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Preparation method of cyanea nozakii kishinouye toxinanti-toxic serum

A technology of white jellyfish and antivenom, applied in the biological field, can solve the problems of unsatisfactory detoxification effect and failure to produce, and achieve the effect of strong detoxification effect, reduced activity loss and high resolution

Active Publication Date: 2019-08-02
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no white jellyfish toxin antivenom serum in China at present, and the research on the preparation of effective white jellyfish toxin antivenom serum is also a blank; moreover, due to the different types of jellyfish, the toxin composition also has great differences, and jellyfish toxin antivenom Venom serum has strong specificity, and the antivenom effect of a jellyfish toxin antivenom on other jellyfish stings is not ideal; the toxin of jellyfish jellyfish is very toxic, how to prepare it into a suitable antigen for animal immunization, both It can produce a better immune effect without poisoning animals; in addition, the toxin composition of the white jellyfish is very complex, and the molecular weight distribution is very wide, ranging from thousands to hundreds of thousands of Daltons, resulting in not all toxin proteins being effective. It has good immunogenicity, so the corresponding antibody may not be produced, so it is also important to choose a suitable adjuvant
Therefore, it is very difficult and challenging to prepare white jellyfish toxin antivenom serum, especially the work of white jellyfish antivenom serum with high potency and good purity

Method used

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  • Preparation method of cyanea nozakii kishinouye toxinanti-toxic serum
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  • Preparation method of cyanea nozakii kishinouye toxinanti-toxic serum

Examples

Experimental program
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Effect test

Embodiment 1

[0027] (1) Attenuation of the toxin of the white jellyfish: add 0.4mL 40% formaldehyde to 100ml white jellyfish toxin, and react at a constant temperature in a 37°C incubator for 1 week; add 0.4mL 40% formaldehyde again after 1 week, and react at a constant temperature at 37°C 1 week; followed by dialysis of 2L, 20mMpH7.0PBS for 48h, centrifugation at 10000rpm for 10min, and filtration with a 0.22μm filter membrane.

[0028] (2) Animal immunization: New Zealand white rabbits were used as immunized animals, and the potency was continuously tested during the period. The antigen used for the first immunization was attenuated white jellyfish toxin + Freund's complete adjuvant, and the immunization dose was 0.35mg / kg ; The second immunization was carried out 3 weeks after the initial immunization, and the immune antigen was attenuated jellyfish toxin + Freund's incomplete adjuvant, and the injection dose was 0.175mg / kg; the third immunization was carried out after 2 weeks, and the i...

Embodiment 2

[0040] (1) Attenuation of the toxin of the white jellyfish: Add 0.5mL of 40% formaldehyde to 100ml of the toxin of the white jellyfish, and react at a constant temperature in a 37°C incubator for 1 week; add 0.5mL of 38% formaldehyde again after 1 week, and react at a constant temperature of 37°C 1 week; followed by dialysis with 3L, 30mMpH7.0PBS for 48h, centrifugation at 12000rpm for 15min, and filtration with a 0.45μm filter membrane.

[0041] (2) Animal immunization: New Zealand white rabbits were used as immunized animals, and the potency was continuously tested during the period. The antigen used for the first immunization was attenuated white jellyfish toxin + Freund's complete adjuvant, and the immunization dose was 0.4mg / kg ; The second immunization was carried out 3 weeks after the initial immunization, and the immune antigen was attenuated jellyfish toxin + Freund's incomplete adjuvant, and the injection dose was 0.2mg / kg; the third immunization was carried out after...

Embodiment 3

[0048] (1) Attenuation of the toxin of the white jellyfish: add 0.6mL 40% formaldehyde to 100ml white jellyfish toxin, and react at a constant temperature in a 37°C incubator for 1 week; add 0.6mL 35% formaldehyde again after 1 week, and keep the temperature at 37°C React for 1 week; then dialyze with 4L, 50mM pH7.0PBS for 48h, centrifuge at 15000rpm for 20min, and filter with a 0.45μm filter membrane.

[0049] (2) Animal immunization: New Zealand white rabbits were used as immunized animals, and the antigen used for the first immunization was attenuated jellyfish toxin + Freund's complete adjuvant, and the immunization dose was 0.45 mg / kg; the first immunization was carried out 3 weeks later For the second immunization, the immune antigen is the attenuated alba jellyfish toxin + Freund's incomplete adjuvant, and the injection dose is 0.225 mg / kg; the third immunization is carried out after 2 weeks, and the immune antigen is the attenuated alba rosa Jellyfish toxin + Freund's ...

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Abstract

The invention relates to the field of biotechnology, and in particular to a preparation method of cyanea nozakii kishinouye toxin anti-toxic serum. The cyanea nozakii kishinouye toxin is attenuated; and after the attenuating treatment, the cyanea nozakii kishinouye toxin is mixed with a Freund's adjuvant for animal immunization, blood is collected after immunization, and an antiserum is separatedand purified, thereby obtaining the cyanea nozakii kishinouye toxin anti-toxic serum. At the same time, the detoxification experiment in the animal shows that the cyanea nozakii kishinouye toxinanti-toxic serum prepared by the invention has the characteristics of high titer, good purity and strong detoxification effect, and lays an important foundation for the in-depth research and application ofthe cyanea nozakii kishinouye toxinanti-toxic serum and antibody thereof.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing an antivenom serum from jellyfish toxin. Background technique [0002] Jellyfish (Jellyfish) is a colloidal zooplankton, which mainly includes four groups: hydromedus, siphonophores, pot jellies and comb jellies of cnidaria. There are many kinds of jellyfish and they are widely distributed. There are about 1,000 species of jellyfish recorded in the world, of which about 400 species are known in my country's sea areas. [0003] The white rosy jellyfish, named Cyanea nozakii Kishinouye in Latin, is a large bowl jellyfish with an umbrella diameter of 40-60cm and its tentacles as long as 2 meters to several meters. In recent years, global jellyfish outbreaks have been increasing year by year, which has aroused widespread concern in the society. Jellyfish outbreaks not only have a huge impact on sea-related activities such as marine fishery production, ecological ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06C07K1/22
CPCC07K16/18C07K16/065C07K2317/20C07K2317/35C07K2317/76
Inventor 李荣锋李鹏程于华华李翱宇于春林
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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