CN102174098B - Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin - Google Patents
Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin Download PDFInfo
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- CN102174098B CN102174098B CN 201110027667 CN201110027667A CN102174098B CN 102174098 B CN102174098 B CN 102174098B CN 201110027667 CN201110027667 CN 201110027667 CN 201110027667 A CN201110027667 A CN 201110027667A CN 102174098 B CN102174098 B CN 102174098B
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Abstract
The invention relates to the technical field of marine organisms, in particular to a method for separating and purifying a cyanea nozakii lethal toxin protein. The method comprises the following steps of: ultrasonically crushing cyanea nozakii nematocyst cells to extract a jellyfish crude toxin, dialyzing and desalting, and separating and purifying by affinity chromatographic separation and an anion exchange chromatographic technology in turn to obtain a jellyfish toxin protein with lethal activity, wherein through measurement, the molecular weight of the toxin protein is 30kDa, and the lethal dose 50 (LD50) of the toxin protein to 1g of zebra fish is 0.56mu g. The method has the characteristics that: the method is quick, simple and convenient and provides important guidance for obtaining the cyanea nozakii lethal toxin protein.
Description
Technical field
The present invention relates to biotechnology, specifically a kind of from rosy clouds jellyfish (Cyanea nozakii) stinging capsule cytotoxin the method for separation and purification lethal toxin albumen.
Background technology
Jellyfish (jellyfish) is a kind of zooplankton of colloidal substance, mainly comprises four big monoids: the Ctenophora of cnidarian hydromedusa, siphonohore class, scyphomedusa and Ctenophora.Jellyfish is of a great variety, and is widely distributed, nearly about 1000 kinds of the jellyfish kind that has recorded in the world, and what wherein China marine site was known has 400 kinds approximately.China is one of very long country in shoreline, extends across cool temperature zone, temperate zone, subtropics and the torrid zone from north to south, and almost all has a large amount of jellyfishes to distribute in the coastland of China, and is wherein particularly abundant with rosy clouds jellyfish quantity.In recent years, jellyfish often breaks out, and causes numerous swimmers to be stung, and fash, redness, itch appear in the lighter's skin, and order agonizing bitten the wounded, and weight person faints, shock even dead.And why jellyfish can bring so big harm to people, and most important reason is: the stinging capsule cell that is present on the jellyfish tentacle contains a large amount of medusocongestins.Medusocongestin mainly is protein matter, wherein comprise the multiple different bioactive toxin proteins that have, as hemolytic activity, enzymic activity, anti-oxidant activity, lethal toxicity, cardiovascular toxicity, hepatotoxicity etc., and medusocongestin is as the albumen of a class formation novelty, for the marine drug of development of new provides important lead compound.
But, up to the present the research to medusocongestin mainly concentrates on the thick malicious aspect of jellyfish, its major cause has following several respects: first, contain many kinds of protein ingredients in the thick toxin of jellyfish, wherein the character between some component (as: molecular weight, electrically charged number, hydrophobicity etc.) is very close, and this has brought very big trouble with regard to the separation and purification of giving medusocongestin; The second, medusocongestin is protein matter, has heat sensitivity, is subjected to Effect of Environmental such as temperature easily and causes its active reduction even forfeiture, and this just causes and be difficult to keep its activity in the separation and purification process.The 3rd, the difference of jellyfish kind, also there are bigger difference in toxin composition and activity contained in its physiological structure and the body, and this just causes the separation and purification work of different sorts medusocongestin to be difficult to directly imitate, and can only grope and could obtain by numerous and diverse experiment.So the separation and purification work of medusocongestin has very big difficulty.
Summary of the invention
The purpose of this invention is to provide a kind of method of separating lethal toxin albumen in the rosy clouds jellyfish stinging capsule toxin.
