CN103233043A - Tetrodotoxin-degrading extract liquid and preparation method thereof - Google Patents
Tetrodotoxin-degrading extract liquid and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing a tetrodotoxin-degrading substance from a tetrodotoxin-producing bacteria fermentation broth, and a preparation and detection method of an extract liquid. The method comprises a method for artificially causing plasmid loss, a tetrodotoxin-degrading substance extraction method, and a tetrodotoxin degradation detection method. According to the invention, for a first time, the existence of a tetrodotoxin-degrading substance in aeromonas molluscorum is found, and an extraction method of the substance is established. The method has certain potential to be used in large-scale productions of the tetrodotoxin-degrading substance. The extract of the bacteria fermentation broth has a tetrodotoxin-degrading effect, and can be used for preparing medicines used for treating tetrodotoxin intoxication. Also, a raw material can be provided for related fields such as scientific research and pharmaceutical industries.
Description
Technical field
The present invention relates to biological technical field, be specifically related to medicinal microorganism development and utilization field, relate more specifically to a kind of method that from ferment product, prepares degradable tetraodotoxin extracting solution, and the tetraodotoxin degradation material extracting solution that is obtained by its extraction.
Background technology
Tetraodotoxin (Tetrodotoxin, TTX), be the alkaloid of the full hydroxyl quinazoline of a kind of amino, be the extremely strong neurotoxin of a kind of toxicity, by the valtage-gated sodium-ion channel on the specific inhibition cytolemma, cause general paralysis, finally die from and breathe and reflux depleted.The TTX latent period of poisoning is very short, is as short as 10-30min, grows to 3-6h morbidity, and morbidity is anxious, if rescue untimely, dead in the fastest 10min after the poisoning, 4-6h death at the latest.That clinical treatment can only depend on is emetic, gastric lavage, catharsis etc. reduce the method for the absorption of poisonous substance, and uses adrenocortical hormone, improves the tolerance of organizing contratoxin.Still there is not effective toxinicide at present.
Have the research report of blocking tetraodotoxin and its acceptor sodium-ion channel by preparation tetraodotoxin antibody at present, but also find to exist the medicine of the tetraodotoxin of directly degrading.
Summary of the invention
The present invention finds that the bacterium of TTX is produced in 1 strain not only can synthesize tetraodotoxin, and has the material of degraded tetraodotoxin in its fermented liquid simultaneously.To the effect that of the present invention: as by manually losing the plasmid that produces the TTX bacterium, to make it lose the ability of producing TTX but can keep the material of producing degraded TTX, lose the ferment product of plasmid by extracting, can obtain the material of degraded tetraodotoxin.The invention provides the method that manually causes plasmid loss, the detection method of plasmid loss and the preparation method of degraded tetraodotoxin material.Degraded tetraodotoxin material can be used as the exploitation and relevant scientific research of the toxicide of tetraodotoxin poisoning.
One object of the present invention is to provide a kind of method of extracting the material of degraded tetraodotoxin, comprises the steps:
(1) fermentation using bacteria of the product tetraodotoxin of plasmid loss is cultivated at least 48 hours (preferred 48-66 hour, more preferably 67-96 hour), centrifugal collecting cell is preferably at 4 ℃ of collecting cells behind the centrifugal 30min of 4000 * g down;
(2) with 0.1%-1% acetic acid (preferably using 1% acetic acid) dissolved cell, ultrasonic disruption cell;
(3) 95-105 ℃ (preferred 100 ℃) boil 20-25min, the centrifugal removal cell debris in cooling back;
(4) filter supernatant;
(5) filtered liquid is crossed active carbon column, the prescription of elutriant is: methyl alcohol: acetic acid solution=20:80 of 1%;
(6) elutriant is in 40-50 ℃ of (preferred 45 ℃) concentrating under reduced pressure, lyophilize;
(7) be dissolved in the aseptic deionized water;
(8) cross Bio-Gel P2 pillar;
(9) with 0.02-0.05M (preferred 0.03M) acetic acid wash-out, collect elutriant;
(10) elutriant is crossed post with C18Sep-Pak cartridges again;
(11) with the acetic acid wash-out of 5-20ml0.3%, preferably use the acetic acid wash-out of 10ml0.3%, collect elutriant;
(12) elutriant is filtered;
(13) after the filtered liquid lyophilize, be dissolved in the aseptic deionized water extracting solution of the material of the tetraodotoxin that obtains degrading.
In one embodiment, method of the present invention further comprises following detection step: as sample to be tested, adjusting the pH value is 6.5-7.4 with filtrate, with the tetraodotoxin in the ELISA method detection sample.
