CN110128534B - Method for preparing sandjellyfish toxin antitoxic serum - Google Patents
Method for preparing sandjellyfish toxin antitoxic serum Download PDFInfo
- Publication number
- CN110128534B CN110128534B CN201910432846.5A CN201910432846A CN110128534B CN 110128534 B CN110128534 B CN 110128534B CN 201910432846 A CN201910432846 A CN 201910432846A CN 110128534 B CN110128534 B CN 110128534B
- Authority
- CN
- China
- Prior art keywords
- toxin
- immunization
- jellyfish
- serum
- weeks
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
-
- 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
-
- 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 invention relates to the technical field of biology, in particular to a method for preparing sandjellyfish toxin antitoxic serum. Carrying out attenuation treatment on the jellyfish toxin, mixing the jellyfish toxin subjected to attenuation treatment with a Freund adjuvant, carrying out animal immunization, collecting blood after immunization, preparing serum, and purifying to obtain the jellyfish toxin antitoxic serum. Meanwhile, animal in-vivo detoxification experiments show that the jellyfish toxin-resistant serum prepared by the invention has the characteristics of high titer, good purity, strong detoxification effect and the like, and lays an important foundation for the deep research and application of the jellyfish toxin-resistant serum and the antibody.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing sandjellyfish toxin antitoxic serum.
Background
Jellyfish (jellyfish) is a colloid zooplankton, mainly comprising four major groups: hydra, medusa, potted medusa and ctenocephalides of Ctenocephalides. The jellyfishes are various in types and widely distributed, about 1000 types of jellyfishes are recorded in the world, and about 400 types of jellyfishes are known in the sea area of China.
Jellyfish, also known as jellyfish or jellyfish, latin, called Nemopilema nomurai, in Chin zoo, samolophus meleagris L. Agassiz, is a large pot jellyfish with an umbrella diameter of 180-980mm. In recent years, the global jellyfish outbreak phenomenon is increased year by year, so that the wide attention of the society is attracted, and China is also one of jellyfish outbreak serious disaster areas. The jellyfish outbreak not only brings great influence to sea-related activities such as marine fishery production, ecological environment, seaside tourism, naval training and the like, but also causes frequent jellyfish sting, and greatly threatens the health and life safety of sea-related personnel such as tourists, fishermen, naval and the like. The skin of a patient with jellyfish sting light causes rash, red swelling and pruritus, so that the patient with jellyfish sting suffers pain, and the patient with jellyfish sting severe fainting, shock and even death. Jellyfish stings are reported to occur as many as 1.5 hundred million people worldwide each year and cause many stinging events. Among them, the box jellyfish alone caused at least 5568 stinging lethal events (1954 to date). In recent years, many jellyfish stings and death events occur in China, including Qingdao, yingkou, dalian and Weihai and other coastal areas, most of which are caused by the stings of the sand sea, so that social attention is paid, and the jellyfish stings become serious public health and safety problems. The jellyfish can cause human stinging, and the most fundamental reason is that the jellyfish contains toxin with severe toxicity, which mainly exists in the stinging sac cells on the upper surface of the tentacle of the jellyfish, is a protein substance with various toxicity, such as hemolytic activity, muscle toxicity, kidney toxicity, cardiovascular toxicity, liver toxicity, lethal toxicity and the like; not only can cause skin and organ damage, but also can cause the death of the person who is hurt.
However, no special medicine for treating jellyfish sting exists in China so far, and no special emergency medicine for treating severe jellyfish sting exists. For jellyfish stings, only symptomatic measures can be taken, such as: dermatitis caused by jellyfish sting is treated by anti-dermatitis medicine; for patients with pulmonary edema, intubation treatment is performed. These measures, although potentially effective, do not essentially destroy the toxin, which continues to cause damage to the body, with serious consequences.