For achieving the above object, the technical solution used in the present invention is:
A kind of method of from rosy clouds jellyfish stinging capsule toxin, separating lethal toxin albumen:
1) rosy clouds jellyfish stinging capsule cell is added fragmentation in the precooling damping fluid, 2～6 ℃ of broken backs are centrifugal, collect supernatant liquor, and are stand-by;
2) with above-mentioned steps 1) supernatant liquor is in 2～6 ℃ of following damping fluid dialysed overnight of using, and low-temperature centrifugation is collected supernatant liquor then, and is stand-by;
3) with step 2) the gained supernatant liquid filtering, then separate with containing Con A Sepharose 4B affinity column, adopt the pH7.4 that contains 0.1～0.3M Alpha-Methyl-D-mannoside and 0.1～0.3MNaCl during separation, the Tris-HCl damping fluid of concentration 10～30mM carries out wash-out and collects elutriant, low-temperature centrifugation, collect supernatant liquor, stand-by;
4) supernatant liquor that step 3) is obtained reinforcing yin essence ion exchange resin Mono Q prepacked column purifies and separates, adopt pH7.8 successively, concentration is the Tris-HCl damping fluid of 10～30mM and the pH7.8 that contains 1M NaCl, concentration is that the Tris-HCl damping fluid of 10～30mM carries out gradient elution, and flow velocity is 0.2～0.5mLmin
-1, collect about 25～40min elutriant, be and from rosy clouds jellyfish stinging capsule toxin, separate lethal toxin albumen.
Described step 1) rosy clouds jellyfish stinging capsule cell is: will place the rosy clouds jellyfish tentacle of-80～-20 ℃ of low temperature in 2～6 ℃ of self-dissolving 12～48h, utilize 20～60 purpose standard sub-sieve elimination tentacle relics after the self-dissolving, then at 2～6 ℃ of following centrifugal 5～20min of 10000～15000g, collection bottom precipitation, 2～6 ℃ of lyophilizes, stand-by.
Described step 1) adds pH7.8 to rosy clouds jellyfish stinging capsule cell, in 2～6 ℃ of following precooling Tris-HCl damping fluids of concentration 10～30mM, then being placed under the ice bath with ultrasonic power is under 200～400kw among broken 20～60min, and work/gap is 5s/30s.
Described step 2) centrifugal condition is under 2～6 ℃, the centrifugal 5～20min of 10000～15000g.
Described step 2) damping fluid is for containing 1～3mM MnCl in
2, 1～3mM CaCl
2With the pH7.4 of 0.1～0.3M NaCl, the Tris-HCl of concentration 10～30mM.
During filtration in the described step 3) with step 2) supernatant liquor collected is the filtering with microporous membrane of 0.22 μ m with the aperture.
In the described step 3) with step 2) the gained supernatant liquid filtering, then separate with containing Con A Sepharose4B affinity column, at first adopt to contain 1～3mM MnCl
2, 1～3mM CaCl
2PH7.4 with 0.1～0.3MNaCl, the Tris-HCl damping fluid of concentration 10～30mM is washed unconjugated albumen off, adopt the pH7.4 that contains 0.1～0.3M Alpha-Methyl-D-mannoside and 0.1～0.3M NaCl then, the Tris-HCl damping fluid of concentration 10～30mM carries out wash-out and collects elutriant, then the elution fraction of collecting is adopted pH7.8, the dialysis of 10～30mM Tris-HCl damping fluid, low-temperature centrifugation in 2～6 ℃, collect supernatant liquor, stand-by.
Advantage of the present invention:
1. the method for the present invention's separation and purification rosy clouds jellyfish lethal toxin albumen from the rosy clouds jellyfish is for the purifying of medusocongestin albumen provides the method guidance.
2. the present invention adopts anionite Con A Sepharose 4B affinity column to separate lethal toxin albumen with reinforcing yin essence ion Mono Q prepacked column, have the advantages that flow velocity is fast, resolving power is high, productive rate is high, and separating step is few, only just can access purer lethal toxin albumen through two step column chromatography for separation, shorten disengaging time effectively, reduced the loss of activity of lethal toxin albumen.
Description of drawings
The tomographic map of the Mono Q separation of C on A Sepharose 4B elutriant that Fig. 1 provides for the embodiment of the invention.