In one embodiment, of the present inventionly provide a kind of method of material of extracting the degraded tetraodotoxin, comprise the steps:
(1) fermentation using bacteria of the product tetraodotoxin of plasmid loss was cultivated 48 hours 4 ℃ of centrifugal 30min collecting cells of 4000 * g down at least;
(2) with 0.1% acetic acid dissolved cell, ultrasonic disruption cell;
(3) 100 ℃ are boiled 20-25min, the centrifugal removal cell debris in cooling back;
(4) filter supernatant;
(5) filtered liquid is crossed active carbon column, the prescription of elutriant is: methyl alcohol: acetic acid solution=20:80 of 1%;
(6) elutriant is in 45 ℃ of concentrating under reduced pressure, lyophilize;
(7) be dissolved in the 2ml aseptic deionized water;
(8) cross Bio-Gel P2 pillar;
(9) with 0.03M acetic acid wash-out, begin to collect from the elutriant outflow, collect 2ml;
(10) elutriant is crossed post with C18Sep-Pak cartridges again;
(11) with the acetic acid wash-out of 10ml0.3%, flow out collection since second column volume elutriant, collect the elutriant of two column volumes;
(12) elutriant is filtered;
(13) after the filtered liquid lyophilize, be dissolved in the 1ml aseptic deionized water;
(14) with filtrate as sample to be tested, adjusting pH value be 6.5-7.4, the tetraodotoxin in the usefulness ELISA method detection sample.
In one embodiment of the present invention, described Aeromonas can be mollusk Aeromonas (Aeromonas molluscorum), also can be other Aeromonas that produces tetraodotoxin, for example from Rhodopseudomonas (Pseudomonas), Shiva Salmonella (Shewanella), replace Zymomonas mobilis (Alteromonas), genus bacillus (Bacillus), acinetobacter (Acinetobacter), Alcaligenes (Alcaligenes), Flavobacterium (Flavobacterium), Plesiomonas (Plesiomonas), serratia (Serratia), nocardia belongs to (Nocardiopsis), micrococcus sp (Micrococcus), Moraxella (Moraxella), Vibrio (Vibrio), actinomycetes (Actinomycetes), the product TTX bacterium of Caulobacter monoids such as (Caulobacter).
In one embodiment of the present invention, cause the bacterium of described product tetraodotoxin to lose artificial fermentation's cultural method of plasmid, comprise the steps:
(1) with the microbionation of described product tetraodotoxin in adding arginic ORI liquid nutrient medium;
(2) under 20-28 ℃ (preferred 23 ℃), 200-450 rev/min (preferred 225 rev/mins) were cultivated 48 hours at least.
In one embodiment of the present invention, consisting of of described ORI liquid nutrient medium: 0.05% arginine, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, the preferred pH8.0 of pH7-8.5().
In one embodiment of the present invention, the preparation method of described ORI liquid nutrient medium (1000ml): in the 800ml deionized water, add 2g Tryptones (OXOID company), 2g soy peptone (OXOID company), 1g yeast extract (OXOID company), 30g sodium-chlor (Yixing City Chemical Reagent Plant No.2) and 0.5g L-arginine (Chemical Reagent Co., Ltd., Sinopharm Group), after the stirring and dissolving, add 0.88g ironic citrate (Chemical Reagent Co., Ltd., Sinopharm Group) again, continue to stir, after dissolving fully, add deionized water and be settled to 1000ml, and regulate the preferred PH8.0 of PH to 7-8.5() after seal sterilization.
In one embodiment of the present invention, the ELISA method in the described detection step detects and comprises the steps:
(1) on the enzyme plate that antigen is fixedly arranged, adds the TTX standard substance respectively, sample to be tested and the sample to be tested that has added the TTX standard substance;
(2) add the specific antibody solution of TTX at enzyme plate;
(3) 20-40 ℃ (preferred room temperature or 37 ℃) hatches to reacting completely, and adds washings detersive enzyme target 3 times;
(4) add ELIAS secondary antibody, 20-40 ℃ (preferred room temperature or 37 ℃) hatches to reacting completely, and adds washings detersive enzyme target 3 times;
(5) add colour developing liquid, 37 ℃ hatch 10-15min after, every hole adds stop buffer with termination reaction;
(6) result detects: enzyme plate is inserted in the microplate reader, detect the light absorption value at 450nm place.
In one embodiment of the present invention, in the step of described ELISA method (1), (Sigma-Aldrich company, production number: sample to be tested T8024-1MG), it is 25-200ng/mL that its TTX standard substance add final concentration to have added the TTX standard substance.
In one embodiment of the present invention, the specific antibody in the step of described ELISA method (2) is specific monoclonal antibody (Beijing centre halfback's food sanitation science and technology company, the production number: 5561) of TTX.
In one embodiment of the present invention, in the step of described ELISA method (3), hatch to the required time that reacts completely be 1.5h; Hatch in the step of described ELISA method (4) and be 1h to reacting completely.
