CN110128534A - A method of it preparing the sea of sand and bites toxin antitoxic serum - Google Patents
A method of it preparing the sea of sand and bites toxin antitoxic serum Download PDFInfo
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- CN110128534A CN110128534A CN201910432846.5A CN201910432846A CN110128534A CN 110128534 A CN110128534 A CN 110128534A CN 201910432846 A CN201910432846 A CN 201910432846A CN 110128534 A CN110128534 A CN 110128534A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Abstract
The present invention relates to field of biotechnology, specifically a kind of method for preparing the sea of sand and biting toxin antitoxic serum.Toxin is bitten to the sea of sand and carries out attenuation treatment, what the sea of sand bit toxin after attenuation treatment is mixed into row animal immune with Freund's adjuvant, takes a blood sample after immune, prepares after serum purifying to get the sea of sand and bites toxin antitoxic serum.Removing toxic substances experiment shows that the sea of sand prepared by the present invention bites toxin antitoxic serum and has the characteristics that potency is high, purity is good and detoxifying effect is strong in animal body simultaneously, bites the further investigation of toxin antitoxic serum and antibody for the sea of sand and important basis has been established in application.
Description
Technical field
The present invention relates to field of biotechnology, specifically a kind of method for preparing the sea of sand and biting toxin antitoxic serum.
Background technique
Jellyfish (jellyfish) is a kind of zooplankter of colloidal substance, mainly includes four big monoids: the water of Cnidaria
The Ctenophora of Xi jellyfish class, Siphonophora, scyphomedusa and Ctenophora.Species of jellyfish is various, widely distributed, in the world
About 1000 kinds or so of the species of jellyfish recorded, wherein there are about 400 kinds known to China sea area.
The sea of sand is bitten also known as sand is bitten, or more preceding jellyfish, the entitled Nemopilema nomurai of Latin, " Fauna Sinica " once
Homonymus or synonymum Stomolophus meleagris L.Agassiz is large-scale scyphomedusa, umbrella diameter 180-980mm.In recent years, entirely
Ball jellyfish breaks out phenomenon and aggravates year by year, causes the extensive concern of society, and China is also one of jellyfish outburst severely afflicated area.Jellyfish is sudden and violent
Hair not only relates to extra large active belt to marine fishery production, ecological environment, seashore tourism and naval training etc. and carrys out tremendous influence, and
And also cause frequent jellyfish and sting, the health and lives for relating to extra large personnel to tourist, fisherman, naval etc. constitute safely very big prestige
The side of body.Jellyfish stings less serious case's skin and fash, redness, itch occurs, and order is bitten, and the wounded is agonizing, and severe one is fainted, shock is even dead
It dies.It is reported that the annual jellyfish in the whole world stings case up to 1.5 hundred million person-times, and many is caused to sting event.Wherein, only case
Jellyfish just at least causes 5568 to sting lethal events (1954 so far).China also occur in recent years multiple jellyfish sting it is lethal
The many places coastal area such as event, including Qingdao, Yingkou, Dalian, Weihai, wherein being risen caused by mostly the sea of sand is stung
The concern of society, jellyfish, which stings, has become serious publilc health and safety problem.Why jellyfish can be caused the mankind to bite
Wound, most basic reason is jellyfish containing toxic violent toxin, it is primarily present in the ecthoaeum bladder cell of jellyfish tentacle upper surface,
Be a kind of protein matter, there are a variety of different toxicity, as hemolytic activity, musclar toxicity, Toxicity of Kidney, cardiovascular toxicity,
Hepatotoxicity and lethal toxicity etc.；It is not only able to cause skin, visceral organ injury, can also result in and bitten the wounded's death.