The SDS-PAGE electrophorogram of the molecular sieve Mono Q lethal toxin protein peak that Fig. 2 embodiment of the invention provides.
The employing Bradford method that Fig. 3 embodiment of the invention provides is measured the typical curve of protein concentration
Embodiment
Embodiment 1
1) preparation of rosy clouds medusocongestin stinging capsule cell: the rosy clouds jellyfish tentacle that 1kg is freezing is after 2 ℃ of following self-dissolvings are spent the night, utilize 20 purpose standard sub-sieve elimination tentacle relics, get supernatant liquor then and at 10000g, 4 ℃ freezing high speed centrifugation 20mi n and collect lower sediment down, use 0.154molL
-1The physiological saline of NaCl solution is clarification in 2 ℃ of repetitive scrubbings 3 times to upper strata liquid, and with after the lyophilize of stinging capsule cell precipitation ,-20 ℃ freezing preservations are standby down.
2) extraction of rosy clouds medusocongestin: get step 1) rosy clouds jellyfish stinging capsule cell 0.5g and place beaker at the 20mL of 2 ℃ of following precoolings pH7.810mM Tris-HCl damping fluid, be under the 200kw among the broken 30min with ultrasonic power under ice bath, work/gap is 5s/30s.Fragmentation finishes the back under 4 ℃, and the centrifugal 20min of 10000g, supernatant liquor are rosy clouds jellyfish stinging capsule cytotoxin, and use the Xylene Brilliant Cyanine G method, are standard with the bovine serum albumin, measures the cytotoxic protein concentration of rosy clouds jellyfish stinging capsule in the supernatant liquor.
3) affinity chromatography Con A Sepharose 4B separates the rosy clouds medusocongestin:
1. go up the preceding The pretreatment of sample: the rosy clouds jellyfish stinging capsule toxin that extracts is adopted down in 2 ℃ contain 1mM MnCl
2, 1mM CaCl
2With the pH7.4 of 0.15M NaCl, 10mM Tris-HCl damping fluid dialysed overnight, 10000g frozen centrifugation 20min abandons precipitation then, collects supernatant liquor.
2. go up sample and wash-out: with Con A Sepharose 4B post (1.0x5cm) on the above-mentioned sample of handling well, flow velocity is 0.2mLmin
-1, to contain the pH7.4 of 0.1M Alpha-Methyl-D-mannoside and 0.1MNaCl, 10mM Tris-HCl buffer solution elution is collected elutriant then.
4) reinforcing yin essence ion Mono Q separates
1. go up the preceding The pretreatment of sample: in 2 ℃ of employing pH7.810mMTris-HCl damping fluid dialysed overnight, 10000g frozen centrifugation 20min abandons precipitation then, collects supernatant liquor with above-mentioned collection component.
2. go up sample and wash-out: get Mono Q prepacked column (0.5/5cm) purifies and separates on the above-mentioned sample of handling well, adopt pH7.820mM Tris-HCl damping fluid and the pH7.810mMTris-HCl damping fluid that contains 1M NaCl to carry out gradient elution successively, flow velocity is 0.2mLmin
-1, collect each elution peak and the utilization molecular weight that dams and for the ultrafiltration pipe of 3kDa pH7.8 20mM Tris-HCl damping fluid carried out concentrating and desalinating (referring to Fig. 1).
5) SDS-PAGE purity detecting: each elution peak that step 4) Mono Q prepacked column is separated to carries out its purity of SDS-PAGE electrophoresis detection (referring to Fig. 2).
6) the active detection that causes death: experimental group is to inject the pH7.820mM Tris-HCl damping fluid that contains single albumen after the above-mentioned detection of 3,5,8,12,15,20 μ L respectively to the zebra fish afterbody with microsyringe, observe the behavior of zebra fish then and survival state in 24 hours, simultaneously respectively to zebra fish afterbody injection 3,5,8,12,15,20 μ L pH7.820mM Tris-HCl damping fluids as blank.Wherein each gradient of experimental group and control group is all used 10 zebra fish parallel laboratory tests.Experiment shows, separation and purification to rosy clouds medusocongestin albumen to zebra fish have cause death active, its Lethal Dose 50 LD
50Be about 0.56 μ g toxin protein/g. fish, wherein the measuring method of protein concentration adopts the Bradford method, utilizes BSA to do typical curve (referring to Fig. 3).