The antitoxic serum is the most effective first-aid medicament for treating bite or sting of toxic animals all over the world at present, such as antitoxin serum and the like; if a patient can inject anti-virus serum at the first time in time, the possibility of survival of the patient is greatly increased, and valuable time is won for subsequent rescue and treatment. However, no jellyfish toxin antitoxic serum exists in China at present, and the preparation research of the effective jellyfish toxin antitoxic serum is a blank; moreover, the toxin composition of jellyfish is very different due to different species of jellyfish, the antitoxic serum of the jellyfish toxin has very strong specificity, and the antitoxic serum of the jellyfish toxin has unsatisfactory detoxification effect on other jellyfish stings; the jellyfish toxin has very strong toxicity, and how to prepare the jellyfish toxin into a proper antigen for animal immunization can not only produce a better immune effect, but also prevent the animal from being killed by poison; in addition, the jellyfish toxin has complex components and very wide molecular weight distribution, and thousands to hundreds of thousands of daltons are distributed, so that not all toxin proteins have better immunogenicity, and corresponding antibodies cannot be generated, and therefore, the selection of a proper adjuvant is also very important. Therefore, the work for preparing the jellyfish toxin antitoxic serum, in particular the antitoxic serum with high titer and good purity, has great difficulty and challenge.
Disclosure of Invention
The invention aims to provide a method for preparing jellyfish toxin antitoxic serum.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preparing anti-toxin serum from saxitoxin comprises subjecting saxitoxin to attenuation treatment, mixing with Freund's adjuvant, immunizing animal, collecting blood, preparing serum, and purifying to obtain the final product.
Further, the jellyfish toxin is attenuated by formaldehyde, the jellyfish toxin after attenuation treatment is mixed with Freund's adjuvant for animal immunization, titer detection is continuously carried out in the process, blood is taken after immunization, and rabbit serum is prepared by centrifugation; then, the CNBr-activated Agarose4B affinity chromatography filler specifically coupled with the saxitoxin is used for carrying out affinity chromatography to purify the serum, and finally, the pure saxitoxin specific antibody is obtained.
The attenuation treatment comprises adding 35-40% formaldehyde into jellyfish toxin, and reacting in 37 deg.C thermostat for 5-7 days; then adding 35-40% formaldehyde again, and reacting at 37 ℃ for 5-7 days; dialyzing to remove formaldehyde, centrifuging at high speed, filtering, and storing the supernatant at-80 deg.C; the dialysate is pH7.0, and the concentration is 20-50mM PBS; the volume ratio of 35-40% formaldehyde to toxin (v: v) is 0.4% -6%.
The animal is immunized: using a new Zealand white rabbit as an immune animal, mixing an attenuated sarjellyfish toxin antigen and a Freund's complete adjuvant for primary immunization, performing three times of boosting immunization after the primary immunization, and immunizing three times by using the sarjellyfish toxin antigen and the Freund's incomplete adjuvant respectively 2 weeks, 4 weeks and 6 weeks after the primary immunization; the volume ratio of the attenuated sarjellyfish toxin antigen to Freund's complete adjuvant or Freund's incomplete adjuvant is 1.
The dose of the immunization for the priming is 0.3-0.4mg/kg; performing a second immunization 3 weeks after the primary immunization, wherein the immunization dose is 0.15-0.2mg/kg; carrying out third immunization after 2 weeks, wherein the immunization dose is 0.15-0.2g/kg, and carrying out fourth immunization after 2 weeks, wherein the immunization dose is 0.15-0.2mg/kg; the titer was determined after the immunization.
And performing chromatography purification on the antitoxic serum obtained by centrifugation by using CNBr-activated agar 4B affinity chromatography filler specifically coupled with the jellyfish toxin protein, thereby obtaining the specific immunoglobulin antibody of the jellyfish toxin protein.
The affinity purification filler is prepared by firstly using 1-3mM HCl to activate CNBr-activated Agarose4B filler, and then reacting the filler with jellyfish toxin under alkaline condition (20-50 mM Na) 2 CO 3 ) Coupling for 8-12h at 4 ℃ to obtain the jellyfish toxin coupled CNBr-activated Agarose4B affinity chromatography filler.
The chromatographic purification uses pH7.0,20-50mM Na2PO4 to wash the chromatographic packing, the antitoxic serum obtained by the method is added, the shaking table is used for overnight at 4 ℃, the supernatant is discarded, and finally, glycine with pH =2.0-3.0 and 20-50mM is used to elute the packing, so that the sandjellyfish toxin protein specific immunoglobulin antibody is obtained.