But the special-purpose medicaments that up to the present China there is no treatment jellyfish to sting, it is stung there are no reply severe jellyfish
Specific first aid medicine.Patient is stung for jellyfish, can only suit the medicine to the illness and take measures, such as: jellyfish, which stings, causes dermatitis, then uses anti-skin
Scorching drug；Pulmonary edema patient then carries out intubation treatment etc..Although these measures may have certain effect, not from basic
On vivotoxin is eliminated, vivotoxin then continues to damage body, ultimately causes serious consequence.
Antitoxic serum is the most effective preferred first aid medicine that current whole world treatment venomous animal bites or stings, such as anti-
Calmette's serum etc.；If patient can will greatly increase a possibility that injecting antitoxic serum in time at the first time, surviving, together
When for it is subsequent rescue and treatment win the quality time.But China there is no the sea of sand to bite toxin antitoxic serum, the effective sea of sand at present
The preparation research for biting toxin antitoxic serum is also blank out；Also, due to the difference of species of jellyfish, toxin composition there is also
Very big difference, medusocongestin antitoxic serum have very strong specificity, and a kind of antitoxic serum of medusocongestin bites other jellyfishes
The detoxifying effect of wound is unsatisfactory；It is very strong that the sea of sand bites toxin toxicity, how to be prepared into suitable antigen and carry out animal exempting from
Epidemic disease not only can produce preferable immune effect, but also be unlikely to poison animal with poison；In addition, the sea of sand bites toxic components sufficiently complex, molecule
Amount distribution it is very wide, be distributed from thousands of to several hundred thousand Dalton, cause and not all toxin protein have have preferably exempt from
Epidemic focus so as to generate corresponding antibodies, therefore selects appropriate adjuvants also most important.So preparing the sea of sand bites poison
The antitoxic serum work that plain antitoxic serum, especially potency are high, purity is good has great difficulty and challenge.
Summary of the invention
The purpose of the present invention is a kind of methods for preparing the sea of sand and biting toxin antitoxic serum.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of sea of sand bites the preparation method of toxin antitoxic serum, bites toxin to the sea of sand and carries out attenuation treatment, after attenuation treatment
What the sea of sand bit toxin is mixed into row animal immune with Freund's adjuvant, takes a blood sample after immune, prepares after serum purifying to get the sea of sand and bites poison
Plain antitoxic serum.
Further, the sea of sand is bitten toxin and is attenuated using formaldehyde, and the sea of sand bites helping with Freund for toxin after attenuation treatment
Agent is mixed into row animal immune, during which constantly carries out bioactivity, takes a blood sample after immune, centrifugation prepares rabbit anteserum；Then using sand
The CNBr-activated Agarose 4B affinity chromatography filler that jellyfish toxin is specifically coupled carries out affinity chromatography and carries out to serum
Purifying, finally obtains the pure sea of sand and bites toxin specific antibody.
The attenuation treatment be bitten to the sea of sand in toxin be added concentration be 35-40% formaldehyde and the constant temperature in 37 DEG C of insulating boxs
Reaction 5-7 days；Then again be added concentration be 35-40% formaldehyde, then at 37 DEG C isothermal reaction 5-7 days；Then dialysis removes first
Supernatant is stored in -80 DEG C for use after high speed centrifugation, filtering by aldehyde；The dialyzate is pH7.0, concentration 20-50mM
PBS；The volume ratio of 35-40% formaldehyde and toxin (v:v) are 0.4%-6%.
The animal immune: using new zealand white rabbit as immune animal, bites Venom antigens using the sea of sand after attenuation
Progress is mixed with Freund's complete adjuvant just to exempt from, and booster immunization three times is carried out after just exempting from, 2 weeks, 4 weeks, 6 weeks after initial immunity
Venom antigens are bitten using the sea of sand and incomplete Freund's adjuvant is immune three times；It is complete with Freund that the sea of sand after the attenuation bites Venom antigens
Full adjuvant or incomplete Freund's adjuvant volume ratio are 1:0.8-1.2.