Another object of the present invention is to provide a kind of artificial fermentation's cultural method that causes the Aeromonas that produces tetraodotoxin to lose plasmid, comprise the steps:
(1) Aeromonas that produces tetraodotoxin is seeded in the arginic ORI liquid nutrient medium of interpolation;
Under (2) 23 ℃, 225 rev/mins, cultivated at least 48 hours.
In one embodiment of the present invention, consisting of of the described ORI liquid nutrient medium that aforesaid method adopts: 0.05% arginine, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, the preferred pH8.0 of pH7-8.5().
Another object of the present invention is to provide a kind of real-time quantitative PCR detection method, it comprises the steps: for detection of the bacterium of above-mentioned arbitrary method employing or the plasmid loss of Aeromonas
(1) quantitative PCR standard curve making: the Ne-1 plasmid that extracts is carried out 10 times of serial dilutions with sterilized water, be diluted to 6 gradients respectively, as template, carry out quantitative fluorescent PCR and set up typical curve, calculate plasmid copy number as follows:
Plasmid copy number (copies μ L
-1)=6.02 * 10
23(copiesmol
-1) * plasmid concentration (g μ L
-1)/plasmid molecule amount (gmol
-1);
(2) real-time quantitative PCR system: testing sample and standard substance are done 3 respectively repeat to place with experiment once and react, press following set of dispense PCR reaction solution processed: iQ SYBR Green Supermix12.5 μ l, PCR forward primer (2 μ mol/L) 1 μ l, PCR reverse primer (2 μ mol/L) 1 μ l, plasmid template 2 μ l add ddH
2O complements to 25 μ l, PCR forward primer sequence 5 '-GACAAGGCTCGGAGACACGCA-3 ', PCR reverse primer sequence 5 '-TCTCTTTCATGCTGCGGTTCAGC-3 ', described testing sample are described Aeromonas or described bacterium of losing plasmid of losing plasmid;
(3) real-time quantitative PCR response procedures: 95 ℃ of pre-denatured plasmid dna templates of 5min, carry out 30 temperature variation circulations with 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30s then, 80 ℃ of 1s, 82 ℃ of 1s carry out 1 circulation, last 59 ℃ to 95 ℃ are extended 20s, at last, utilize accompanying software to carry out melting curve analysis and the analysis of CT value, determine plasmid copy number;
(4) utilization contained arginic ORI culture medium culturing after at least 48 hours, and plasmid is lost fully.
Another object of the present invention is to provide a kind of extracting solution that extracts the tetraodotoxin degradation material for preparing according to aforesaid method.
The application of the extracting solution of the tetraodotoxin degradation material that another object of the present invention is to provide above-mentioned in the medicine of poisoning for the preparation of the treatment tetraodotoxin.
The present invention first discloses by changing the method for medium component, manually causes Aeromonas Aeromonas molluscorum plasmid loss, makes it lose the ability of the former synthetic TTX that possesses, it is characterized in that, comprises the steps:
(1) with microbionation add arginic ORI liquid nutrient medium (0.05% arginine, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, pH8.0) in; Under 23 ℃, 225 rev/mins, cultivated 48 hours.
(2) quantitative PCR standard curve making: the standard plasmid that extracts is carried out 10 times of serial dilutions with sterilized water, be diluted to 6 gradients respectively, as template (the time standby liquid-transfering gun piping and druming 20 times of dilution, centrifugal behind the vibration 5s, make the abundant mixing of solution), carry out quantitative fluorescent PCR and set up typical curve.Calculate plasmid copy number as follows: plasmid copy number (copies μ L
-1)=6.02 * 10
23(copiesmol
-1) * plasmid concentration (g μ L
-1)/plasmid molecule amount (gmol
-1).
(3) real-time quantitative PCR system: testing sample and standard substance are done 3 respectively repeat to place with experiment once and react, press following set of dispense PCR reaction solution processed: iQ SYBR Green Supermix12.5 μ l, PCR forward primer (2 μ mol/L) 1 μ l, PCR reverse primer (2 μ mol/L) 1 μ l, plasmid template 2 μ l add ddH
2O complements to 25 μ l.PCR forward primer sequence 5 '-GACAAGGCTCGGAGACACGCA-3 '; PCR reverse primer sequence 5 '-TCTCTTTCATGCTGCGGTTCAGC-3 '.
(4) real-time quantitative PCR response procedures: 95 ℃ of pre-sex change of 5min, carry out 30 circulations with 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30s then, 80 ℃ of 1s, 82 ℃ 1s1 circulation, last 59 ℃ to 95 ℃ are extended 20s.At last, utilize accompanying software to carry out melting curve analysis and the analysis of CT value, determine plasmid copy number.
(5) utilize and to contain arginic ORI culture medium culturing at least 48 hours, Aeromonas Aeromonas after molluscorum48 hour plasmid lose fully.