A jellyfish toxin antitoxic serum is prepared by the method.
The invention has the advantages that:
the preparation method of the anti-toxin serum containing the jellyfish toxin provides important method guidance for developing jellyfish sting treatment medicines; the method comprises the following specific steps:
the invention adopts attenuated toxin immunity experimental animals to prepare antitoxic serum, and then adopts CNBr-activated Agarose4B affinity chromatography filler coupled with jellyfish toxin to purify toxin antibody in rabbit serum, which can effectively remove non-specific hybrid protein and prepare purer toxin antibody protein. The antitoxic serum prepared by the invention has the characteristics of high titer, good purity, strong detoxification effect and the like.
Drawings
FIG. 1 is a graph showing the potency test of crude sandjellyfish toxin-resistant serum after completion of immunization, provided in example 1 of the present invention; wherein 1-12 represent negative control (1), blank (2) and dilution 1000-5.12x10 respectively 5 Fold serum (3-12);
FIG. 2 is a graph showing the titer of the anti-toxin serum from saxitoxin obtained by affinity chromatography purification according to example 1 of the present invention; wherein 1-12 represent negative control (1), blank (2) and dilution 1000-5.12x10, respectively 5 Fold serum (3-12);
FIG. 3 is a SDS-PAGE electrophoresis purity test chart of purified jellyfish toxin-resistant serum provided in example 1 of the present invention; wherein M is standard molecular weight protein, AV is specific affinity purified antitoxic serum;
FIG. 4 shows the results of the in vivo detoxification experiments of the jellyfish toxin-resistant serum provided in example 1 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are only for purposes of illustration and explanation and are not intended to be limiting.
Example 1:
(1) Attenuation of saxitoxin: adding 0.4mL of 40% formaldehyde into 100mL of jellyfish toxin, and reacting for 1 week in a constant temperature oven at 37 ℃; after 1 week, adding 0.4mL of 40% formaldehyde again, and reacting at the constant temperature of 37 ℃ for 1 week; then dialyzing with 2L,20mM pH7.0PBS for 48h, centrifuging at 10000rpm for 10min, filtering with 0.22 μm filter membrane, and collecting supernatant as attenuated saxitoxin which can be used as antigen for next animal immunization.
(2) Animal immunization: using a new zealand white rabbit as an immune animal, wherein the antigen for the first immunization is attenuated jellyfish toxin + Freund's complete adjuvant (v: v =1: 0.8), and the immune dose is 0.3mg/kg; 3 weeks after the primary immunization, the secondary immunization was carried out, and the immunizing antigen was attenuated sarjellyfish toxin + freund's incomplete adjuvant (v: v = 1; after 2 weeks, a third immunization was carried out, wherein the immunizing antigen was attenuated sajellyfish toxin + freund's incomplete adjuvant (v: v =1: 0.8) at an injection dose of 0.15mg/kg, and after 2 weeks, the fourth immunization was carried out, wherein the attenuated sajellyfish toxin + freund's incomplete adjuvant (v: v =1: 0.8) at an injection dose of 0.15mg/kg; titer detection was performed after the immunization was finished (see fig. 1); the titer detection adopts an ELISA method to determine the titer of the antitoxic serum, and comprises the following steps:
(1) coating antigen: antigen was treated with 50mM Na 2 CO 3 pH =9.6, plate at 0.2 ug/well, 100 ul/well, incubate overnight at 4 ℃; (2) washing the plate: 0.05% of Tween-20, 20Mm NaH for extraction 2 PO 4 Three washes at pH7.4 (PBST), 3 min/time; (3) and (3) sealing: adding 150ul of 5% skimmed milk powder sealing solution into each hole, and sealing at 37 deg.C for 60 min; (4) washing the plate: taking out and washing with PBST for three times, 3 minutes/time; (5) adding a primary antibody: the rabbit sera were diluted according to 1; (6) washing the plate: taking out and washing with PBST for three times, 3 minutes/time; (7) adding a secondary antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H + L), diluted at 8000, incubated at 37 ℃ for 45min; (8) washing the plate: take out and wash with PBST five times, 3 min/time; (9) color development: adding 100ul of substrate solution into the reaction kettle, reacting for 15min, and finally adding 100ul of 2mol/L of sulfuric acid to stop the reaction; OD value in the measurement of R: OD was measured at a wavelength of 450nm using a microplate reader.
