Cryoprotectant and preparation method thereof, and application of cryoprotectant in coccidiosis oocysts

A cryopreservation agent and insect egg technology, applied in the application, preservation of human or animal bodies, animal husbandry, etc., can solve the problems of increasing the cost of vaccines, time-consuming and labor-intensive, etc., to inhibit the recrystallization process of ice crystals, reduce production costs, and inhibit Effects of icing and ice crystal recrystallization

Active Publication Date: 2020-09-29
SONGSHAN LAKE MATERIALS LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, Eimeria and live coccidia vaccines are stored at 4°C, but the storage period is usually no more than 6 months, and manual subculture intervention is required every two months for the preservation of coccidia vaccines, which is time-consuming and laborious, which increases vaccine cost

Method used

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  • Cryoprotectant and preparation method thereof, and application of cryoprotectant in coccidiosis oocysts
  • Cryoprotectant and preparation method thereof, and application of cryoprotectant in coccidiosis oocysts
  • Cryoprotectant and preparation method thereof, and application of cryoprotectant in coccidiosis oocysts

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Embodiment 1

[0036] Embodiment 1: This embodiment is illustrated by taking the cryopreservation and recovery of Eimeria maxima oocysts as an example, but it is not used to limit the scope of the present invention.

[0037](1) Preparation of cryopreservative: take 90ml of water for injection and put it into a beaker placed in a water bath at 4°C, put a stirrer, and adjust the rotation speed to 500rpm; add 0.02g of lauric acid and 0.2g of hydrogenated phosphatidylcholine, and stir for 10 Minutes; Add sodium fluoride 3g, ammonium chloride 3g, aspartic acid 0.3g, proline 7g, sucrose 8g, standard PBS buffer 5ml, sodium dihydrogen phosphate 0.3g and disodium hydrogen phosphate 0.7g, stir 30 minutes.

[0038] (2) pretreatment oocysts, need to carry out following purification pretreatment before adding Eimeria maxima oocysts:

[0039] 1. Take 2ml of Eimeria maxima oocyst fluid and centrifuge (3000rpm, 5min) to precipitate;

[0040] 2. Remove the supernatant, add 2ml saturated sodium chloride aqu...

Embodiment 2

[0054] Embodiment 2: This embodiment is basically the same as Embodiment 1, the only difference is that the cryopreservation agent includes the following components in mass percentage: lauric acid 0.03%, hydrogenated phosphatidylcholine 0.2%, sodium fluoride 1.7% , ammonium chloride 1.5%, aspartic acid 0.2%, proline 5%, sucrose 5%, sodium dihydrogen phosphate 0.2%, disodium hydrogen phosphate 0.5%, PBS buffer 3%, and the balance is water for injection.

Embodiment 3

[0055] Embodiment 3: This embodiment is basically the same as Embodiment 1, the only difference is that the cryopreservation agent includes the following components in mass percentage: lauric acid 0.02%, hydrogenated phosphatidylcholine 0.05%, sodium fluoride 3.6% , ammonium chloride 4.1%, aspartic acid 0.1%, proline 3%, sucrose 7%, sodium dihydrogen phosphate 0.4%, disodium hydrogen phosphate 0.2%, PBS buffer 2%, and the balance is water for injection.

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Abstract

The invention discloses a cryoprotectant and a preparation method thereof, and application of the cryoprotectant in coccidiosis oocysts. The cryoprotectant comprises the following components: lauric acid, hydrogenated phosphatidylcholine, sodium fluoride, ammonium chloride, asparaginic acid, proline, cane sugar, sodium dihydrogen phosphate, disodium hydrogen phosphate, a PBS buffer solution and water for injection. The cryoprotectant is reasonable in formula, ice crystal recrystallization in the cryopreservation process can be effectively inhibited, ice crystal morphology is regulated and controlled, the ice crystal growth rate is reduced, and damage of ice crystals to coccidiosis oocysts is reduced; and meanwhile, phospholipid molecules can be adsorbed to the walls of the coccidiosis oocysts, so the ice damage resistance of the coccidiosis oocysts is improved, and the coccidiosis oocysts can be stored in liquid nitrogen for a long time. Compared with a traditional cryoprotectant, thecryoprotectant of the invention has the advantages that the cryoprotectant does not contain toxic molecules such as DMSO and components such as expensive serum, avoids the toxic damage of DMSO to coccidiosis oocysts, and is reduced in production cost, effectively improved in applicability in the field of coccidiosis vaccines and beneficial to wide popularization and application.

Description

technical field [0001] The invention relates to the technical field of cryopreservation, in particular to a cryopreservation agent, a preparation method thereof and an application in coccidia oocysts. Background technique [0002] In recent years, cryopreservation of cells, tissues and organs has attracted much attention, and breakthroughs in cryopreservation technology will provide strong theoretical and experimental support for the development of regenerative medicine. Active substances that are separated from the maternal environment, such as cells, consume their own nutrients in the cells due to metabolism and other life activities during preservation, and their activity will inevitably weaken with the preservation time until they are completely inactivated. The extremely low temperature, usually 77K (-196°C, the boiling point of liquid nitrogen), can effectively inhibit or even stop life activities such as metabolism in cells, prolong the life of cells and achieve long-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 王健君赵立山吕健勇
Owner SONGSHAN LAKE MATERIALS LAB
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