Embodiment 2
1) preparation of rosy clouds medusocongestin stinging capsule cell: the rosy clouds jellyfish tentacle that 2kg is freezing is after 4 ℃ of following self-dissolvings are spent the night, utilize 40 purpose standard sub-sieve elimination tentacle relics, get supernatant liquor then and at 12000g, 4 ℃ freezing high speed centrifugation 10min and collect lower sediment down, use 0.154molL
-1The physiological saline of NaCl solution is clarification in 4 ℃ of repetitive scrubbings 3 times to upper strata liquid, and with after the lyophilize of stinging capsule cell precipitation ,-20 ℃ freezing preservations are standby down.
2) extraction of rosy clouds medusocongestin: get step 1) rosy clouds jellyfish stinging capsule cell 1.0g and place beaker at the 30mL of 4 ℃ of following precoolings pH7.820mM Tris-HCl damping fluid, be under the 300kw among the broken 30min with ultrasonic power under ice bath, work/gap is 5s/30s.Fragmentation finishes the back under 4 ℃, and the centrifugal 10min of 12000g, supernatant liquor are rosy clouds jellyfish stinging capsule cytotoxin, and use the Xylene Brilliant Cyanine G method, are standard with the bovine serum albumin, measures the cytotoxic protein concentration of rosy clouds jellyfish stinging capsule in the supernatant liquor.
3) affinity chromatography Con A Sepharose 4B separates the rosy clouds medusocongestin:
1. go up the preceding The pretreatment of sample: the rosy clouds jellyfish stinging capsule toxin that extracts is adopted down in 4 ℃ contain 2mM MnCl
2, 2mM CaCl
2The pH7.4 of nuclear 0.2M NaCl, 20mM Tris-HCl damping fluid dialysed overnight, 12000g frozen centrifugation 10min abandons precipitation then, collects supernatant liquor.
2. go up sample and wash-out: with Con A Sepharose 4B post (1.6x10cm) on the above-mentioned sample of handling well, flow velocity is 0.3mLmin
-1, to contain the pH7.4 of 0.2M Alpha-Methyl-D-mannoside and 0.2M NaCl, 20mM Tris-HCl buffer solution elution is collected elutriant then.
4) reinforcing yin essence ion Mono Q separates
1. go up the preceding The pretreatment of sample: 4 ℃ of said components are adopted pH7.820mM Tris-HCl damping fluid dialysed overnight, and 12000g frozen centrifugation 10min abandons precipitation then, collects supernatant liquor.
2. go up sample and wash-out: get Mono Q prepacked column (0.5/5cm) purifies and separates on the above-mentioned sample of handling well, adopt pH7.820mM Tris-HCl damping fluid and the pH7.810mMTris-HCl damping fluid that contains 1M NaCl to carry out gradient elution successively, flow velocity is 0.2mLmin
-1, collect each elution peak and the utilization molecular weight that dams and for the ultrafiltration pipe of 3kDa pH7.820mM Tris-HCl damping fluid carried out concentrating and desalinating.
5) SDS-PAGE purity detecting: each elution peak that step 4) Mono Q prepacked column is separated to carries out its purity of SDS-PAGE electrophoresis detection.
6) deadly active detection: experimental group is to inject the above-mentioned pH7.820mM Tris-HCl damping fluid that contains single albumen of 3,5,8,12,15,20 μ L respectively to the zebra fish afterbody with microsyringe, observe the behavior of zebra fish then and survival state in 24 hours, simultaneously respectively to zebra fish afterbody injection 3,5,8,12,15,20 μ L pH7.820mM Tris-HCl damping fluids as blank.Wherein each gradient of experimental group and control group is all used 10 zebra fish parallel laboratory tests.Experiment shows, separation and purification to rosy clouds medusocongestin albumen to zebra fish have cause death active, its Lethal Dose 50 LD
50Be about 0.58 μ g toxin protein/g. fish, wherein the measuring method of protein concentration adopts the Bradford method, utilizes BSA to do typical curve.