The present invention second discloses the extracting method of the material of degraded tetraodotoxin in the described product tetraodotoxin fermentation using bacteria process, it is characterized in that, comprises the steps:
(1) fermentation culture is after 66 hours, and 4 ℃ are descended the centrifugal 30min collecting cells of 4000 * g;
(2) with 0.1% acetic acid dissolved cell, ultrasonic disruption cell;
(3) 100 ℃ are boiled 20-25min, the centrifugal removal cell debris in cooling back;
(4) supernatant diameter 0.25 μ m filter paper filtering;
(5) filtered liquid is crossed active carbon column, and with elutriant wash-out activated carbon, the prescription of elutriant is: methyl alcohol: acetic acid solution=20:80 of 1%;
(6) elutriant is decompressed to 0.001-0.003M Pa for 45 ℃ and concentrates lyophilize;
(7) be dissolved in the 2ml aseptic deionized water;
(8) the Bio-Gel P2 pillar of mistake 1 * 80cm;
(9) with 0.03M acetic acid wash-out, begin to collect from the elutriant outflow, collect 2ml;
(10) elutriant is crossed post with C18Sep-Pak cartridges again;
(11) with the acetic acid wash-out of 10ml0.3%, flow out collection since second column volume elutriant, collect the elutriant of two column volumes;
(12) elutriant is filtered;
(13) after the filtered liquid lyophilize, be dissolved in the 1ml aseptic deionized water.
The present invention the 3rd discloses the detection method of tetraodotoxin in the described extraction sample, it is characterized in that, comprises the steps:
(1) on the enzyme plate that antigen is fixedly arranged, adds the TTX standard substance respectively, sample to be tested and the sample to be tested that has added the TTX standard substance;
(2) add the specific antibody solution of TTX at enzyme plate;
(3) room temperature or 37 ℃ are hatched to reacting completely, and add washings detersive enzyme target 3 times;
(4) add ELIAS secondary antibody, room temperature or 37 ℃ are hatched to reacting completely, and add washings detersive enzyme target 3 times;
(5) add colour developing liquid, 37 ℃ hatch 10-15min after, every hole adds stop buffer with termination reaction;
(6) result detects: enzyme plate is inserted in the microplate reader, detect the light absorption value at 450nm place.
In the described step (1), added the sample to be tested of TTX standard substance, it is 25-200ng/mL that its TTX standard substance add final concentration.
Specific antibody in the described step (2) is the specific monoclonal antibody of TTX.
In the described step (3), hatch to the required time that reacts completely be 1.5h; Hatch in the described step (4) and be 1h to reacting completely.
The preparation method (1000ml) of " ORI liquid nutrient medium " of the present invention: in the 800ml deionized water, add 2g Tryptones (OXOID company), 2g soy peptone (Britain OXOID company), 1g yeast extract (Britain OXOID company), 30g sodium-chlor (Yixing City Chemical Reagent Plant No.2) and 0.5g L-arginine (Chemical Reagent Co., Ltd., Sinopharm Group), after the stirring and dissolving, add 0.88g ironic citrate (Chemical Reagent Co., Ltd., Sinopharm Group) again, continue to stir, after dissolving fully, add deionized water and be settled to 1000ml, and seal sterilization after regulating PH to 8.0.
The application adopts the Aeromonas (Aeromonas molluscorum) of the product tetraodotoxin of record on March 30th, 2011 disclosed Chinese patent application numbers 201010179363.8, and this culture presevation is in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.Preservation date: on 04 23rd, 2010, deposit number: CGMCC No.3760.
Cause the cultural method of the plasmid loss that produces the tetraodotoxin bacterium can repetition.Microbionation (consisting of of ORI liquid nutrient medium: 0.05% arginine in adding arginic ORI liquid nutrient medium of tetraodotoxin will be produced, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, pH8.0), under 23 ℃, 225 rev/mins, cultivate 48 hours at least after, can cause the plasmid loss that produces the tetraodotoxin bacterium.
Reagent of the present invention, sample are can be by the commercially available prod of commercial sources acquisition, and major part is available from Britain OXOID company and Chemical Reagent Co., Ltd., Sinopharm Group.
Beneficial effect of the present invention:
1) the present invention has the extracting solution of degradation effect by preparation from ferment product to tetraodotoxin, and this extracting solution can be used as the medicine that clinical treatment tetraodotoxin is from now on poisoned.
2) extracting method of filefish degradation material of the present invention can be used as the large-scale production of degraded tetraodotoxin material, can be association areas such as scientific research, pharmaceutical industries and supplies raw materials.
Description of drawings
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Fig. 1 is Aeromonas Aeromonas molluscorum bacterial plasmid drop-out time sequence.
Fig. 2 detects the typical curve of TTX for ELISA.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect is easy to understand, below in conjunction with concrete diagram, further set forth the present invention.
