H-11)，1.60(1H，m，H-11)，1.49(1H，m，H-16)，1.45(1H，m，H-24)，1.27(1H，m，H-12)，1.23(1H，m，H-17)，1.05(3H，d，J＝6.7Hz，H-21)，0.98(3H，s，H-19)，0.95(3H，s，H-18)，0.92(3H，d，J＝6.7Hz，H-28)，0.83(3H，d，J＝6.7Hz，H-27)，0.81(3H，d，J＝6.7Hz，H-26)。 ¹³C-NMR(100MHz，CDCl ₃)δ：199.5(C-3)，164.3(C-5)，156.1(C-14)，134.9(C-22)，134.0(C-7)，132.4(C-23)，124.4(C-8)，124.4(C-6)，122.9(C-4)，55.6(C-17)，44.2(C-9)，43.9(C-13)，42.8(C-25)，39.3(C-20)，36.7(C-10)，35.5(C-12)，34.1(2C，C-1，2)，33.0(C-24)，27.7(C-16)，25.3(C-15)，21.2(C-21)，19.9(C-27)，19.6(C-26)，18.9(C-11)，18.9(C-18)，17.6(C-28)，16.6(C-19)。

Through antitumor activity screening, formula I compound has stronger antitumor action to the K562 cell, to the half-inhibition concentration (IC of K562 cell ₅₀) be 7.8 μ g/mL.

To sum up, even the fermentation of original fungi wild strain G59 slightly gets sample product under the high density of 1000 μ g/mL to the K562 cell also without any restraining effect, although and the fermentation of the neomycin resistance mutant strain 2-2-3 of the G59 product that slightly get sample have certain antitumor action to the K562 cell, but in the mtt assay antitumor activity screening to the restraining effect of K562 cell also a little less than, the screening active ingredients that begins from fermentation culture repeatedly independently to carry out, 100 μ g/mL samples to the inhibiting rate of K562 cell always between 20%～30%.However, the present embodiment but separates from the fermented product of 2-2-3 and has obtained that the K562 cell is had more remarkable anti-tumor activity (IC ₅₀Be 7.8 μ g/mL) mutant strain 2-2-3 newly produce anti-tumor activity product formula I compound.

Above experimental result shows, the anti-tumor activity mutant strain of the present invention through the antibiotics resistance screening of non-activity fungi wild strain G59 is obtained can be used as the screening study of going deep into that the active bacterial strain new resources are used for the new product of medicine source activity, even 100 μ g/mL slightly get sample product to the inhibiting rate of K562 cell less than the weak mutant strain of 30% activity, from its fermented product, also can obtain the stronger mutant strain of anti-tumor activity and newly produce the anti-tumor activity product, therefore, the 100 μ g/mL product that slightly get sample also are good medicine source activity bacterial strain new resources to the active mutant strain of inhibiting rate between 20%～30% of K562 cell, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The neomycin resistance anti-tumor activity mutant strain PDN-f-2 of embodiment 16.G59 newly produces the anti-tumor activity Study on product

Experimental strain

Original bacterium G59 and the neomycin resistance anti-tumor activity mutant strain PDN-f-2 that in above-described embodiment 5, the DMSO of G59 is mediated neomycin resistance screening and the G59 that the antitumor activity screening experiment screening obtains in above-described embodiment 6.

The screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, product do not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL even the fermentation of original bacterium G59 slightly gets sample, and 1000 μ g/mL or 100 μ g/mL samples all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell; And the fermentation product that slightly get sample of the neomycin resistance anti-tumor activity mutant strain PDN-f-2 of G59 are stablized the anti-tumor activity that reappears the K562 cell substantially, be 61.6% such as 100 μ g/mL samples in above-described embodiment 6 to the inhibiting rate of K562 cell, and in the repeatedly repeated screening afterwards the inhibiting rate of 100 μ g/mL samples always greater than 60%.