As can be seen from FIG. 1, the serum was diluted 5.12x10 5 After doubling, the OD450 is more than 2.5 times of negative value; therefore, the first and second electrodes are formed on the substrate,the obtained anti-virus serum has high titer.
(3) Purification of the anti-virus serum:
CNBr-activated Agarose4B specific purified toxin protein antibody
(1) Preparing an affinity purification column: 1ml of CNBr-activated Agarose4B filler was activated for 1min with 1mM HCl and then with pH7.0,20mM Na 2 PO 4 Washing, adding Na 2 CO 3 Coupling with jellyfish toxin at 4 ℃ for 8h;
(2) pH7.0,20mM Na was used 2 PO 4 Washing CNBr-activated Agarose4B, adding the collected eluent, shaking overnight at 4 ℃, removing the supernatant containing the non-specific hybrid antibody, and finally eluting the filler by using glycine with pH =3.0 to obtain the corresponding antibody of the toxin protein. Then, the titer was determined by ELISA and the purity was checked by non-reducing SDS-PAGE (see FIG. 2 and FIG. 3).
As can be seen from FIG. 3, the anti-toxin serum of sandjellyfish toxin after affinity purification is diluted to 5.12x10 5 After the dilution, the OD value measured at the wavelength of 450nm is 0.129, > 2.5x0.037 (negative control) =0.0925, which indicates that the titer of the purified sandjellyfish toxin antitoxic serum is high; in addition, as can be seen from fig. 4, the sandjellyfish toxin antitoxic serum after affinity purification shows a single band in non-reduction SDS-PAGE electrophoresis, the molecular weight is about 110kDa, and the purity of the sandjellyfish toxin antitoxic serum after purification is very high. Therefore, the method can prepare the anti-toxin serum of the sandjellyfish toxin with high price and high purity.
(4) Determination of toxin-neutralizing Effect of antitoxic serum
About 1g of healthy small grass carp is used as an animal model to detect the neutralizing effect of the purified antitoxic serum on the jellyfish toxin, and three groups are set in an experiment and respectively comprise a blank control group, a toxin group and a toxin and antitoxic serum group; each group was tested using 12 small grass carps, to which a blank group of 30. Mu.L of pH7.0,20mM Na, respectively, was injected 2 PO 4 Buffer, toxin group 30 μ L toxin and toxin + antitoxic serogroup 30 μ L toxin and antitoxic serum, wherein, toxin: anti-toxin serum 1)。
As can be seen from figure 4, the sandjellyfish antitoxic serum prepared by the method can improve the survival rate of animals by one time, can effectively neutralize sandjellyfish toxin and has good detoxification effect in the bodies of the animals.
Example 2:
(1) Attenuation of saxitoxin: adding 0.5mL of 40% formaldehyde into 100ml of jellyfish toxin, and reacting for 1 week in a constant temperature oven at 37 ℃; after 1 week, adding 0.5mL of 38% formaldehyde again, and reacting at the constant temperature of 37 ℃ for 1 week; then 3L,30mM, pH7.0PBS dialysis for 48h,12000rpm centrifugation for 15min,0.45 mu m filter membrane filtration, the supernatant fluid is the attenuated sajellyfish toxin which can be used as antigen for the next step of animal immunization.
(2) Animal immunization: the new Zealand white rabbit is used as an immunized animal, the antigen for the first immunization is the attenuated jellyfish toxin and Freund complete adjuvant, and the immunization dose is 0.35mg/kg; performing secondary immunization 3 weeks after the primary immunization, wherein the immune antigen is attenuated jellyfish toxin and Freund's incomplete adjuvant, and the injection dose is 0.175mg/kg; carrying out third immunization after 2 weeks, wherein the immunization antigen is attenuated sajellyfish toxin and Freund incomplete adjuvant, the injection dosage is 0.175mg/kg, and the fourth immunization is carried out after 2 weeks, the attenuated sajellyfish toxin and Freund incomplete adjuvant, and the injection dosage is 0.175mg/kg; wherein, the white cyanea nozakii toxin in each immune process: immune adjuvant ratio =1 (v: v), titer test was performed after each immunization; the detection result shows that the serum is diluted by 5.12x10 5 After doubling, the OD450 is more than 2.5 times of negative value; therefore, the titer of the obtained anti-virus serum is high.