Embodiment 1
1, utilize the improvement substratum to cause the method for Aeromonas Aeromonas molluscorum plasmid loss, can operate according to following steps:
(1) with microbionation in adding arginic ORI liquid nutrient medium, ORI liquid nutrient medium preparation method (1000ml) is: add 2g Tryptones (OXOID company) in the 800ml deionized water, 2g soy peptone (OXOID company), 1g yeast extract (OXOID company), 30g sodium-chlor (Yixing City Chemical Reagent Plant No.2) and 0.5g L-arginine (Chemical Reagent Co., Ltd., Sinopharm Group), after the stirring and dissolving, add 0.88g ironic citrate (Chemical Reagent Co., Ltd., Sinopharm Group) again, continue to stir, after dissolving fully, add deionized water and be settled to 1000ml, and seal sterilization after regulating PH to 8.0.
Under (2) 23 ℃, 225 rev/mins, cultivated at least 48 hours.
2, utilize the method for real-time quantitative PCR bacterial detection Aeromonas molluscorum plasmid loss, can operate according to following steps:
(1) quantitative PCR standard curve making: the standard plasmid that extracts is carried out 10 times of serial dilutions with sterilized water, be diluted to 6 gradients respectively, as template (the time standby liquid-transfering gun piping and druming 20 times of dilution, centrifugal behind the vibration 5s, make the abundant mixing of solution), carry out quantitative fluorescent PCR and set up typical curve.Calculate plasmid copy number as follows: plasmid copy number (copies μ L
-1)=602 * 1023 (copiesmol
-1) * plasmid concentration (g μ L
-1)/plasmid molecule amount (gmol
-1).
(2) with microbionation in adding arginic ORI liquid nutrient medium, be cultured to 18,24,48,72,96 hours respectively after, collect bacterium, with the plasmid of each of alkaline process extracting simultaneously bacterium phase.
(3) real-time quantitative PCR system: plasmid sample to be measured and standard substance are done 3 respectively repeat to place with experiment once and react, press following set of dispense PCR reaction solution processed: 12.5 μ l iQ SYBR Green Supermix(Bio-Rad company), PCR forward primer (2 μ mol/L) 1 μ l, PCR reverse primer (2 μ mol/L) 1 μ l, plasmid template 2 μ l add ddH
2O complements to 25 μ l.PCR forward primer sequence 5 '-GACAAGGCTCGGAGACACGCA-3 '; PCR reverse primer sequence 5 '-TCTCTTTCATGCTGCGGTTCAGC-3 '.
(4) real-time quantitative PCR response procedures: 95 ℃ of pre-sex change of 5min, carry out 30 circulations with 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30s then, 80 ℃ of 1s, 82 ℃ 1s1 circulation, last 59 ℃ to 95 ℃ are extended 20s.At last, utilize accompanying software to carry out melting curve analysis and the analysis of CT value, determine plasmid copy number.
3, the extracting method of the material of degraded tetraodotoxin comprises the steps:
(1) the Aeromonas fermentation culture that will lose plasmid is after 48 hours, and 4 ℃ are descended the centrifugal 30min collecting cells of 4000 * g;
(2) with 0.1% acetic acid dissolved cell, ultrasonic disruption cell;
(3) 100 ℃ are boiled 20-25min, the centrifugal removal cell debris in cooling back;
(4) supernatant diameter 0.25 μ m filter paper filtering;
(5) filtered liquid is crossed active carbon column, and with elutriant wash-out activated carbon, the prescription of elutriant is: methyl alcohol: acetic acid solution=20:80 of 1%;
(6) elutriant is decompressed to 0.001-0.003M Pa for 45 ℃ and concentrates lyophilize;
(7) be dissolved in the 2ml aseptic deionized water;
(8) cross 1 * 80cm Bio-Gel P2 pillar (Bio-Rad Lab, Richmond, VA, USA);
(9) with 0.03M acetic acid wash-out, begin to collect from the elutriant outflow, collect 2ml;
(10) elutriant again with C18Sep-Pak cartridges cross post (Waters, Milford, MA);
(11) with the acetic acid wash-out of 10ml0.3%, flow out collection since second column volume elutriant, collect the elutriant of two column volumes;
(12) elutriant is filtered;
(13) after the filtered liquid lyophilize, be dissolved in the 1ml aseptic deionized water.
(14) with filtrate as sample to be tested, adjusting pH value be 6.5-7.4, the tetraodotoxin in the usefulness ELISA method detection sample.