The immunizing dose just exempted from is 0.3-0.4mg/kg；Second of immune, immunizing dose is carried out after initial immunity 3 weeks
For 0.15-0.2mg/kg；It carries out third time after 2 weeks to be immunized, immunizing dose 0.15-0.2g/kg exempt from for the 4th time after 2 weeks
Epidemic disease, immunizing dose 0.15-0.2mg/kg；Its potency is measured after immune.
The centrifugation obtains antitoxic serum and bites the CNBr-activated that toxin protein is specifically coupled using the sea of sand
Agarose 4B affinity chromatography filler carries out chromatographic purifying, and then obtains the sea of sand to bite toxin protein specific immunoglobulin anti-
Body.
The affinity purification filler preparation is first filled out using 1-3mM HCl activation CNBr-activated Agarose 4B
Material, then it is bitten into toxin (20-50mM Na under alkaline condition with the sea of sand2CO3), it is bitten in 4 DEG C of coupling 8-12h to get to the sea of sand
Toxin conjugated CNBr-activated Agarose4B affinity chromatography filler.
The chromatographic purifying uses pH7.0,20-50mM Na2PO4 to rinse chromatographic stuffing, the antitoxin blood of above-mentioned acquisition is added
Clearly, it is stayed overnight in 4 DEG C of shaking tables, abandons supernatant, finally use pH=2.0-3.0,20-50mM glycine elution filler, to obtain
The sea of sand bites toxin protein specific immunoglobulin antibody.
A kind of sea of sand bites toxin antitoxic serum, prepares the gained sea of sand according to the method and bites toxin antitoxic serum.
Advantages of the present invention:
The sea of sand of the present invention bites the preparation method of toxin antitoxic serum, stings therapeutic agent for development jellyfish and provides important side
Method guidance；Specifically:
The present invention prepares antitoxic serum using attenuation toxin immunity experimental animal, is then bitten using the sea of sand toxin conjugated
CNBr-activated Agarose 4B affinity chromatography filler purified rabbit serum intoxication element antibody, can effectively remove non-specificity
Foreign protein prepares purer toxin antibody albumen, has the characteristics that specific good and high resolution, only passes through the affine layer of a step
Analysis separation can obtain purer antibody protein, be effectively shortened disengaging time, reduce the loss of activity of antibody protein.
Antitoxic serum prepared by the present invention has the characteristics that potency is high, purity is good and detoxifying effect is strong.
Detailed description of the invention
Fig. 1 be the embodiment of the present invention 1 provide it is immune after the sea of sand bite the bioactivity of toxin antitoxic serum semifinished product
Figure；Wherein, 1-12 negative control (1), blank (2) and dilution 1000-5.12x10 are respectively represented5Serum (3-12) again；
The sea of sand, which is obtained, after the affinitive layer purification that Fig. 2 provides for the embodiment of the present invention 1 bites toxin antitoxic serum bioactivity
Figure；Wherein, 1-12 negative control (1), blank (2) and dilution 1000-5.12x10 are respectively represented5Serum (3-12) again；
Fig. 3 provides the sea of sand after purification for the embodiment of the present invention 1 and bites the detection of toxin antitoxic serum SDS-PAGE electrophoresis purity
Figure；Wherein, M: standard molecular weight albumen, AV: antitoxic serum after special affinity purification；
Fig. 4 provides the sea of sand for the embodiment of the present invention 1 and bites experimental result of detoxifying in toxin antitoxic serum body.
Specific embodiment
A specific embodiment of the invention is described further below in conjunction with example, it is noted that retouch in this place
The specific embodiment stated is simply to illustrate that with the present invention is explained, it is not limited to the present invention.
Embodiment 1:
(1) sea of sand bites the attenuation of toxin: addition 40% formaldehyde of 0.4mL in toxin is bitten to the 100ml sea of sand, in 37 DEG C of constant temperature
Isothermal reaction 1 week in case；Be added 40% formaldehyde of 0.4mL after 1 week again, 37 DEG C isothermal reaction 1 week；Subsequent 2L, 20mM
PH7.0PBS dialysis 48h, 10000rpm are centrifuged 10min, and 0.22 μm of membrane filtration, the sea of sand bites toxin after supernatant is as attenuated,
It can be used as antigen and carry out next step animal immune.