Embodiment 3
1) preparation of rosy clouds medusocongestin stinging capsule cell: the rosy clouds jellyfish tentacle that 3kg is freezing is after 6 ℃ of following self-dissolvings are spent the night, utilize 60 purpose standard sub-sieve elimination tentacle relics, get supernatant liquor then and at 15000g, 6 ℃ freezing high speed centrifugation 5min and collect lower sediment down, use 0.154molL
-1The physiological saline of NaCl solution is clarification in 6 ℃ of repetitive scrubbings 3 times to upper strata liquid, and with after the lyophilize of stinging capsule cell precipitation ,-20 ℃ freezing preservations are standby down.
2) extraction of rosy clouds medusocongestin: get step 1) rosy clouds jellyfish stinging capsule cell 2.0g and place beaker at the 50mL of 6 ℃ of following precoolings pH7.830mM Tris-HCl damping fluid, be under the 400kw among the broken 60min with ultrasonic power under ice bath, work/gap is 5s/30s.Fragmentation finishes the back under 6 ℃, and the centrifugal 5min of 15000g, supernatant liquor are rosy clouds jellyfish stinging capsule cytotoxin, and use the Xylene Brilliant Cyanine G method, are standard with the bovine serum albumin, measures the cytotoxic protein concentration of rosy clouds jellyfish stinging capsule in the supernatant liquor.
3) affinity chromatography Con A Sepharose 4B separates the rosy clouds medusocongestin:
1. go up the preceding The pretreatment of sample: the rosy clouds jellyfish stinging capsule toxin that extracts is adopted down in 6 ℃ contain 3mM MnCl
2, 3mM CaCl
2With the pH7.4 of 0.3M NaCl, 30mM Tris-HCl damping fluid dialysed overnight, 15000g frozen centrifugation 5min abandons precipitation then, collects supernatant liquor.
2. go up sample and wash-out: with Con A Sepharose 4B post on the above-mentioned sample of handling well, flow velocity is 0.5mLmin
-1, adopting the pH7.4 that contains 0.3M Alpha-Methyl-D-mannoside nuclear 0.3M NaCl then, 30mM Tris-HCl buffer solution elution is collected elutriant.
4) reinforcing yin essence ion Mono Q separates
1. go up The pretreatment before the sample: to pH7.830mM Tris-HCl damping fluid dialysed overnight, 15000g frozen centrifugation 5min abandons precipitation then, collects supernatant liquor with 6 ℃ of said components.
2. go up sample and wash-out: get Mono Q prepacked column (0.5/5cm) purifies and separates on the above-mentioned sample of handling well, adopt pH7.820mM Tris-HCl damping fluid and the pH7.810mMTris-HCl damping fluid that contains 1M NaCl to carry out gradient elution successively, flow velocity is 0.2mLmin
-1, collect each elution peak and the utilization molecular weight that dams and for the ultrafiltration pipe of 3kDa pH7.820mM Tris-HCl damping fluid carried out concentrating and desalinating.
5) SDS-PAGE purity detecting: each elution peak that step 4) Mono Q prepacked column is separated to carries out its purity of SDS-PAGE electrophoresis detection.