4, the detection of tetraodotoxin in the fermented extracted sample comprises the steps:
(1) adopts commercially available tetraodotoxin ELISA detection kit (Beijing centre halfback's food sanitation science and technology company, production number: 5561), on the enzyme plate that antigen is fixedly arranged, add 50 μ L TTX standard substance (Sigma-Aldrich companies respectively, production number: T8024-1MG) (0,10,20,50,200ng/ml), (final concentration is 50ng/mL to sample to be tested, sample to be tested 100ng/mL) and check sample (substratum) with having added the TTX standard substance;
(2) add the specific antibody solution (50 μ L/ hole) of TTX at enzyme plate;
Hatch 1.5h for (3) 37 ℃, add washings detersive enzyme target 3 times;
(4) every hole adds 100 μ L ELIAS secondary antibody, hatches 1h for 37 ℃, adds washings detersive enzyme target 3 times;
(5) every hole adds 100 μ L colour developing liquid, hatches 12min for 37 ℃, and every hole adds stop buffer 50 μ L;
(6) result detects: enzyme plate is inserted in the microplate reader, detect the light absorption value at 450nm place.
(7) ratio with Ai/Ao is ordinate zou, and the logarithmic value of TTX standard substance concentration is that X-coordinate is set up typical curve, obtains the concentration of tetraodotoxin in the sample according to typical curve.Wherein Ai is the light absorption value of sample, the light absorption value when Ao is 0ng/mL for standard substance concentration.The result as shown in Figure 2, the logarithmic value of absorbance ratio (Ai/Ao) and tetraodotoxin standard substance concentration is proportional, the equation of its fitting a straight line is y=-0.393x+1.2543, wherein " y " is the Ai/Ao value, " x " is tetraodotoxin concentration.According to this formula, can calculate tetraodotoxin concentration in the sample according to detecting sample gained light absorption value.
Embodiment 2
The working method of present embodiment and step are with embodiment 1, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 66 hours.
Embodiment 3
The working method of present embodiment and step are with embodiment 1, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 96 hours.
Embodiment 4
The working method of present embodiment and step are as embodiment 1, and difference is to change the arginic ORI liquid nutrient medium of interpolation into interpolation filefish liver crude extract.ORI liquid nutrient medium preparation method (1000ml) is: add 2g Tryptones (OXOID company) in the 800ml deionized water, 2g soy peptone (OXOID company), 1g yeast extract (OXOID company), 30g sodium-chlor (Yixing City Chemical Reagent Plant No.2), after the stirring and dissolving, add 0.88g ironic citrate (Chemical Reagent Co., Ltd., Sinopharm Group) again, continue to stir, after dissolving fully, add 10ml filefish liver crude extract, add deionized water again and be settled to 1000ml, and seal sterilization after regulating PH to 8.0.
Embodiment 5
The working method of present embodiment and step are as embodiment 4, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 66 hours.
Embodiment 6
The working method of present embodiment and step are as embodiment 4, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 96 hours.
Embodiment 7
The working method of present embodiment and step are as embodiment 1, and difference is to change the arginic ORI liquid nutrient medium of interpolation into interpolation Fugu ocellatus ovary crude extract.ORI liquid nutrient medium preparation method (1000ml) is: add 2g Tryptones (OXOID company) in the 800ml deionized water, 2g soy peptone (OXOID company), 1g yeast extract (OXOID company), 30g sodium-chlor (Yixing City Chemical Reagent Plant No.2), after the stirring and dissolving, add 0.88g ironic citrate (Chemical Reagent Co., Ltd., Sinopharm Group) again, continue to stir, after dissolving fully, add 10ml Fugu ocellatus ovary crude extract, add deionized water again and be settled to 1000ml, and seal sterilization after regulating PH to 8.0.
Embodiment 8
The working method of present embodiment and step are as embodiment 7, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 66 hours.
Embodiment 9
The working method of present embodiment and step are as embodiment 7, and difference is that the Aeromonas fermented incubation time that will lose plasmid is 96 hours.
Detect the variation of the amount of the TTX in the sample by contrast contrast and each, can draw extracting solution to the degradation effect of TTX.Table 1 is the TTX degradation effect comparison to different fermentations time extracting solution.
The TTX degradation effect of table 1 different fermentations time extracting solution relatively
As shown in Table 1, compared to control medium (ORI) sample, the TTX content in 66h and the 96h nutrient solution all is lower than check sample; Increase along with TTX standard substance addition, TTX amount in check sample, 66h and the 96h nutrient solution sample also increases thereupon, but the TTX content in 66h and the 96h nutrient solution still is lower than check sample, and the TTX content in the 96h fermentation sample is lower than 66h fermentation sample.The above results shows the material that contains the TTX that degrades in 66h and the 96h fermentation sample, adopts extracting method of the present invention and detection method can successfully detect content in the tetraodotoxin degradation material extracting solution.