Fermentation culture and sample preparation

The neomycin resistance mutant strain PDN-f-2 that gets G59 from the PDA test tube slant is an amount of, and streak inoculation is on the PDA plate culture medium, and activation culture is 5 days in 28 ℃ of incubators, newborn spore on the scraping PDA flat board, and be mixed with spore suspension with an amount of sterilized water.An amount of respectively equivalent of this spore suspension is inoculated in 100 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium 28 ℃, 200rpm shaker fermentation 12 days.Fermented product (approximately 20L) is divided into supernatant liquor and thalline two portions through the centrifugal 20min of 3000rpm.Supernatant liquor is directly used the equal-volume ethyl acetate extraction 3 times, gets the acetic acid ethyl acetate extract of supernatant liquor.Thalline then adds an amount of 80% (v/v) acetone water, and the ultrasonication thalline extracts, the centrifuging and taking aqueous acetone, and concentrating under reduced pressure boils off acetone, and remaining water layer gets the acetic acid ethyl acetate extract of thallus extract with equal-volume ethyl acetate extraction 3 times.Merge supernatant liquor and thalline source acetic acid ethyl acetate extract, concentrating under reduced pressure gets the ethyl acetate extract (20g) of mutant strain PDN-f-2, newly produces active result for follow the tracks of the separation mutant strain in following experiment.In addition, adopt identical method, through to the 200mL of G59 fermentation with extract operation, make the ethyl acetate extract (96mg) of G59, in following experiment in contrast.

The tracking that mutant strain PDN-f-2 newly produces the anti-tumor activity product separates

In following experiment, antitumor activity screening all adopts identical experimental technique described in above-described embodiment 2, and specimen is active to the inhibition of K562 cell.Each step lock out operation of the following stated all adopts the experiment model that antitumor activity screening is combined with the thin-layer chromatography chemical monitoring, micro-prerun guide → amplification test prepares, by directly comparing with original bacterium G59 sample, follow the tracks of the mutant strain PDN-f-2 that does not have in the original bacterium G59 sample of separation and newly produce the anti-tumor activity product.

With the ethyl acetate extract (20g) of mutant strain PDN-f-2 with an amount of chloroform-methanol (v/v=1: 1) dissolving, add 40g silica gel (100～200 order) adsorption dry, add the glass decompression post that 100g silica gel (100-200 order) is housed to, use 60～90 ℃ of sherwood oil → chloroforms of bp → methanol solvate system, gradient elution reduces pressure again.The every 500mL of elutriant is a stream part, accesses altogether 60 stream parts, and again according to the silica gel thin-layer chromatography detected result, merging obtains 9 component Fr-1～Fr-9.Wherein, first component Fr-1 (3.8g) by sherwood oil → chloroform wash-out is active ingredient, then (v/v=9: recrystallization 1), the mutant strain PDN-f-2 that obtains not having in the original bacterium G59 sample newly produces anti-tumor activity product formula II compound (2.1g) at chloroform-methanol with component Fr-1 (3.8g).

Formula II

Arabic numerals represent mark among the formula II

The spectral data of formula II compound and anti-tumor activity

Formula II compound: lemon yellow needle (CHCl ₃), [α] _D ²¹-14.9 ° of (c1, CHCl ₃).Positive ion ESI-MS m/z:251[M+H] ⁺, 273[M+Na] ⁺Negative ion ESI-MS m/z:249[M-H] ^- ¹H-NMR(400MHz，CDCl ₃)δ：15.89(1H，s，HO-12)，15.10(1H，s，HO-8)，8.22(1H，s，H-1)，4.76(1H，q，J＝6.2Hz，H-3)，2.97(1H，q，J＝7.2Hz，H-4)，1.99(3H，s，H-11)，1.32(3H，d，J＝6.2Hz，H-9)，1.20(3H，d，J＝7.2Hz，H-10)。 ¹³C-NMR(100MHz，CDCl ₃)δ：183.8(C-6)，177.1(C-8)，174.4(C-12)，162.9(C-1)，139.1(C-4a)，122.9(C-5)，107.3(C-8a)，100.2(C-7)，81.6(C-3)，34.5(C-4)，18.4(C-11)，18.1(C-9)，9.4(C-10)。

Through antitumor activity screening, formula II compound has stronger antitumor action to the K562 cell, to the half-inhibition concentration (IC of K562 cell ₅₀) be 48 μ g/mL.