(3) Purification of the anti-virus serum:
CNBr-activated Agarose4B specific purified saxitoxin protein antibody
(1) Preparing an affinity purification column: 1ml of CNBr-activated Agarose4B filler was activated for 1min with 2mM HCl and then with pH7.0,30mM Na 2 PO 4 Washing, adding Na 2 CO 3 Coupling with jellyfish toxin at 4 ℃ for 10h;
(2) pH7.0,30mM Na was used 2 PO 4 Washing CNBr-activated Agarose4B, adding prepared rabbit serum eluateShaking overnight at 4 deg.C, removing the supernatant containing non-specific antibody, and eluting the filler with glycine having pH =2.5 to obtain the corresponding antibody to the toxin protein. Then, the titer of the sample was measured by the ELISA method described above.
(4) Determination of toxin-neutralizing Effect of antitoxic serum
About 1g of healthy small grass carp is used as an animal model to detect the neutralizing effect of the purified antitoxic serum on the jellyfish toxin, and three groups are set in an experiment and respectively comprise a blank control group, a toxin group and a toxin and antitoxic serum group; each group was tested using 12 small grass carps, to which 30. Mu.L of a blank control group of 30mM Na, pH7.0, was injected 2 PO 4 Buffer, toxin group 30 μ L toxin and toxin + antitoxic serogroup 30 μ L toxin and antitoxic serum, wherein toxin: 1 (m: m) of the anti-toxin serum, and observing the survival condition of the small grass carps after 2 hours. The result shows that the jellyfish antitoxic serum can obviously improve the survival rate of animals, can effectively neutralize the jellyfish toxin and has good detoxification effect in the bodies of the animals.
Example 3:
(1) Attenuation of saxitoxin: adding 0.6mL of 40% formaldehyde into 100ml of jellyfish toxin, and reacting for 1 week in a constant temperature oven at 37 ℃; after 1 week, adding 0.6mL of 35% formaldehyde again, and reacting at the constant temperature of 37 ℃ for 1 week; then 4L,50mM, pH7.0PBS dialysis for 48h,15000rpm centrifugation for 20min,0.45 μm filter membrane filtration, the supernatant is attenuated saxitoxin, and can be used as antigen for next animal immunization.
(2) Animal immunization: new Zealand white rabbits are used as immunized animals, the antigen for the first immunization is attenuated jellyfish toxin and Freund's complete adjuvant, and the immunization dose is 0.4mg/kg; performing secondary immunization 3 weeks after the primary immunization, wherein the immune antigen is attenuated jellyfish toxin and Freund's incomplete adjuvant, and the injection dose is 0.2mg/kg; carrying out third immunization after 2 weeks, wherein the immune antigen is attenuated sandjellyfish toxin and Freund incomplete adjuvant, the injection dose is 0.2mg/kg, and the fourth immunization is carried out after 2 weeks, the attenuated sandjellyfish toxin and Freund incomplete adjuvant, and the injection dose is 0.2mg/kg; wherein, the white cyanea nozakii toxin in each immune process: immune adjuvant ratio = 1.2 (v: v), immuneThen carrying out titer detection; the detection result shows that the serum is diluted by 5.12x10 5 After doubling, its OD 450 Greater than 2.5 times negative value; therefore, the titer of the obtained anti-virus serum is high.
(3) Purification of the anti-virus serum:
CNBr-activated Agarose4B specific purified toxin protein antibody
(1) Preparing an affinity purification column: 1ml of CNBr-activated Agarose4B filler was activated for 1min with 3mM HCl and then pH7.0,50mM Na 2 PO 4 Washing, adding Na 2 CO 3 Coupling with jellyfish toxin at 4 ℃ for 12h;
(2) the pH7.0,50mM Na was used 2 PO 4 Washing CNBr-activated Agarose4B, adding the prepared rabbit serum, shaking overnight at 4 ℃, removing the supernatant containing the non-specific hybrid antibody, and finally eluting the filler by using glycine with pH =2.0 to obtain the corresponding antibody of the toxin protein. Then the titer of the antibody is determined by the ELISA method.