In the present invention, adopt the method that changes medium component to cause Aeromonas Aeromonas molluscorum plasmid loss, utilize real-time quantitative PCR to detect the plasmid loss situation, from the bacterium of plasmid loss, extract the material of degraded tetraodotoxin, adopt the competitiveness enzyme linked immunosorbent assay to measure the ability of degraded tetraodotoxin.The material of the degraded tetraodotoxin that the present invention extracts can be used for preparing the medicine of clinical treatment tetraodotoxin poisoning from now on.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (16)
1. a method of extracting the material of degraded tetraodotoxin is characterized in that, comprises the steps:
(1) fermentation using bacteria of the product tetraodotoxin of plasmid loss was cultivated centrifugal collecting cell at least 48 hours;
(2) with 0.1%-1% acetic acid dissolved cell, ultrasonic disruption cell;
(3) 95-105 ℃ is boiled 20-25min, the centrifugal removal cell debris in cooling back;
(4) filter supernatant;
(5) filtered liquid is crossed active carbon column, the prescription of elutriant is: methyl alcohol: acetic acid solution=20:80 of 1%;
(6) elutriant is in 40-50 ℃ of concentrating under reduced pressure, lyophilize;
(7) be dissolved in the aseptic deionized water;
(8) cross Bio-Gel P2 pillar;
(9) with 0.02-0.05M acetic acid wash-out, collect elutriant;
(10) elutriant is crossed post with C18Sep-Pak cartridges again;
(11) with the acetic acid wash-out of 5-20ml0.3%, collect elutriant;
(12) elutriant is filtered;
(13) after the filtered liquid lyophilize, be dissolved in the aseptic deionized water extracting solution of the material of the tetraodotoxin that obtains degrading.
2. the method for the material of extraction degraded tetraodotoxin as claimed in claim 1 it is characterized in that the product tetraodotoxin bacterium of described plasmid loss is the mollusk Aeromonas, or other is produced the bacterium of tetraodotoxin.
3. the method for the material of extraction as claimed in claim 1 or 2 degraded tetraodotoxin is characterized in that described method further comprises following detection step:
As sample to be tested, adjusting the pH value is 6.5-7.4 with filtrate, with the tetraodotoxin in the ELISA method detection sample.
4. the method for the material of extraction degraded tetraodotoxin as claimed in claim 1 or 2 is characterized in that, causes described product tetraodotoxin bacterium to lose artificial fermentation's cultural method of plasmid, comprises the steps:
(1) with the microbionation of described product tetraodotoxin in adding arginic ORI liquid nutrient medium;
(2) under 20-28 ℃, 200-450 rev/min, cultivated at least 48 hours.
5. the method for the material of extraction as claimed in claim 4 degraded tetraodotoxin, it is characterized in that, consisting of of described ORI liquid nutrient medium: 0.05% arginine, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, pH7-8.5.
6. want the method for the material of 4 described extraction degraded tetraodotoxin as right, it is characterized in that described ORI liquid nutrient medium preparation method: in the 800ml deionized water, add 2g Tryptones, 2g soy peptone, the 1g yeast extract, 30g sodium-chlor and 0.5g L-arginine after the stirring and dissolving, add the 0.88g ironic citrate again, continue to stir, after dissolving fully, add deionized water and be settled to 1000ml, and seal sterilization after regulating PH to 7-8.5.
7. the method for the material of extraction degraded tetraodotoxin as claimed in claim 3 is characterized in that the ELISA method in the described detection step detects and comprises the steps:
(1) on the enzyme plate that antigen is fixedly arranged, adds the tetraodotoxin standard substance respectively, sample to be tested and the sample to be tested that has added the tetraodotoxin standard substance;
(2) add the specific antibody solution of tetraodotoxin at enzyme plate;
(3) hatch to reacting completely for 20-40 ℃, add washings detersive enzyme target 3 times;
(4) add ELIAS secondary antibody, hatch to reacting completely for 20-40 ℃, add washings detersive enzyme target 3 times;
(5) add colour developing liquid, 37 ℃ hatch 10-15min after, every hole adds stop buffer with termination reaction;
(6) result detects: enzyme plate is inserted in the microplate reader, detect the light absorption value at 450nm place.
8. the method for the material of extraction degraded tetraodotoxin as claimed in claim 7 is characterized in that, in the step of described ELISA method (1), added the sample to be tested of tetraodotoxin standard substance, it is 25-200ng/mL that its tetraodotoxin standard substance add final concentration.
9. the method for the material of extraction degraded tetraodotoxin as claimed in claim 7 is characterized in that the specific antibody in the step of described ELISA method (2) is the specific monoclonal antibody of tetraodotoxin.
10. the method for the material of extraction as claimed in claim 7 degraded tetraodotoxin is characterized in that, in the step of described ELISA method (3), hatch to the required time that reacts completely be 1.5h; Hatch in the step of described ELISA method (4) and be 1h to reacting completely.
11. the artificial fermentation's cultural method that makes the bacterium that produces tetraodotoxin lose plasmid is characterized in that, comprises the steps:
(1) with the microbionation of described product tetraodotoxin in adding arginic ORI liquid nutrient medium;
Under (2) 23 ℃, 225 rev/mins, cultivated at least 48 hours.