To sum up, even the fermentation of original fungi wild strain G59 slightly gets sample product under the high density of 1000 μ g/mL to the K562 cell also without any restraining effect, and the fermentation of the neomycin resistance mutant strain PDN-f-2 of the G59 product that slightly get sample have remarkable anti-tumor activity to the K562 cell, and 100 μ g/mL reach 61.6% to the inhibiting rate of K562 cell.The present embodiment extracts through simple several steps from the fermented product of PDN-f-2 and separates and purification refine operates just to separate and obtained a large amount of the K562 cell being had more remarkable anti-tumor activity (IC ₅₀Be 48 μ g/mL) mutant strain PDN-f-2 newly produce anti-tumor activity product formula II compound.

Above experimental result shows, from the 100 μ g/mL product that slightly get sample the inhibiting rate of K562 cell is newly produced the anti-tumor activity product greater than obtaining with comparalive ease the stronger mutant strain of a large amount of anti-tumor activities 50% the high reactivity mutant strain fermented product, thereby experiment confirm, the anti-tumor activity mutant strain of the present invention through the antibiotics resistance screening of non-activity fungi wild strain G59 is obtained can be used as the screening study of going deep into that the active bacterial strain new resources are used for the new product of medicine source activity, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The ethyl sulfate mutagenesis anti-tumor activity mutant strain BD-1-6 of embodiment 17.G59 newly produces the anti-tumor activity Study on product

Experimental strain

The ethyl sulfate mutagenesis anti-tumor activity mutant strain BD-1-6 of original bacterium G59 and the G59 that in above-described embodiment 8, antitumor activity screening experiment screening described in the ethyl sulfate mutagenesis of G59 and the embodiment 9 obtained.

The screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, product do not show any anti-tumor activity to the K562 cell yet under the high density of 1000 μ g/mL even the fermentation of original bacterium G59 slightly gets sample, and 1000 μ g/mL or 100 μ g/mL samples all fluctuate in less than 6.9% experimental error scope to the inhibiting rate of K562 cell; And the fermentation product that slightly get sample of the ethyl sulfate mutagenesis anti-tumor activity mutant strain BD-1-6 of G59 are stablized the anti-tumor activity that reappears the K562 cell substantially, be 60.1% such as 100 μ g/mL samples in above-described embodiment 9 to the inhibiting rate of K562 cell, and in the repeatedly repeated screening afterwards the inhibiting rate of 100 μ g/mL samples always greater than 50%.

Fermentation culture and sample preparation

The ethyl sulfate mutagenic mutant BD-1-6 that gets G59 from the PDA test tube slant of Refrigerator store is an amount of, and streak inoculation is on the PDA plate culture medium, and activation culture is 5 days in 28 ℃ of incubators.The BD-1-6 that gets activation culture with transfering loop is an amount of, is inoculated in 1 500mL Erlenmeyer flask that includes the 200mL liquid nutrient medium, carries out the seed culture 3 days first time in 28 ℃, 200rpm shaking table.This seed culture fluid is inoculated in 7 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium by 10mL/ bottle inoculum size, places 28 ℃, 200rpm shaking table, again carry out the seed culture 3 days second time.The gained seed culture fluid is inoculated in 105 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium by 10mL/ bottle inoculum size again, and 28 ℃, 200rpm shaker fermentation 11 days get altogether 21L of fermented product.Gained fermented product (21L) is divided into filtrate approximately 18L and thalline two portions through four layers of filtered through gauze.Thalline adds an amount of 70% (v/v) acetone water, the ultrasonication thalline extracts, the leaching aqueous acetone, concentrating under reduced pressure boils off acetone, remaining water layer equal-volume ethyl acetate extraction, identical extraction operation 3 times, the combined ethyl acetate extraction liquid is concentrated, gets BD-1-6 thalline source acetic acid ethyl ester extract 17g.And filtrate (18L) is used equal-volume ethyl acetate extraction 3 times, merges concentrated acetic acid ethyl acetate extract, gets the ethyl acetate extract 4.8g of mutant strain BD-1-6 ferment filtrate, newly produces active result for follow the tracks of the separation mutant strain in following experiment.In addition, adopt identical method, through to the 200mL of G59 fermentation with extract operation, make the ethyl acetate extract (112mg) of G59, in following experiment in contrast.