(4) Determination of toxin-neutralizing Effect of antitoxic serum
Using about 1g of healthy small grass carp as an animal model to detect the neutralizing effect of the purified anti-toxin serum on the jellyfish toxin, and setting three groups in an experiment, namely a blank control group, a toxin group and a toxin and anti-toxin serum group; no group was tested using 12 small grass carp, to which 30. Mu.L of pH7.0,50mM Na as a blank control group was injected 2 PO 4 Buffer, toxin group 30 μ L toxin and toxin + antitoxic serogroup 30 μ L toxin and antitoxic serum, wherein, toxin: 1 (m: m) of the anti-virus serum, and observing the survival condition of the small grass carps after 2 hours. The result shows that the sandjellyfish antitoxic serum can obviously improve the survival rate of animals, can effectively neutralize sandjellyfish toxin and has good detoxification effect in the bodies of the animals.
Claims (2)
1. A preparation method of jellyfish toxin antitoxic serum is characterized in that:
the jellyfish toxin is attenuated by formaldehyde, the attenuated jellyfish toxin is mixed with Freund's adjuvant for animal immunization, titer detection is continuously carried out during the animal immunization, blood is taken after the animal immunization, and rabbit serum is prepared by centrifugation; then, purifying the serum by affinity chromatography by using CNBr-activated agar 4B affinity chromatography filler specifically coupled with the saxitoxin, and finally obtaining a pure saxitoxin specific antibody;
the attenuation treatment comprises adding 35-40% formaldehyde into jellyfish toxin, and reacting in 37 deg.C thermostat for 5-7 days; then adding 35-40% formaldehyde again, and reacting for 5-7 days at the constant temperature of 37 ℃; dialyzing to remove formaldehyde, centrifuging at high speed, filtering, and storing the supernatant at-80 deg.C; the dialysate is pH7.0, and the concentration is 20-50mM PBS;35-40% of formaldehyde and toxin with the volume ratio of 0.4% -6%;
performing chromatography purification on the antitoxic serum obtained by centrifugation by using CNBr-activated agar 4B affinity chromatography filler specifically coupled with the jellyfish toxin protein, thereby obtaining the specific immunoglobulin antibody of the jellyfish toxin protein;
the affinity purification filler is prepared by firstly using 1-3mM HCl to activate CNBr-activated Agarose4B filler, and then mixing the filler with the jellyfish toxin in 20-50mM Na 2 CO 3 Under the alkaline condition, coupling for 8-12h at 4 ℃ to obtain the CNBr-activated Agarose4B affinity chromatography filler coupled with the sajatoxin;
the animal is immunized: using a new Zealand white rabbit as an immune animal, mixing an attenuated sarjellyfish toxin antigen and a Freund's complete adjuvant for primary immunization, performing three times of boosting immunization after the primary immunization, and immunizing three times by using the sarjellyfish toxin antigen and the Freund's incomplete adjuvant respectively 2 weeks, 4 weeks and 6 weeks after the primary immunization; the volume ratio of the attenuated sarjellyfish toxin antigen to Freund's complete adjuvant or Freund's incomplete adjuvant is 1;
the dose of the immunization for the priming is 0.3-0.4mg/kg; performing a second immunization 3 weeks after the primary immunization, wherein the immunization dose is 0.15-0.2mg/kg; carrying out third immunization after 2 weeks, wherein the immunization dose is 0.15-0.2g/kg, and carrying out fourth immunization after 2 weeks, wherein the immunization dose is 0.15-0.2mg/kg; the titer was determined after the immunization.