(2) animal immune: using new zealand white rabbit as immune animal, and the sand after being attenuation with antigen is immunized for the first time
Jellyfish toxin+Freund's complete adjuvant (v:v=1:0.8), immunizing dose 0.3mg/kg；It is carried out after initial immunity 3 weeks second
Immune, immunizing antigen is that the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant (v:v=1:0.8), and injection dosage is
0.15mg/kg；It is immune that third time is carried out after 2 weeks, immunizing antigen is that the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant (v:v
=1:0.8), injection volume dosage is 0.15mg/kg, and the 4th time is carried out after 2 weeks and is immunized, the sea of sand after attenuation bites toxin+Freund not
Freund's complete adjuvant (v:v=1:0.8), injection dosage 0.15mg/kg；Carry out bioactivity after immune (referring to Fig. 1)；Potency
Detection measures antitoxic serum potency using ELISA method, specific as follows:
1. envelope antigen: by antigen 50mM Na2CO3PH=9.6, by the hole 0.2ug/ wrapper sheet, the hole 100ul/, 4 DEG C of incubations
Overnight；2. board-washing: taking out and use 0.05%Tween-20,20Mm NaH2PO4, pH7.4 (PBST) wash three times, 3 minutes/time；③
Closing: 5% skimmed milk power 150ul confining liquid is added in every hole, and 37 DEG C are closed 60 minutes；4. board-washing: taking-up is washed three times with PBST,
3 minutes/time；5. plus primary antibody: the serum of rabbit being diluted according to 1:1000 respectively, then doubling dilution, 37 DEG C are incubated for 1 hour；
6. board-washing: taking-up is washed three times with PBST, 3 minutes/time；7. plus secondary antibody: horseradish enzyme marks goat anti-rabbit igg (H+L), 1:8000
Dilution, 37 DEG C of incubation 45min；8. board-washing: taking-up is washed five times with PBST, 3 minutes/time；9. colour developing: substrate solution is added
15min is reacted in the hole 100ul/, is eventually adding 100ul 2mol/L sulfuric acid and terminates reaction；10. surveying OD value: with microplate reader in 450nm
OD value is measured under wavelength.
As seen from Figure 1, serum dilutes 5.12x105After times, 2.5 times of OD450 > negative values；So resulting antitoxin blood
Clear potency is high.
(3) purifying of antitoxic serum:
CNBr-activated Agarose 4B specificity purified toxins protein antibodies
1. preparing affinity purification column: taking 1ml CNBr-activated Agarose 4B filler, activated using 1mM HCl
1min uses pH7.0,20mM Na afterwards2PO4Na is added after flushing2CO34 DEG C of coupling 8h of toxin are bitten with the sea of sand；
2. using pH7.0,20mM Na2PO4CNBr-activated Agarose 4B is rinsed, above-mentioned collection is added and obtains
Eluent is stayed overnight in 4 DEG C of shaking tables, and then supernatant of the removal containing non-specific miscellaneous antibody, finally uses pH=3.0 glycine
Filler is eluted to get toxin protein corresponding antibodies are arrived.Then its potency and non-reduced SDS- are measured using above-mentioned ELISA method again
Its purity (referring to fig. 2, Fig. 3) of PAGE electrophoresis detection.
As seen from Figure 3, the sea of sand after affinity purification bites toxin antitoxic serum in dilution 5.12x105After times, 450nm wave
Long lower measurement OD value is 0.129, > 2.5x0.037 (negative control)=0.0925, and it is antitoxin that the sea of sand for illustrating after purification bites toxin
The potency of serum is high；In addition, the sea of sand after affinity purification bites toxin antitoxic serum in non-reduced SDS-PAGE electrophoresis as seen from Figure 4
In be shown as single band, for molecular weight about in 110kDa or so, the sea of sand for illustrating after purification bites the purity of toxin antitoxic serum
It is very high.Therefore, high-titer has been prepared in this method, the sea of sand of high-purity bites toxin antitoxic serum.