The tracking that mutant strain BD-1-6 newly produces the anti-tumor activity product separates

In following experiment, antitumor activity screening all adopts identical experimental technique described in above-described embodiment 2, and specimen is active to the inhibition of K562 cell.Each step lock out operation of the following stated all adopts the experiment model that antitumor activity screening is combined with the thin-layer chromatography chemical monitoring, micro-prerun guide → amplification test prepares, by directly comparing with original bacterium G59 sample, follow the tracks of the mutant strain BD-1-6 that does not have in the original bacterium G59 sample of separation and newly produce the anti-tumor activity product.

With the ethyl acetate extract (4.8g) of mutant strain BD-1-6 ferment filtrate with an amount of chloroform-methanol (v/v=1: 1) dissolving, add 10g silica gel (100～200 order) adsorption dry, add the glass normal pressure post that 70g silica gel (100-200 order) is housed to, with methylene dichloride → acetone (v/v=100: 0 → 0: 100) gradient elution, through stream part of TLC combining data detection same blob, be divided into 12 component Fr-1～Fr-12.

Will (v/v=98: 2 → 97: 3) the component Fr-5 of wash-out (240mg) dissolves with an amount of chloroform by methylene dichloride-acetone, add 1g silica gel (100～200 order) adsorption dry, add on the glass normal pressure post that 14g silica gel (100-200 order) is housed, with methylene dichloride → acetone (v/v=97: 3) Gradient elution, intercepting contains stream part of formula III compound, merge concentrated, again through Sephadex LH-20 column chromatography repeatedly (methylene chloride-methanol v/v=1: 1 wash-out) refining, get the mutant strain BD-1-6 that does not have in the original bacterium G59 sample and newly produce anti-tumor activity product formula III compound 12mg.

(v/v=97: the 3) component of wash-out, total amount amounts to 1g by methylene dichloride-acetone in component Fr-7 system.Get Fr-7 sample 31mg, recrystallization in an amount of methyl alcohol, the mutant strain BD-1-6 that obtains not having in the original bacterium G59 sample newly produces anti-tumor activity product formula IV compound 14mg.

Formula III formula IV

Arabic numerals represent mark among formula III and the formula IV

Spectral data and the anti-tumor activity of formula III compound and formula IV compound

Formula III compound: crystalline powder (methyl alcohol), [α] _D ²¹-12.9 ° (c0.9, EtOH).Positive ion ESI-MS m/z:291[M+H] ⁺, 313[M+Na] ⁺, negative ion ESI-MS m/z:289[M-H] ^- ¹H-NMR(400MHz，acetone-d ₆)δ：6.79(1H，d，J＝15.2Hz，H-9)，6.58(1H，m，H-8)，6.36(1H，d，J＝2.4Hz，H-14)，630(1H，d，J＝2.4Hz，H-12)，4.72(1H，m，H-4)，4.09(1H，d，J＝18Hz，H-1a)，3.62(1H，d，J＝18Hz，H-1b)，2.43(1H，m，H-7a)，2.36(1H，m，H-7b)，1.97(1H，m，H-5a)，1.85(1H，m，H-6a)，1.65(2H，m，H-5b，6b)，1.18(3H，d，J＝6.4Hz，4-CH ₃)。 ¹³C-NMR(100MHz，acetone-d ₆)δ：196.8(C-10)，171.5(C-2)，165.7(C-11)，162.7(C-13)，149.3(C-8)，139.2(C-15)，132.2(C-9)，115.0(C-16)，113.3(C-14)，102.5(C-12)，72.5(C-4)，43.3(C-1)，34.3(C-5)，32.7(C-7)，24.6(C-6)，19.9(4-CH ₃)。