6) deadly active detection: experimental group is to inject the above-mentioned pH7.820mM Tris-HCl damping fluid that contains single albumen of 3,5,8,12,15,20 μ L respectively to the zebra fish afterbody with microsyringe, observe the behavior of zebra fish then and survival state in 24 hours, simultaneously respectively to zebra fish afterbody injection 3,5,8,12,15,20 μ L pH7.820mM Tris-HCl damping fluids as blank.Wherein each gradient of experimental group and control group is all used 10 zebra fish parallel laboratory tests.Experiment shows, separation and purification to rosy clouds medusocongestin albumen to zebra fish have cause death active, its Lethal Dose 50 LD
50Be about 0.54 μ g toxin protein/g. fish, wherein the measuring method of protein concentration adopts the Bradford method, utilizes BSA to do typical curve.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. method of separating lethal toxin albumen from white rosy clouds jellyfish (Cyanea nozakii) stinging capsule toxin is characterized in that:
1) white rosy clouds jellyfish stinging capsule cell is added fragmentation in the precooling damping fluid, 2～6 ℃ of broken backs are centrifugal, collect supernatant liquor, and are stand-by;
2) with above-mentioned steps 1) supernatant liquor is in 2～6 ℃ of following damping fluid dialysed overnight of using, and low-temperature centrifugation is collected supernatant liquor then, and is stand-by;
Described damping fluid is for containing 1～3mM MnCl
2, 1～3mM CaCl
2With the pH7.4 of 0.1～0.3M NaCl, the Tris-HCl of concentration 10～30mM;
3) with step 2) the gained supernatant liquid filtering, be that the Con A Sepharose4B affinity column of 1.0 * 5cm separates with specification then, be 0.2mLmin with the flow velocity
-1Employing contains the pH7.4 of 0.1～0.3M Alpha-Methyl-D-mannoside and 0.1～0.3M NaCl, the Tris-HCl damping fluid of concentration 10～30mM carries out wash-out, and collection elutriant, then the elution fraction of collecting is adopted pH7.8 in 2～6 ℃, the dialysis of 10～30mM Tris-HCl damping fluid, low-temperature centrifugation is collected supernatant liquor, and is stand-by;
4) the supernatant liquor specification that step 3) is obtained is the reinforcing yin essence ion exchange resin Mono Q prepacked column purifies and separates of 0.5 * 5cm, adopt pH7.8 successively, concentration is the Tris-HCl damping fluid of 20mM and the pH7.8 that contains 1M NaCl, concentration is that the Tris-HCl damping fluid of 10mM carries out gradient elution, and flow velocity is 0.2mLmin
-1, collect 25～40min elutriant, be and from white rosy clouds jellyfish stinging capsule toxin, separate lethal toxin albumen.
2. by the described method of from white rosy clouds jellyfish stinging capsule toxin, separating lethal toxin albumen of claim 1, it is characterized in that: the white rosy clouds jellyfish stinging capsule cell of described step 1) adopts following method to obtain: will place the white rosy clouds jellyfish tentacle of-80～-20 ℃ of low temperature in 2～6 ℃ of self-dissolving 12～48h, utilize 20～60 purpose standard sub-sieve elimination tentacle relics after the self-dissolving, then at 2～6 ℃ of following centrifugal 5～20min of 10000～15000g, collection bottom precipitation, 2～6 ℃ of lyophilizes, stand-by.
3. by claim 1 or 2 described methods of from white rosy clouds jellyfish stinging capsule toxin, separating lethal toxin albumen, it is characterized in that: in described step 1) white rosy clouds jellyfish stinging capsule cell, add pH7.8, the Tris-HCl damping fluid of 2～6 ℃ of following precoolings of concentration 10～30mM, then be placed in the ice bath, be broken 20～60min under 200～400kw condition at ultrasonic power, work/gap is 5s/30s.
4. by the described method of separating lethal toxin albumen from white rosy clouds jellyfish stinging capsule toxin of claim 1, it is characterized in that: described step 2) centrifugal condition is under 2～6 ℃, the centrifugal 5～20min of 10000～15000g.
5. by the described method of from white rosy clouds jellyfish stinging capsule toxin, separating lethal toxin albumen of claim 1, it is characterized in that: during filtration in the described step 3) with step 2) supernatant liquor collected is the filtering with microporous membrane of 0.22 μ m with the aperture.
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| CN106749590A (en) * | 2016-12-22 | 2017-05-31 | 中国科学院海洋研究所 | A kind of method for isolating and purifying rosy clouds medusocongestin albumen |
| CN109096382A (en) * | 2018-09-06 | 2018-12-28 | 中国科学院海洋研究所 | A method of separating thioredoxin from jellyfish nematocyst extracting solution |
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