(4) the toxin neutralization measurement of antitoxic serum
Use the small grass carp of 1g or so health to detect above-mentioned antitoxic serum after purification as animal model and toxin is bitten to the sea of sand
Neutralization, three groups of experimental setup, respectively blank control group, toxin group and toxin+antitoxic serum group；Every group using 12 small
Grass carp is tested, and injects 30 μ L pH7.0,20mM Na of blank group thereto respectively2PO4Buffer, 30 μ L toxin of toxin group and
The toxin and antitoxic serum of 30 μ L of toxin+antitoxic serum group, wherein toxin: antitoxic serum 1:4 (m:m) observes small grass carp after 2h
Survival condition (referring to fig. 4).
From fig. 4, it can be seen that the sea of sand of this method preparation bites antitoxic serum and animal survival rate is made to be doubled, can effectively neutralize
The sea of sand bites toxin, has detoxifying effect in good animal body.
Embodiment 2:
(1) sea of sand bites the attenuation of toxin: addition 40% formaldehyde of 0.5mL in toxin is bitten to the 100ml sea of sand, in 37 DEG C of constant temperature
Isothermal reaction 1 week in case；Be added 38% formaldehyde of 0.5mL after 1 week again, 37 DEG C isothermal reaction 1 week；Subsequent 3L, 30mM
PH7.0PBS dialysis 48h, 12000rpm are centrifuged 15min, and 0.45 μm of membrane filtration, the sea of sand bites toxin after supernatant is as attenuated,
It can be used as antigen and carry out next step animal immune.
(2) animal immune: using new zealand white rabbit as immune animal, and the sand after being attenuation with antigen is immunized for the first time
Jellyfish toxin+Freund's complete adjuvant, immunizing dose 0.35mg/kg；It carries out being immunized for second after initial immunity 3 weeks, be immunized anti-
Originally be attenuation after the sea of sand bite toxin+incomplete Freund's adjuvant, injection dosage 0.175mg/kg；Third time is carried out after 2 weeks to exempt from
Epidemic disease, immunizing antigen are that the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant, and injection volume dosage is 0.175mg/kg, and 2 weeks laggard
Row the 4th time immune, and the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant, injection dosage 0.175mg/kg；Wherein, above-mentioned
Cyanea nozakii toxin in each immunologic process: immunologic adjuvant ratio=1:1 (v:v) carries out bioactivity after being immunized every time；By examining
It surveys serum as the result is shown and dilutes 5.12x105After times, 2.5 times of OD450 > negative values；So the potency of resulting antitoxic serum
It is high.
(3) purifying of antitoxic serum:
The CNBr-activated Agarose 4B specificity purifying sea of sand bites toxin protein antibody
1. preparing affinity purification column: taking 1ml CNBr-activated Agarose 4B filler, activated using 2mM HCl
1min uses pH7.0,30mM Na afterwards2PO4Na is added after flushing2CO34 DEG C of coupling 10h of toxin are bitten with the sea of sand；
2. using pH7.0,30mM Na2PO4CNBr-activated Agarose 4B is rinsed, the rabbit anteserum of preparation is added
Eluent is stayed overnight in 4 DEG C of shaking tables, and then supernatant of the removal containing non-specific miscellaneous antibody, finally uses pH=2.5 glycine
Filler is eluted to get toxin protein corresponding antibodies are arrived.Then its potency is measured using above-mentioned ELISA method again.
