CN107253974A - A kind of thick pure method of live body box jellyfish protein toxin - Google Patents
A kind of thick pure method of live body box jellyfish protein toxin Download PDFInfo
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- CN107253974A CN107253974A CN201710677264.4A CN201710677264A CN107253974A CN 107253974 A CN107253974 A CN 107253974A CN 201710677264 A CN201710677264 A CN 201710677264A CN 107253974 A CN107253974 A CN 107253974A
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- starch
- jellyfish
- solidifying body
- toxin
- box jellyfish
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Engineering & Computer Science (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The present invention proposes a kind of thick pure method of live body box jellyfish protein toxin, using preparations of starch solidifying body, overlay film, toxin collections, enzymolysis, centrifugation, nickel column chromatography, ultrafiltration removal of impurities method, acquisition box jellyfish protein toxin slightly pure liquid.Compared with prior art, the beneficial effects of the invention are as follows：During operation, operating personnel are not directly contacted with box jellyfish, it is ensured that personal security；Only box jellyfish nematocyst and its toxicant are acquired, small is damaged to jellyfish, its recovery time is short, it is only necessary to 2 days, produces sustainability very advantageous, condition is provided using medusocongestin for futurity industryization；It is shorter compared with the jellyfish self-dissolving time using the enzyme process starch-splitting solidifying body time, beneficial to the shortening production time；The nematocyst and its toxin of collection, small by extraneous protein contamination possibility, the albumen that preliminary purification is isolated is only medusocongestin albumen, is that follow-up be further purified creates good basis.
Description
Technical field
The invention belongs to field of marine biotechnology, and in particular to a kind of thick pure side of live body box jellyfish protein toxin
Method.
Background technology
Box jellyfish（Chironex fleckeri）Belong to Coelenterata, Scyphozoa, Cubomedusae, box jellyfish
Section.Box jellyfish is a kind of nattier blue transparent jellyfish, and shape picture chest has 4 obvious sides, there is tens of antennas,
It has been covered with the nematoblast of storage venom on every antenna, has been toxicity most strong jellyfish in the world, according to national science foundation of the US
The data that can be provided, are singly, in Philippine, just to have 20~40 people to die from the telson of box jellyfish every year.Box jellyfish toxin has
Hemolytic activity, neurotoxicity, integumentary musculature toxicity, are that the marine drug of great potential is exploited natural resources, but due to its huge poison
Property, it is that further research and following produce bring difficulty, how safety obtains the emphasis that its toxin is also scientists from all over the world's research
One of.
The method of currently acquired medusocongestin mainly separates nematocyst with automatic edman degradation Edman, by jellyfish feeler at 4 DEG C
Under, placed 1~4 day in a certain amount of seawater or NaCl solution, wait after the automatic degraded of jellyfish, reclaimed using centrifugal process and obtain small
Spherical nematocyst, the ecthoaeum bladder cell that this method is obtained is most pure, and activity is best, Chinese Patent Application No. 201010149779.5
A kind of method of entitled removing hemolytic toxicity from physaliatoxin, i.e., obtain the thick malicious extract solution of jellyfish using the method.But this method has
More shortcoming, such as jellyfish feeler can only be utilized disposably, it is impossible to which circulation continuous is used, even if jellyfish tentacle can regenerate, also be needed
Completion could be regenerated within 7-10 days, it is time-consuming longer, it is unfavorable for industrializing the realization of medusocongestin collection.And jellyfish is in actinal feelings
Under condition, regeneration nematocyst only needs 2 day time, if nematocyst can be gathered individually, jellyfish tentacle is not damaged, and that will shorten collection week
Phase, it is advantageously implemented serialization, industrialized production, the present invention is exactly to realize that this purpose is produced.
The content of the invention
The present invention provides a kind of thick pure method of live body box jellyfish protein toxin.
For achieving the above object, technical scheme feature disclosed by the invention is comprised the following steps：
A) prepared by starch solidifying body：Starch 20~30%（w/w）, thickener 0~20%（w/w）, purified water polishing to 100%（w/
w）, heating is boiled, does not during which stop to stir, when without precipitation, and stopping heating entering mould（Lay nylon gauze in mould bottom）Dry in the air to room temperature,
As starch solidifying body；
B) net is covered：When starch solidifying body does not solidify completely, exterior measuring is fixed using nylon gauze, to increase the anti-of starch solidifying body
Damaged performance；
C) toxin is collected：Jellyfish food is imitated using net starch solidifying body is covered, close to box jellyfish, induction jellyfish is prevented it
Imperial and hunting, reclaims after waiting for a period of time and covers net starch solidifying body；
D) digest：The net starch solidifying body that covers of recovery is dissected, takes outer layer thickness 1~5mm starch solidifying body to crush, uses
The amylase of histidine modification, is digested in 20~35 DEG C, adds a certain amount of purified water, enzymolysis is to without bulk starch solids
Stop during residual, obtain enzymolysis liquid；
E) centrifuge：Kept for 4 DEG C, enzymolysis liquid is centrifuged, to remove the residue not digested, retain centrifuged supernatant；
F) nickel column chromatography：Kept for 4 DEG C, supernatant progress circulation is splined on nickel column chromatography post, retains final filter liquor, removes
Amylase, last elution chromatography post reclaims amylase；
G) ultrafiltration removal of impurities：Kept for 4 DEG C, using the aqueous film that interception is 1kD to step f）In filter liquor carry out ultrafiltration, remove
The small molecular weight impurities such as salt, glucose, and medusocongestin is concentrated, it is the thick pure liquid of medusocongestin to retain ultrafiltrate.