Formula IV compound: white needle (methyl alcohol), [α] _D ²¹-34.6 ° (c3.8, EtOH).Positive ion ESI-MS m/z:293[M+H] ⁺, 315[M+Na] ⁺Negative ion ESI-MS m/z:291[M-H] ^- ¹H-NMR(400MHz，acetone-d ₆)δ：6.38(1H，d，J＝2.4Hz，H-12)，6.35(1H，d，J＝2.4?Hz，H-14)，4.91(1H，m，H-4)，3.78(1H，d，J＝15.6Hz，H-1a)，3.70(1H，d，J＝15.6Hz，H-1b)，3.10(1H，m，H-9a)，2.78(1H，m，H-9b)，1.75(1H，m，H-11a)，1.20～1.66(7H，m)，1.18(3H，d，J＝6.4Hz，4-CH ₃)。 ¹³C-NMR(100MHz，acetone-d ₆)δ：206.6(C-10)，171.0(C-2)，160.1(C-13)，158.2(C-11)，136.9(C-15)，121.2(C-16)，112.2(C-14)，102.5(C-12)，72.5(C-4)，43.9(C-9)，39.6(C-1)，32.8(C-5)，27.4(C-7)，24.5(C-6)，23.4(C-8)，20.5(4-CH ₃)。

Through antitumor activity screening, the present embodiment separates the above-mentioned formula III compound and the formula IV compound that obtain the K562 cell is all had stronger antitumor action, and 100 μ g/mL are respectively 70.1% and 52.9% to the inhibiting rate of K562 cell.

To sum up, even the fermentation of original fungi wild strain G59 slightly gets sample product under the high density of 1000 μ g/mL to the K562 cell also without any restraining effect, and the fermentation of the ethyl sulfate mutagenesis anti-tumor activity mutant strain BD-1-6 of the G59 product that slightly get sample have remarkable anti-tumor activity to the K562 cell, and 100 μ g/mL reach 60.1% to the inhibiting rate of K562 cell.The present embodiment from the fermented product of BD-1-6 by easier several steps extract separate and the purification refine operation just separates the mutant strain PDN-f-2 that obtained the K562 cell is had a more remarkable anti-tumor activity newly 2 anti-tumor activity products of product be formula III compound and formula IV compound.

Above experimental result shows, from the ethyl sulfate mutagenesis high reactivity mutant strain fermented product of G59, also can separate and obtain the stronger mutant strain of a plurality of anti-tumor activities and newly produce active result, thereby experiment confirm, the anti-tumor activity mutant strain that the induced mutations that the present invention mediates through the DMSO to non-activity fungi wild strain G59 obtains also can be used as the further investigation that the active bacterial strain new resources are used for the new product of medicine source activity, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

The disease-resistant fungal pathogens active result research of the new product of the streptomycin resistance anti-mycotic activity mutant strain G592DS20Z of embodiment 18.G59

Experimental strain

Original bacterium G59 and the active mutant strain G592DS20Z of the disease-resistant fungal pathogens of streptomycin resistance that in above-described embodiment 1, the DMSO of G59 is mediated streptomycin resistance screening and the G59 that the screening of anti-mycotic activity screening experiment obtains in above-described embodiment 3.

The screening active ingredients repeatedly that the bacterial strain with different passage numbers begins from fermentation culture independently to carry out, the fermentation of original bacterium G59 slightly get sample product in the concentration range of the trial 1500 μ g/ scraps of paper～75 μ g/ scraps of paper to tested 3 pathogen strain fungi clinical separation strain Q-1, W-1, Y-1 and 1 strain Candida albicans type strain ATCC10231 all without any anti-microbial activity, inhibition zone is zero.And the fermentation of the active mutant strain G592DS20Z of the streptomycin resistance of the G59 product (the 750 μ g/8mm scraps of paper) that slightly get sample are respectively 25mm, 10mm and 10mm to the inhibition zone of pathogenic fungi clinical separation strain Q-1, W-1 and Y-1, and the basicly stable reproduction of bacteriostatic action in test repeatedly.