(4) the toxin neutralization measurement of antitoxic serum
Use the small grass carp of 1g or so health to detect above-mentioned antitoxic serum after purification as animal model and toxin is bitten to the sea of sand
Neutralization, three groups of experimental setup, respectively blank control group, toxin group and toxin+antitoxic serum group；Every group using 12 small
Grass carp is tested, and injects 30 μ L pH7.0,30mM Na of blank control group thereto respectively2PO4Buffer, 30 μ L poison of toxin group
The toxin and antitoxic serum of element and 30 μ L of toxin+antitoxic serum group, wherein toxin: small after antitoxic serum 1:3 (m:m), observation 2h
The survival condition of grass carp.As a result the sea of sand, which bites antitoxic serum, significantly improves animal survival rate, can effectively neutralize the sea of sand and bite toxin, tool
There is detoxifying effect in good animal body.
Embodiment 3:
(1) sea of sand bites the attenuation of toxin: addition 40% formaldehyde of 0.6mL in toxin is bitten to the 100ml sea of sand, in 37 DEG C of constant temperature
Isothermal reaction 1 week in case；Be added 35% formaldehyde of 0.6mL after 1 week again, 37 DEG C isothermal reaction 1 week；Subsequent 4L, 50mM
PH7.0PBS dialysis 48h, 15000rpm are centrifuged 20min, and 0.45 μm of membrane filtration, the sea of sand bites toxin after supernatant is as attenuated,
It can be used as antigen and carry out next step animal immune.
(2) animal immune: using new zealand white rabbit as immune animal, and the sand after being attenuation with antigen is immunized for the first time
Jellyfish toxin+Freund's complete adjuvant, immunizing dose 0.4mg/kg；Second of immune, immunizing antigen is carried out after initial immunity 3 weeks
Toxin+incomplete Freund's adjuvant, injection dosage 0.2mg/kg are bitten for the sea of sand after attenuation；It is immune that third time is carried out after 2 weeks, is exempted from
Epidemic disease antigen is that the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant, and injection volume dosage is 0.2mg/kg, and the 4th is carried out after 2 weeks
Secondary immune, the sea of sand after attenuation bites toxin+incomplete Freund's adjuvant, injection dosage 0.2mg/kg；Wherein, above-mentioned each to be immunized
Cyanea nozakii toxin in journey: immunologic adjuvant ratio=1:1.2 (v:v) carries out bioactivity after immune；It is shown by testing result
Serum dilutes 5.12x105After times, OD4502.5 times of > negative values；So the potency of resulting antitoxic serum is high.
(3) purifying of antitoxic serum:
CNBr-activated Agarose 4B specificity purified toxins protein antibodies
1. preparing affinity purification column: taking 1ml CNBr-activated Agarose 4B filler, activated using 3mM HCl
1min uses pH7.0,50mM Na afterwards2PO4Na is added after flushing2CO34 DEG C of coupling 12h of toxin are bitten with the sea of sand；
2. using pH7.0,50mM Na2PO4CNBr-activated Agarose4B is rinsed, the rabbit anteserum of preparation is added,
It is stayed overnight in 4 DEG C of shaking tables, then supernatant of the removal containing non-specific miscellaneous antibody, is finally filled out using pH=2.0 glycine elution
Expect to get toxin protein corresponding antibodies are arrived.Then its potency is measured using above-mentioned ELISA method again.
(4) the toxin neutralization measurement of antitoxic serum
Use the small grass carp of 1g or so health to detect above-mentioned antitoxic serum after purification as animal model and toxin is bitten to the sea of sand
Neutralization, three groups of experimental setup, respectively blank control group, toxin group and toxin+antitoxic serum group；It does not organize small using 12
Grass carp is tested, and injects 30 μ L pH7.0,50mM Na of blank control group thereto respectively2PO4Buffer, 30 μ L poison of toxin group
The toxin and antitoxic serum of element and 30 μ L of toxin+antitoxic serum group, wherein toxin: small after antitoxic serum 1:2 (m:m), observation 2h
The survival condition of grass carp.The sea of sand, which bites antitoxic serum, as the result is shown significantly improves animal survival rate, can effectively neutralize the sea of sand and bite poison
Element has detoxifying effect in good animal body.
