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Exosome extraction and separation method of seawater parasitic ciliate cryptocaryon irritans and application

A technology that stimulates Cryptocaryonia and separates it. It is applied in the field of extraction and separation of exosomes. It can solve the problems of high mortality, fast spread, and destructive death of fish, and achieve the effect of improving purity and reducing the difficulty of separation.

Pending Publication Date: 2021-05-14
自然资源部第四海洋研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stimulate the outbreak of Cryptocaryoniasis without symptoms, spread quickly, and have high mortality, which can lead to catastrophic fish death in aquaculture areas in a short period of time

Method used

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  • Exosome extraction and separation method of seawater parasitic ciliate cryptocaryon irritans and application
  • Exosome extraction and separation method of seawater parasitic ciliate cryptocaryon irritans and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] A method for extracting and separating exosomes from seawater parasitic ciliates stimulating Cryptocaryon, comprising the following steps:

[0031] Step 1. After collecting Cryptocaryonia stimuli cysts, the cysts are cleaned and incubated in sterile seawater containing antibiotics;

[0032] Step 2: Cyst hatching, after the infectious larvae escape, use a sterile filter to collect the infectious larvae, and incubate in seawater isotonic Leibovitz's L-15 cell culture medium containing antibiotics and exosome-free fetal bovine serum ;

[0033] Step 3, centrifuging to remove the infected stage larvae, and collecting the supernatant;

[0034] Step 4, the supernatant is sterilized through a 0.22 μm filter membrane, and the filtrate is concentrated to obtain a concentrate;

[0035] Step 5, the concentrated solution is centrifuged to collect the coarse precipitate of exosomes;

[0036] Step 6. The crude exosome pellet was resuspended in sterile pre-cooled PBS, and 1×HALT pro...

Embodiment 2

[0039] A method for extracting and separating exosomes from seawater parasitic ciliates stimulating Cryptocaryon, comprising the following steps:

[0040] Step 1. After collecting Cryptocaryon stimuli cysts, clean the cysts and place them in sterile seawater containing antibiotics for incubation; wherein, the antibiotics are 100I.U.ml –1 Penicillin, 80 μg ml –1 streptomycin sulfate;

[0041] Step 2: Cyst hatching, after the infectious larvae escape, use a sterile filter to collect the infectious larvae, and incubate in seawater isotonic Leibovitz's L-15 cell culture medium containing antibiotics and exosome-free fetal bovine serum ;

[0042] Step 3, centrifuging to remove the infected stage larvae, and collecting the supernatant;

[0043] Step 4, the supernatant is sterilized through a 0.22 μm filter membrane, and the filtrate is concentrated to obtain a concentrate;

[0044] Step 5, the concentrated solution is centrifuged to collect the coarse precipitate of exosomes;

...

Embodiment 3

[0048] A method for extracting and separating exosomes from seawater parasitic ciliates stimulating Cryptocaryon, comprising the following steps:

[0049] Step 1. After collecting Cryptocaryon stimuli cysts, clean the cysts and place them in sterile seawater containing antibiotics for incubation; wherein, the antibiotics are 80I.U.ml –1 Penicillin, 80 μg ml –1 streptomycin sulfate;

[0050]Step 2: Cyst hatching, after the infectious larvae escape, use a sterile filter to collect the infectious larvae, and incubate in seawater isotonic Leibovitz's L-15 cell culture medium containing antibiotics and exosome-free fetal bovine serum ; Among them, the larvae in the infecting period were collected by a sterile filter and diluted to 1×10 6 id / ml concentration cultivation, the mass fraction of exosome-free fetal bovine serum in the medium is 15%, the incubation temperature is 25°C, and the incubation is 4h;

[0051] Step 3, centrifuging to remove the infected stage larvae, and coll...

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Abstract

The invention discloses an exosome extraction and separation method of seawater parasitic ciliate cryptocaryon irritans, which comprises the following steps of: 1, collecting cysts of cryptocaryon irritans, cleaning the cysts, and incubating the cysts in sterile seawater containing antibiotics; 2, collecting the larvae in the infection period by using a sterile filter screen, and incubating and culturing the larvae in a culture medium containing antibiotics and exosome-free fetal calf serum; 3, centrifuging to remove larvae in an infection period, and collecting supernate; 4, sterilizing the supernate through a filter membrane, and concentrating the filtrate; 5, centrifuging the concentrated solution and collecting exosome coarse precipitate; 6, resuspending the exosome coarse precipitate by using sterile precooled PBS, and adding a protease inhibitor; and 7, centrifuging to remove supernate, and carrying out resuspension precipitation to obtain the exosome. According to the method, the exosome of cryptocaryon irritans can be effectively separated and extracted, the extracted exosome has a typical dish-shaped or saucer-shaped vesicle-shaped structure, the overall particle size meets the standard, and the extraction concentration of the exosome is high.

Description

technical field [0001] The invention belongs to the field of exosome extraction. More specifically, the present invention relates to a method for extracting and separating exosomes stimulated by seawater parasitic ciliates. Background technique [0002] Cryptocaryon irritans Brown (1951), commonly known as seawater melon worm, belongs to Class Prostomatea, Order Proreodonisa, Family Cryptocaryonidae, and Genus Cryptocaryon. Cryptocaryon stimuli, as a parasitic seawater ciliate, are widely distributed in tropical and subtropical waters such as the Atlantic Ocean, the Caribbean Sea, the Persian Gulf, the Indian Ocean, the East my country Sea and the South China Sea, and have strong effects on a few cartilaginous fishes and most bony fishes. Strong infectivity. Stimulate the dorsal skin, fin rays and gill flaps of the main parasitic hosts of Cryptocaryonia, as well as exposed sites such as the cornea and the mouth. The skin and gills of infected fish are stimulated to secrete...

Claims

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Application Information

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IPC IPC(8): C12N1/10C12N1/02A61K47/46C12R1/90
CPCC12N1/10C12N1/02A61K47/46
Inventor 乔莹马笑晚陈旭阳陈佳潘莹
Owner 自然资源部第四海洋研究所
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