Fermentation culture and sample preparation

From the PDA test tube slant of the 4th preservation of going down to posterity, the streptomycin resistant mutation strain G592DS20Z that gets G59 is an amount of, is inoculated in 3 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium, carries out seed culture 2 days in 28 ℃, 200rpm shaking table.This seed culture fluid is inoculated in 60 500mL Erlenmeyer flasks that include the 200mL liquid nutrient medium 28 ℃, 200rpm shaker fermentation 13 days by every bottle of 10mL inoculum size.Fermented product (approximately 12L) is divided into filtrate and thalline two portions through six layers of filtered through gauze.Filtrate is directly used the equal-volume ethyl acetate extraction 3 times, gets the acetic acid ethyl acetate extract of filtrate.Thalline then adds approximately 60% (v/v) acetone water of 2 times of volumes, the ultrasonication thalline extracts and the leaching aqueous acetone, identical extraction operation is carried out 5 times, merge the aqueous acetone concentrating under reduced pressure and boil off acetone, remaining water layer gets the acetic acid ethyl acetate extract of thallus extract with equal-volume ethyl acetate extraction 3 times.The acetic acid ethyl acetate extract that merges ferment filtrate and thalline source, concentrating under reduced pressure gets the ethyl acetate extract (14.4g) of mutant strain G592DS20Z, newly produces active result for follow the tracks of the separation mutant strain in following experiment.In addition, adopt identical method, through to the 400mL of G59 fermentation with extract operation, make the ethyl acetate extract (203mg) of G59, in following experiment in contrast.

The new tracking of producing disease-resistant fungal pathogens active result of mutant strain G592DS20Z separates

In following experiment, identical experimental technique described in above-described embodiment 3 is all adopted in the anti-mycotic activity screening, and specimen is active to the inhibition of pathogenic fungi clinical separation strain Q-1, W-1 and Y-1.Each step lock out operation of the following stated all adopts the experiment model that the anti-mycotic activity screening is combined with the thin-layer chromatography chemical monitoring, micro-prerun guide → amplification test prepares, by directly comparing with original bacterium G59 sample, follow the tracks of and separate the new anti-mycotic activity product that produces of the mutant strain G592DS20Z that does not have in the original bacterium G59 sample.

With the ethyl acetate extract (14.4g) of mutant strain G592DS20Z with an amount of chloroform-methanol (v/v=9: 1) dissolving, add 30g silica gel (100～200 order) adsorption dry, add glass decompression post that silica gel (100-200 order) is housed to (on strain bed: the 6.5cm * 15cm), use hexanaphthene → chloroform → methanol solvate system, gradient elution reduces pressure again.According to the silica gel thin-layer chromatography detected result, collect the corresponding elutriant of merging concentrated, obtain 10 component Fr-1～Fr-10.Wherein, (v: v=9: 1) the 5th of wash-out the component Fr-5 (2.8g) is active ingredient by chloroform-methanol.With component Fr-5 (2.8g) with an amount of chloroform-methanol (v/v=3: 1) dissolving, add 10g silica gel (100～200 order) adsorption dry, add glass decompression post that 100-200 order silica gel is housed to (on strain bed: the 5cm * 13cm), with chloroform → methanol solvate system decompression gradient elution, elutriant and concentrated is closed in collection according to the thin layer detected result, is divided into 6 component Fr-51～Fr-56.Wherein (v: v=9: 1) the 3rd of wash-out the component Fr-53 (0.8g) is active ingredient by chloroform-methanol.Component Fr-53 (0.8g) with an amount of chloroform-methanol (v/v=1: 1) dissolving, chloroform-methanol on the wet method (v/v=1: the Sephadex LH-20 post that installs 1) (post bed: 3cm * 150cm), with chloroform-methanol (v/v=1: 1) wash-out, collect the elutriant merging according to the thin layer detected result and concentrate, obtain containing the target components Fr-533 (240mg) of formula V compound.With component Fr-533 (240mg) with an amount of chloroform-methanol (v/v=1: 1) dissolving, add 2g reverse phase silica gel ODS adsorption dry, decompression reverse phase silica gel ODS post on the dry method (post bed: 3cm * 12cm), with Methanol+Water in successively by newly increasing the mode gradient elution of methyl alcohol ratio in the water, collect to merge the elutriant that contains formula V compound according to the thin layer detected result concentrated, and recrystallization obtains the new anti-mycotic activity product formula V compound (15mg) that produces of mutant strain G592DS20Z that do not have in the original bacterium G59 sample from methyl alcohol.

Formula V

Arabic numerals represent mark among the formula V

The spectral data of formula V compound and anti-mycotic activity

Formula V compound: white, needle-shaped crystals (methyl alcohol), [α] _D ²²-47.3 ° (c 1.0, EtOH).Positive ion ESI-MS m/z:267[M+H] ⁺, 289[M+Na] ⁺Negative ion ESI-MS m/z:265[M-H] ^-IR(KBr)v：3402(OH)，3250～2500brs(CO OH)，2930，2864，2823(CH ₂&CH ₃)，1722(CO)，1461，1439，1325，1242，1191，1063，1018cm ^-1。 ¹H-NMR(400MHz，CD ₃OD)δ：4.30(1H，d，J＝8.4Hz，H-2)，3.70(1H，d，J＝8.0Hz，Hb-13)，3.35(1H，dd，J＝8.0，0.8Hz，Ha-13)，2.38-2.44(2H，m，Hb-4and?Hb-8)，2.27-2.33(2H，m，H-6and?Hb-9)，2.16(1H，dd，J＝12.6，8.4Hz，Hb-1)，2.01-2.04(2H，m，Ha-1and?Ha-9)，1.74(3H，s，H3-15)，1.66-1.72(2H，m，Hb-5and?Ha-8)，1.60(3H，s，?H ₃-14)，1.49(1H，dd，J＝11.8，7.6Hz，Ha-5)，1.41(1H，m，Ha-4)。 ¹³C-NMR(100MHz，CD ₃OD)δ：177.8(C-12)，136.7(C-10)，125.3(C-11)，84.9(C-2)，79.4(C-13)，76.5(C-3)，56.7(C-6)，51.3(C-7)，36.7(C-1)，35.3(C-4)，35.1(C-5)，33.1(C-9)，24.8(C-8)，23.4(C-15)，21.0(C-14)。

Screen through anti-mycotic activity, formula V compound has stronger antifungic action to pathogenic fungi clinical separation strain Q-1, the 375 μ g/8mm scraps of paper reach 30mm to the antibacterial circle diameter of Q-1, and the 375 μ g/8mm scraps of paper are to W-1 and then unrestraint effect of Y-1, and antibacterial circle diameter is zero.

To sum up, the fermentation of original bacterium G59 slightly get sample product in the concentration range of the 1500 μ g/ scraps of paper～75 μ g/ scraps of paper to tested 4 pathogen strain fungi Q-1, W-1, Y-1 and Candida albicans type strain ATCC10231 all without any anti-microbial activity, antibacterial circle diameter is zero.And the fermentation of the active mutant strain G592DS20Z of the streptomycin resistance of the G59 product (the 750 μ g/8mm scraps of paper) that slightly get sample are respectively 25mm, 10mm and 10mm to the antibacterial circle diameter of pathogenic fungi clinical separation strain Q-1, W-1 and Y-1, and the basicly stable reproduction of bacteriostatic action in test repeatedly.The present embodiment separates the anti-mycotic activity product formula V compound that has obtained pathogenic fungi clinical separation strain Q-1 is had the new product of obvious inhibiting mutant strain G592DS20Z from the fermented product of G592DS20Z, the formula V compound 375 μ g/8mm scraps of paper reach 30mm to the antibacterial circle diameter of Q-1.

Above experimental result shows, the active mutant strain with disease-resistant fungal pathogens activity that the present invention obtains from the G59 conversion can be used as medicine source activity bacterial strain and newly produces active result research for mutant strain, and the new anti-mycotic activity product that produces of mutant strain that therefrom obtains having disease-resistant fungal pathogens activity, thereby experiment confirm, the present invention can be used as the further investigation that the active bacterial strain new resources are used for the new product of medicine source activity through the active mutant strain of disease-resistant fungal pathogens that the secondary metabolism function modifications to non-activity fungi wild strain G59 obtains, thereby further experiment confirmed, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

Conclusion:

The antibiotics resistance screening of DMSO mediation of the present invention and the artificial chemistry mutagenesis experimental technique of DMSO mediation are embodied as the merit transformation to the fungal secondary metabolic function, antitumor, disease-resistant fungal pathogens isoreactivity mutant strain is obtained in the effective conversion and the efficient screening that can be used for non-activity fungi wild strain, the active mutant strain that obtains can be for carrying out the research of active result in a deep going way, and find that therefrom mutant strain newly produces medicine source activity compound.Therefore, experimental technique method of the present invention has the actual utility value of non-activity fungi wild strain as the new strain resource of original bacterium resource efficient extn medicine source activity.

Claims

1. the method for the transformation fungal secondary metabolic function of DMSO mediation, it is characterized in that, in DMSO solution, with processed original fungi and the microbiotic that can act on microorganism rrna performance anti-microbial effect interact 0.5～200 day during, and in during this period in good time sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

2. the method for the transformation fungal secondary metabolic function of DMSO mediation, it is characterized in that, in DMSO solution, with original fungi and chemical mutagen interact 0.5～200 day during, and in during this period in good time sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed.

3. DMSO mediation transforms to obtain the method for the active mutant strain of anti-tumor pathogenic fungi by fungi, it is characterized in that, in DMSO solution, with described fungi and the microbiotic that acts on microorganism rrna performance anti-microbial effect and/or chemical mutagen interact 0.5～200 day during, and in during this period in good time sampling, select and separation and Culture through the dull and stereotyped coated plate of PDA and single bacterium colony repeatedly, purifying obtains the mutant strain that the secondary metabolism gain-of-function is transformed, through the anti-tumor pathogenic fungi screening active ingredients of mutant strain, obtain the mutant strain with anti-tumor pathogenic fungi activity again.

4. the method for claim 3, wherein said fungi is the fungi wild strain.

5. the method for claim 3, wherein said fungi is the fungi wild strain of non-activity.

6. the described method of claim 1, it is characterized in that following one or more: i) described DMSO solution is the DMSO aqueous solution; Ii) the described microbiotic that acts on microorganism rrna performance anti-microbial effect is to be selected from Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, gentamicin, paraxin, the Rifampin one or more.

7. method claimed in claim 2, it is characterized in that following one or more: i) described DMSO solution is the DMSO aqueous solution; Ii) described chemical mutagen is to be selected from ethyl sulfate, the nitrosoguanidine one or more.

8. method claimed in claim 3, it is characterized in that following one or more: i) described DMSO solution is the DMSO aqueous solution; Ii) the described microbiotic that acts on microorganism rrna performance anti-microbial effect is to be selected from Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, gentamicin, paraxin, the Rifampin one or more; And iii) described chemical mutagen is to be selected from ethyl sulfate, the nitrosoguanidine one or more.

9. the fungi mutant strain of secondary metabolism function through transforming that obtain of each described method of claim 1 to the 8 fast separating and purifying preparation method that newly produces active result, it is characterized in that, original bacterium and active mutant strain thereof ferment respectively, the gained fermented product is extracted with aqueous acetone solution, then to this aqueous acetone extracting solution reclaim under reduced pressure acetone, then with residual water layer ethyl acetate extraction, obtain respectively original bacterium acetic acid ethyl ester extract and contain the mutant strain acetic acid ethyl ester extract that mutant strain newly produces active result, then all adopt active the tracking with chemical detection to analyze combination to each step lock out operation of mutant strain acetic acid ethyl ester extract, trace prerun guide, then the experiment model of amplification test preparation, described active the tracking with chemical detection analyzed combination, trace prerun guide, then the experiment model of amplification test preparation refers to by mutant strain tunning and original bacterium tunning are directly compared, determine fast that in micro-prerun mutant strain newly produces on the basis of the TLC spot of active result or HPLC elution peak, amplification test under TLC or HPLC guidance or detection, sharp separation prepares mutant strain and newly produces active result.

10. the fungi activity mutant strain of secondary metabolism function through transforming that obtain of each described method of claim 1 to 8 purposes of newly producing medicine source activity compound for the preparation of mutant strain, described active mutant strain refers to have the mutant strain of antitumor, disease-resistant fungal pathogens activity, and described active compound refers to have the compound of antitumor, disease-resistant fungal pathogens activity.

Thereby 11. each described method of claim 1 to 8 in the secondary metabolism function that be used for to transform non-activity fungi wild strain with non-activity fungi wild strain as original bacterium resource to obtain the purposes of the new bacterial strain of medicine source activity, it is active that described activity refers to have antitumor, disease-resistant fungal pathogens.