Claims (9)
1. the preparation method that a kind of sea of sand bites toxin antitoxic serum, it is characterised in that: bite toxin to the sea of sand and carry out attenuation treatment, subtract
What the sea of sand bit toxin after poison processing is mixed into row animal immune with Freund's adjuvant, takes a blood sample after immune, prepare after serum purifying to get
The sea of sand bites toxin antitoxic serum.
2. the preparation method that the sea of sand according to claim 1 bites toxin antitoxic serum, it is characterised in that: the sea of sand bites toxin use
Formaldehyde is attenuated, and what the sea of sand bit toxin after attenuation treatment is mixed into row animal immune with Freund's adjuvant, is during which constantly imitated
Valence detection, takes a blood sample, centrifugation prepares rabbit anteserum after immune；Then the CNBr-activated that toxin is specifically coupled is bitten using the sea of sand
Agarose4B affinity chromatography filler carries out affinity chromatography and purifies to serum, finally obtains the pure sea of sand and bites toxin specificity
Antibody.
3. the preparation method that the sea of sand as described in claim 1 or 2 bites toxin antitoxic serum, it is characterised in that: at the attenuation
To bite to the sea of sand, concentration is added in toxin is 35-40% formaldehyde and isothermal reaction 5-7 days in 37 DEG C of insulating boxs to reason；Then again
Addition concentration be 35-40% formaldehyde, then at 37 DEG C isothermal reaction 5-7 days；Then dialysis removes formaldehyde, after high speed centrifugation, filtering
Supernatant is stored in -80 DEG C for use；The dialyzate is pH7.0, and concentration is 20-50mM PBS；35-40% formaldehyde and poison
The volume ratio of plain (v:v) is 0.4%-6%.
4. the preparation method that the sea of sand as described in claim 1 or 2 bites toxin antitoxic serum, it is characterised in that: the animal is exempted from
Epidemic disease: it uses new zealand white rabbit as immune animal, Venom antigens is bitten using the sea of sand after attenuation and are mixed with Freund's complete adjuvant
Just exempt from, carry out booster immunization three times after just exempting from, 2 weeks after initial immunity, bites Venom antigens using the sea of sand within 4 weeks, 6 weeks
It is immune three times with incomplete Freund's adjuvant；The sea of sand after the attenuation bites Venom antigens and Freund's complete adjuvant or Freund are incomplete
Adjuvant volume ratio is 1:0.8-1.2.
5. the preparation method that the sea of sand according to claim 4 bites toxin antitoxic serum, it is characterised in that: that exempts from the beginning of described is immune
Dosage is 0.3-0.4mg/kg；Second of immune, immunizing dose 0.15-0.2mg/kg is carried out after initial immunity 3 weeks；After 2 weeks
It is immune to carry out third time, immunizing dose 0.15-0.2g/kg carries out the 4th immune, immunizing dose 0.15- after 2 weeks
0.2mg/kg；Its potency is measured after immune.
6. the preparation method that the sea of sand as described in claim 1 or 2 bites toxin antitoxic serum, it is characterised in that: the centrifugation obtains
Antitoxic serum using the sea of sand bite CNBr-activated Agarose4B affinity chromatography filler that toxin protein is specifically coupled into
Row chromatographic purifying, and then obtain the sea of sand and bite toxin protein specific immunoglobulin antibody.
7. the preparation method that the sea of sand according to claim 6 bites toxin antitoxic serum, it is characterised in that: the affinity purification is filled out
Material preparation, first activates CNBr-activated Agarose 4B filler using 1-3mM HCl, then it is bitten toxin with the sea of sand and is existed
(20-50mM Na under alkaline condition2CO3), in 4 DEG C of coupling 8-12h to get biting toxin conjugated CNBr-activated to the sea of sand
Agarose4B affinity chromatography filler.