Described starch includes but is not limited to cornstarch, farina, starch from sweet potato.
Described thickener includes but is not limited to xanthans, carragheen, gelatin.
Compared with prior art, the beneficial effects of the invention are as follows：Only box jellyfish nematocyst and its toxicant are carried out
Collection, damages small to jellyfish, its recovery time is short, it is only necessary to 2 days, produces sustainability very advantageous, is that futurity industryization is utilized
Medusocongestin provides condition；It is shorter compared with the jellyfish self-dissolving time using the enzyme process starch-splitting solidifying body time, produced beneficial to shortening
Time；The nematocyst and its toxin of collection, albumen that preliminary purification isolate only jellyfish small by extraneous protein contamination possibility
Toxin protein, is that follow-up be further purified creates good basis.
Embodiment
Embodiment 1：
Cornstarch 25g, xanthans 3g, purified water 72g are added to heat in stainless-steel pan and boiled, does not stop stirring to without precipitation
Afterwards, pouring into cleaned mesh aperture on cooled and solidified in long 20cm, wide 12cm, deep 2cm stainless steel square groove, square groove middle berth is
2mm nylon gauze, when not solidifying complete, then covers last layer nylon gauze, after solidification, the careful demoulding.Net starch gel will be covered
Solid is fixed on stainless steel or plastic extension rod, in access expansion waters or the live body box jellyfish 8 propagated artificially, slightly
Agitation, induction jellyfish is on the defensive behavior to it, and every is collected after 10min, and net starch solidifying body is covered in recovery, is slightly rushed with purified water
Wash, carefully tear and remove nylon wire, the starch solidifying body of two sides 5mm thickness is cut with stainless steel knife, using stainless steel plate by its
Crushing is complete, adds 200ml purified waters, adds the alpha-amylase 10000U of histidine modification, sets 25 DEG C of temperature, digests 5h,
Without bulk starch solids residual, 4 DEG C of centrifugations retain supernatant, Tris-HCl are added into supernatant, is made final concentration of
20mmol/L, is added NaCl, makes final concentration of 5mol/L, purified using metallic nickel affinity chromatography, flow velocity 1ml/min,
Post was repeated once, to remove amylase.It is concentrated by ultrafiltration, is gone out with removing amylorrhexis in 4 DEG C of post liquid of crossing to recovery
Monose and polysaccharide and buffer solution in salt, using aqueous fiber plain film of the interception for 1kD, purifying is added into ultrafiltrate
Water 1L, stops concentration, as medusocongestin thick pure liquid when being concentrated into volume for 50ml.
Embodiment 2：
Potato starch 26g, carragheen 2g, purified water 72g are added to heat in stainless-steel pan and boiled, does not stop stirring to without precipitation
Afterwards, pouring into cleaned mesh aperture on cooled and solidified in long 20cm, wide 12cm, deep 2cm stainless steel square groove, square groove middle berth is
2mm nylon gauze, when not solidifying complete, then covers last layer nylon gauze, after solidification, the careful demoulding.Net starch gel will be covered
Solid is fixed on stainless steel or plastic extension rod, in access expansion waters or the live body box jellyfish 6 propagated artificially, slightly
Agitation, induction jellyfish is on the defensive behavior to it, and every is collected after 10min, and net starch solidifying body is covered in recovery, is slightly rushed with purified water
Wash, carefully tear and remove nylon wire, the starch solidifying body of two sides 5mm thickness is cut with stainless steel knife, using stainless steel plate by its
Crushing is complete, adds 300ml purified waters, adds the alpha-amylase 10000U of histidine modification, sets 25 DEG C of temperature, digests 4h,
Without bulk starch solids residual, 4 DEG C of centrifugations retain supernatant, Tris-HCl are added into supernatant, is made final concentration of
20mmol/L, is added NaCl, makes final concentration of 5mol/L, purified using metallic nickel affinity chromatography, flow velocity 1ml/min,
Post was repeated once, to remove amylase.It is concentrated by ultrafiltration, is gone out with removing amylorrhexis in 4 DEG C of post liquid of crossing to recovery
Monose and polysaccharide and buffer solution in salt, using aqueous fiber plain film of the interception for 1kD, purifying is added into ultrafiltrate
Water 1L, stops concentration, as medusocongestin thick pure liquid when being concentrated into volume for 50ml.
Claims (3)
1. a kind of thick pure method of live body box jellyfish protein toxin, its feature is as follows：
A) prepared by starch solidifying body：Starch 20~30%（w/w）, thickener 0~20%（w/w）, purified water polishing to 100%（w/
w）, heating is boiled, does not during which stop to stir, when without precipitation, and stopping heating entering mould（Lay nylon gauze in mould bottom）Dry in the air to room temperature,
As starch solidifying body；
B) net is covered：When starch solidifying body does not solidify completely, exterior measuring is fixed using nylon gauze, to increase the anti-of starch solidifying body
Damaged performance；
C) toxin is collected：Jellyfish food is imitated using net starch solidifying body is covered, close to box jellyfish, induction jellyfish is prevented it
Imperial and hunting, reclaims after waiting for a period of time and covers net starch solidifying body；
D) digest：The net starch solidifying body that covers of recovery is dissected, takes outer layer thickness 1~5mm starch solidifying body to crush, uses
The amylase of histidine modification, is digested in 20~35 DEG C, adds a certain amount of purified water, enzymolysis is to without bulk starch solids
Stop during residual, obtain enzymolysis liquid；
E) centrifuge：Kept for 4 DEG C, enzymolysis liquid is centrifuged, to remove the residue not digested, retain centrifuged supernatant；
F) nickel column chromatography：Kept for 4 DEG C, supernatant progress circulation is splined on nickel column chromatography post, retains final filter liquor, removes
Amylase, last elution chromatography post reclaims amylase；
G) ultrafiltration removal of impurities：Kept for 4 DEG C, using the aqueous film that interception is 1kD to step f）In filter liquor carry out ultrafiltration, remove
The small molecular weight impurities such as salt, glucose, and medusocongestin is concentrated, it is the thick pure liquid of medusocongestin to retain ultrafiltrate.
2. a kind of thick pure method of live body box jellyfish protein toxin according to claim 1, it is characterised in that：It is described
Starch includes but is not limited to cornstarch, farina, starch from sweet potato.
3. a kind of thick pure method of live body box jellyfish protein toxin according to claim 1, it is characterised in that：It is described
Thickener includes but is not limited to xanthans, carragheen, gelatin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710677264.4A CN107253974A (en) | 2017-08-09 | 2017-08-09 | A kind of thick pure method of live body box jellyfish protein toxin |
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|---|---|---|---|
| CN201710677264.4A CN107253974A (en) | 2017-08-09 | 2017-08-09 | A kind of thick pure method of live body box jellyfish protein toxin |
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| CN201710677264.4A Pending CN107253974A (en) | 2017-08-09 | 2017-08-09 | A kind of thick pure method of live body box jellyfish protein toxin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841813A (en) * | 2018-07-13 | 2018-11-20 | 中国科学院海洋研究所 | It is a kind of for fixing the carrier and its preparation method and application of jellyfish nematocyst |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181305A (en) * | 2007-11-07 | 2008-05-21 | 淮海工学院 | Extract of acaleph toxin as well as preparation method and usage thereof |
| CN102002094A (en) * | 2010-10-10 | 2011-04-06 | 中国科学院海洋研究所 | Method for protecting bioactivity of cyanea nozakii toxin |
-
2017
- 2017-08-09 CN CN201710677264.4A patent/CN107253974A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181305A (en) * | 2007-11-07 | 2008-05-21 | 淮海工学院 | Extract of acaleph toxin as well as preparation method and usage thereof |
| CN102002094A (en) * | 2010-10-10 | 2011-04-06 | 中国科学院海洋研究所 | Method for protecting bioactivity of cyanea nozakii toxin |
Non-Patent Citations (4)
| Title |
|---|
| DAVID A.BLOOM等: "Parial purification of box jellyfish (Chironex fleckeri) nematocyst venom isolated at the beachside", 《TOXICON》 * |
| 于华华等: "水母毒素的研究现状", 《海洋科学》 * |
| 李荣锋: "水母毒素的分离纯化及蛋白质组学研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 肖良等: "水母毒素的研究进展", 《中国海洋药物杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841813A (en) * | 2018-07-13 | 2018-11-20 | 中国科学院海洋研究所 | It is a kind of for fixing the carrier and its preparation method and application of jellyfish nematocyst |
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Application publication date: 20171017 |