12. as described in claim 11, cause the bacterium that produces tetraodotoxin to lose artificial fermentation's cultural method of plasmid, it is characterized in that the bacterium of described product tetraodotoxin is for producing the Aeromonas of tetraodotoxin.
13. as described in claim 11 or 12, cause the bacterium that produces tetraodotoxin to lose artificial fermentation's cultural method of plasmid, it is characterized in that, consisting of of described ORI liquid nutrient medium: 0.05% arginine, 0.2% Tryptones, 0.2% soy peptone, 0.1% yeast extract, 0.088% ironic citrate, 3%NaCl, surplus is distilled water, pH7-8.5.
14. a real-time quantitative PCR detection method, it is characterized in that for detection of the plasmid loss as the claim 1-13 bacterium that method adopts as described in each, comprises the steps:
(1) quantitative PCR standard curve making: the Ne-1 plasmid that extracts is carried out 10 times of serial dilutions with sterilized water, be diluted to 6 gradients respectively, as template, carry out quantitative fluorescent PCR and set up typical curve, calculate plasmid copy number as follows:
Plasmid copy number (copies μ L
-1)=6.02 * 10
23(copiesmol
-1) * plasmid concentration (g μ L
-1)/plasmid molecule amount (gmol
-1);
(2) real-time quantitative PCR system: testing sample and standard substance are done 3 respectively repeat to place with experiment once and react, press following set of dispense PCR reaction solution processed: iQ SYBR Green Supermix12.5 μ l, PCR forward primer 1 μ l, PCR reverse primer 1 μ l, plasmid template 2 μ l add ddH
2O complements to 25 μ l, PCR forward primer sequence 5 '-GACAAGGCTCGGAGACACGCA-3 ', and PCR reverse primer sequence 5 '-TCTCTTTCATGCTGCGGTTCAGC-3 ', described testing sample are described bacterium of losing plasmid;
(3) real-time quantitative PCR response procedures: 95 ℃ of pre-denatured plasmid dna templates of 5min, carry out 30 temperature variation circulations with 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30s then, 80 ℃ of 1s, 82 ℃ of 1s carry out 1 circulation, last 59 ℃ to 95 ℃ are extended 20s, at last, utilize accompanying software to carry out melting curve analysis and the analysis of CT value, determine plasmid copy number;
(4) utilization contains arginic ORI culture medium culturing at least 48 hours, and plasmid is lost fully.
15. extracting solution that extracts the tetraodotoxin degradation material for preparing according to each described method among the claim 1-10.
16. the application of the extracting solution of tetraodotoxin degradation material as claimed in claim 15 in the medicine of poisoning for the preparation of the treatment tetraodotoxin.
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| CN104391061A (en) * | 2014-10-31 | 2015-03-04 | 浙江省海洋水产研究所 | Method for determining tetrodotoxin in marine organisms by utilizing immunoaffinity column purification-liquid phase chromatography-tandem mass spectrometry |
| CN106365327A (en) * | 2016-10-25 | 2017-02-01 | 嘉善蓝欣涂料有限公司 | Usage of a distiller's yeast in degrading tetrodotoxin and performing purification treating on water body having tetrodotoxin |
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| CN108977505A (en) * | 2018-08-08 | 2018-12-11 | 浙江海洋大学 | A kind of quick screening generate tetraodotoxin microbial strains method and Digoxigenin labeled DNA probe used |
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| CN104391061B (en) * | 2014-10-31 | 2016-01-06 | 浙江省海洋水产研究所 | The method of tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life |
| CN106365327A (en) * | 2016-10-25 | 2017-02-01 | 嘉善蓝欣涂料有限公司 | Usage of a distiller's yeast in degrading tetrodotoxin and performing purification treating on water body having tetrodotoxin |
| CN108576577A (en) * | 2018-06-13 | 2018-09-28 | 上海海洋大学 | The method and medicament of fast degradation tetraodotoxin |
| CN108576577B (en) * | 2018-06-13 | 2021-06-25 | 上海海洋大学 | Method and medicament for rapidly degrading tetrodotoxin |
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| CN108977505B (en) * | 2018-08-08 | 2022-03-22 | 浙江海洋大学 | Method for rapidly screening microbial strains generating tetrodotoxin and digoxin labeled DNA probe used by same |
| CN115772488A (en) * | 2022-12-20 | 2023-03-10 | 中国水产科学研究院黄海水产研究所 | Shewanella decolorationis for producing tetrodotoxin and application thereof |
| US12116568B2 (en) | 2022-12-20 | 2024-10-15 | Yellow Sea Fisheries Research Institute, Chinese Academy Of Fishery Sciences | Shewanella decolorationis producing tetrodotoxin and application thereof |
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