8. the preparation method that the sea of sand according to claim 6 bites toxin antitoxic serum, it is characterised in that: the chromatographic purifying makes
Chromatographic stuffing is rinsed, above-mentioned acquisition antitoxic serum is added with pH7.0,20-50mM Na2PO4, is stayed overnight in 4 DEG C of shaking tables, supernatant is abandoned
Liquid finally uses pH=2.0-3.0,20-50mM glycine elution filler, so that obtaining the sea of sand bites toxin protein specific immunity
Globulin antibody.
9. a kind of claim 1 the method, which prepares the sea of sand, bites toxin antitoxic serum, it is characterised in that: press the claim 1
The method preparation gained sea of sand bites toxin antitoxic serum.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111748031A (en) * | 2020-06-24 | 2020-10-09 | 宁波毓昌生物技术有限公司 | High-purity antivenin and preparation method thereof |
| CN112920273A (en) * | 2020-12-30 | 2021-06-08 | 中国人民解放军海军军医大学 | Anti-aequorin nano antibody COZO32, preparation method and application |
| CN113559259A (en) * | 2021-07-19 | 2021-10-29 | 上海赛伦生物技术股份有限公司 | Method for preparing bee venom antitoxic serum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6613326B1 (en) * | 1989-10-31 | 2003-09-02 | Promega Corporation | Antitoxins and methods for making antitoxins |
| CN102516380A (en) * | 2011-12-13 | 2012-06-27 | 中国科学院海洋研究所 | Method for separating hemolytic toxin proteins from Stomolophus meleagris L. Agassiz nematocyst toxins |
| CN106580995A (en) * | 2016-12-12 | 2017-04-26 | 中国科学院海洋研究所 | Application of phenazine-1-carboxylic acid serving as jellyfish venom fibrinogen hydrolase inhibitor |
-
2019
- 2019-05-23 CN CN201910432846.5A patent/CN110128534B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6613326B1 (en) * | 1989-10-31 | 2003-09-02 | Promega Corporation | Antitoxins and methods for making antitoxins |
| CN102516380A (en) * | 2011-12-13 | 2012-06-27 | 中国科学院海洋研究所 | Method for separating hemolytic toxin proteins from Stomolophus meleagris L. Agassiz nematocyst toxins |
| CN106580995A (en) * | 2016-12-12 | 2017-04-26 | 中国科学院海洋研究所 | Application of phenazine-1-carboxylic acid serving as jellyfish venom fibrinogen hydrolase inhibitor |
Non-Patent Citations (4)
| Title |
|---|
| CHANGKEUN KANG ET AL: "Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody", 《TOXICON》 * |
| NARGES SAFARI FOROUSHANI ET AL: "Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice", 《TOXICON》 * |
| NUNEZ RANGEL V ET AL: "Development of a sensitive enzyme immunoassay (ELISA) for specific identification of Lachesis acrochorda venom", 《THE JOURNAL OF VENOMOUS ANIMALS AND TOXINS INCLUDING TROPICAL DISEASES》 * |
| 徐金涛等: "沙蜇和白色霞水母刺丝囊毒素的细胞毒性比较研究", 《海洋科学进展》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748031A (en) * | 2020-06-24 | 2020-10-09 | 宁波毓昌生物技术有限公司 | High-purity antivenin and preparation method thereof |
| CN111748031B (en) * | 2020-06-24 | 2021-12-10 | 浙江毓昌生物技术有限公司 | High-purity antivenin and preparation method thereof |
| CN112920273A (en) * | 2020-12-30 | 2021-06-08 | 中国人民解放军海军军医大学 | Anti-aequorin nano antibody COZO32, preparation method and application |
| CN113559259A (en) * | 2021-07-19 | 2021-10-29 | 上海赛伦生物技术股份有限公司 | Method for preparing bee venom antitoxic serum |
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