2. The method of producing a jellyfish toxin-resistant serum according to claim 1, wherein: the chromatographic purification uses pH7.0,20-50mM Na2PO4 to wash the chromatographic packing, the antitoxic serum obtained by the method is added, the shaking table is used for overnight at 4 ℃, the supernatant is discarded, and finally, glycine with pH =2.0-3.0 and 20-50mM is used to elute the packing, so that the sandjellyfish toxin protein specific immunoglobulin antibody is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910432846.5A CN110128534B (en) | 2019-05-23 | 2019-05-23 | Method for preparing sandjellyfish toxin antitoxic serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910432846.5A CN110128534B (en) | 2019-05-23 | 2019-05-23 | Method for preparing sandjellyfish toxin antitoxic serum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110128534A CN110128534A (en) | 2019-08-16 |
| CN110128534B true CN110128534B (en) | 2023-01-24 |
Family
ID=67572513
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910432846.5A Active CN110128534B (en) | 2019-05-23 | 2019-05-23 | Method for preparing sandjellyfish toxin antitoxic serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110128534B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112920273B (en) * | 2020-12-30 | 2022-07-01 | 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 |
|---|
| Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody;Changkeun Kang et al;《Toxicon》;20140419;第86卷;摘要，第117页左栏第1段，右栏2.4 * |
| Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice;Narges Safari Foroushani et al;《Toxicon》;20180706;第152卷;摘要，第31页2.2，第35页左栏第1段 * |
| Development of a sensitive enzyme immunoassay (ELISA) for specific identification of Lachesis acrochorda venom;Nunez Rangel V et al;《The Journal of Venomous Animals and Toxins including Tropical Diseases》;20121231;第18卷(第2期);第174页左栏第4段，右栏第3段-第175页左栏第1段 * |
| 沙蜇和白色霞水母刺丝囊毒素的细胞毒性比较研究;徐金涛等;《海洋科学进展》;20141015;第32卷(第04期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110128534A (en) | 2019-08-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110128534B (en) | Method for preparing sandjellyfish toxin antitoxic serum | |
| CN110078824B (en) | Preparation method of white cyanea nozakii toxin antitoxic serum | |
| Chavez-Olortegui et al. | In vivo protection against scorpion toxins by liposomal immunization | |
| Morais et al. | Snake antivenoms: adverse reactions and production technology | |
| Lamanna et al. | The botulinal, tetanal, and enterostaphylococcal toxins: a review | |
| JP2013532182A (en) | Combination pharmaceutical composition and method for treating diseases or conditions associated with neurodegenerative diseases | |
| RU2517084C2 (en) | Method and means for inhibiting production or enhancing protein p24 elimination | |
| CN105198989A (en) | Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof | |
| CN102504023B (en) | Method for preparing egg yolk antibody, egg yolk antibody prepared by method and application of egg yolk antibody | |
| AT503297B1 (en) | Use of antibody or its derivative for preparation of medicament for passive immunization of individual for preventing and/or treating allergic reactions in individual caused by exposure to birch pollen allergen | |
| DE69434665T2 (en) | A HAUSMILBEALLERGEN (THE P III), THEIR USE AND NUCLEIC ACIDS CODED FOR IT | |
| RU2114636C1 (en) | Antidote, method of its preparing, pharmaceutical composition, method of counteraction to venom and a set | |
| CN112745388B (en) | Preparation method of refined antitoxin from white cyanea nozakii | |
| CN103864930A (en) | Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2 | |
| TWI639615B (en) | Method of antivenom serum production by disulfide scrambling and product thereof | |
| Shelke et al. | Equine rabies immunoglobulin: A review | |
| Xian et al. | Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus | |
| Yamauchi et al. | IgG2 antibodies block IgE antibody-induced asthma in guinea pigs | |
| Liu et al. | Preparation and characterization of immunoglobulin yolk against the venom of Naja naja atra | |
| CN111620947B (en) | Preparation method of divalent sea snake poison resisting serum | |
| de Souza et al. | Production of IgY against iron permease Ftr1 from Candida albicans and evaluation of its antifungal activity using Galleria mellonella as a model of systemic infection | |
| WO1984001897A1 (en) | Babesiosis antigen and method for preparation | |
| US9546200B2 (en) | Peptide for protection of allergic respiratory disorders | |
| Mori et al. | Venom and antivenom of the redback spider (Latrodectus hasseltii) in Japan. Part II. Experimental production of equine antivenom against the redback spider | |
| TWI653049B (en) | Synthetic polypeptide, vaccine composition comprising the same, and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |