A method of obtaining a cryoprotectant for crossed

 

(57) Abstract:

The invention relates to biotechnology and Cryobiology. The method includes the use of a jellyfish, for example, Beroe ovata, in the source glikoproteid for crossed. The method improves the quality of crossed with significant simplification and cheapening of the process for their preparation.

The invention relates to biotechnology and Cryobiology, in particular a process for the production of cryoprotectants for cryopreservation, and can be used as additives in criosray for long-term storage of frozen cells, embryos, semen and tissue samples.

A known method of manufacturing a solution for preservation of cells by freezing [1], in which the basic solution as a cryoprotectant add glycerin or ethylene glycol. The amount of glycerin is about 10-20% (vol./vol.). The amount of ethylene glycol is about 5-10% (wt./vol.).

To provide more complete viability in the deep freeze objects in basic medium containing glycerol, dimethyl sulfoxide or ethylene glycol, as well as the buffer nutrient solution as cryoprotectant impose additional antifreeze CH the ical (nototheniidae) fish. The glycoproteins isolated from the serum of these fish by the method of differential deposition and purification by gelfiltration or isoelectric focusing [2].

The composition of the carbohydrate portion of antifreeze glycoproteins allows you to associate and special way to structure water molecules, preventing the formation of large damaging intracellular patterns of ice crystals in a deep freeze cells. This effect is used in Cryobiology.

A disadvantage of the known methods of obtaining of antifreeze glycoproteins are the complexity and economiccost associated with prior blood serum, which causes the rise in the cost of the final product.

In addition, the use of glycoproteins in the blood of Antarctic fishes currently difficult due to the decline in notothenia in their natural habitat. Arctic fish more accessible as a source of antifreeze glycoproteins of cryoprotectants, however, upon receipt of these glycoproteins require a large number of raw materials.

In connection with the foregoing used glycoproteins roads and inaccessible for broad public use.

Task cottage is achieved by as a source of antifreeze glikoproteid use the jellyfish, for example, Beroe ovata, and glycoprotein emit from his body.

The novelty of the method and the main significant feature of the invention is the use of new, unconventional for Cryobiology source of raw material for producing glycoproteins of Grebenikov, for example, Beroe ovata.

The choice of Grebenikov, for example, Beroe ovata as a source of glikoproteid related to the fact that they can live in Arctic waters at low temperatures of the sea water, the area of their latitudinal distribution and temperature tolerance coincides with treskovye fish whose blood is used to obtain the cryoprotectants. The biochemical composition of the body of Grebenikov are predominantly glycoproteins and glycosaminoglycans. Thus, grabdevice are more accessible by mass and number of glycoproteins object to obtain cryoprotectants. Important critisicim effect for use as a cryoprotectant is also a property of the glycoproteins of the body of Grebenikov to act as a “structural antioxidants that provides additional security of the viability of the objects when thawed. the toproceed use of different sized Grebenikov Beroe ovata in length from 40 to 95 mm Biomass of Grebenikov homogenize until a homogeneous viscous solution. The obtained homogenate was centrifuged at 2500-3000 rpm for deposition of a relatively dense formations. Adosados is opalescense transparent slabovskyy jelly-like liquid, which is used for the selection of cryoprotectant. The glycoproteins in the fluid ranges from 110 to 160 mcg; seromukoidov from 60 to 85 µg; glycosaminoglycans from 105 to 160 µg balance of glucose in 1 ml of homogenate depending on the size of the used species. In adosados add ethanol to a final concentration of 28-30% (vol./about.) and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 2500-3000 rpm for 15 min, the precipitate discarded, and adosados added dropwise 0,8-1N model HC1 solution to create pH 5.0, then add cetyltrimethylammonium bromide to a final concentration of 0.1% and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 2500-3000 rpm for 15 min, the precipitate discarded, and adosados with stirring, add 0,5-0,7 N solution of perchloric acid to a final concentration of 0.3 N and after 15 min, centrifuged at 5500-6000 rpm for 10 minutes In the last 10 minutes The precipitate washed twice with 96% ethanol by centrifuging and dissolved in Tris-Hcl buffer pH of 8.2. The resulting solution of antifreeze glikoproteid Beroe clear of trace contaminants by the gel filtration method on a chromatographic column filled with Sephadex G-25. Fraction glikoproteid dried by vacuum evaporation or lyophilization. The output is the average of 45-55 mg glikoproteid of 1 kg is taken mass Beroe.

Example 1.

For the preparation of cryoprotective of glycoproteins use of Grebenikov Beroe ovata, which homogenize. The obtained homogenate was centrifuged at 1800 rpm Adosados used for selection of cryoprotectant. In adosados add ethanol to a final concentration of 48% (V/V) and the mixture is cooled at a temperature of 4-6°C for 3 h, and then centrifuged at 5000 rpm for 15 min, the precipitate discarded. To the supernatant was added with stirring a 0.4 N solution of chloric acid to the ratio 1:2 rpm and about 15 min, centrifuged at 6000 rpm for 10 min, the precipitate drop, add chilled acetone in the ratio 1:3 V/V and centrifuged at 6000 rpm for 10 minutes the Precipitate is dried by the method of vacuu

Example 2.

Grabdevice Beroe length 40-95 mm homogenize until a homogeneous viscous solution. The obtained homogenate was centrifuged at 2500 rpm for deposition of a relatively dense formations. In adosados add ethanol to a final concentration of 28% (V/V) and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 2500 rpm for 15 min, the precipitate discarded, and adosados added dropwise to 0.8 N Hcl solution to create pH 5.0, then add cetyltrimethylammonium bromide to a final concentration of 0.1% and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 2500 rpm for 15 min, the precipitate discarded, and adosados added with stirring 0.5 N solution of perchloric acid to a final concentration of 0.3 N and after 15 min, centrifuged at 5500 rpm for 10 min. the supernatant add 5% solution of acetone in the ratio 1:1 rpm rpm and centrifuged at 5500 rpm for 10 min. the Precipitate washed twice with 96% ethanol by centrifuging and dissolved in Tris-Hcl buffer pH of 8.2. The resulting solution of antifreeze glikoproteid Beroe, as in example 1, was purified from traces and dried. oratorie grabdevice Beroe ovata in length from 40 to 95 mm homogenize until a homogeneous viscous solution. The obtained homogenate was centrifuged at 3000 rpm for deposition of a relatively dense formations. In adosados add ethanol to a final concentration of 30% (V/V) and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 3000 rpm for 15 min, the precipitate discarded, and adosados added dropwise 1N aqueous HCl to create pH 5.0, then add cetyltrimethylammonium bromide to a final concentration of 0.1% and the mixture is cooled at a temperature of 4-6°C for 6-8 hours, the Mixture was centrifuged at 3000 rpm for 15 min, the precipitate discarded, and adosados with stirring 0,7 N solution of perchloric acid to a final concentration of 0.3 N and after 15 min, centrifuged at 6000 rpm for 10 min. the supernatant add 5% solution of acetone in the ratio 1:1 rpm rpm and centrifuged at 6000 rpm for 10 minutes Further, as in example 2, the precipitate washed twice with ethanol and dissolved in Tris-Hcl buffer pH of 8.2. The resulting solution of antifreeze glikoproteid Beroe clear of trace contaminants, and then dried. Output averaged 55 mg glikoproteid of 1 kg is taken mass Beroe.

Example 4.

To prepare cryopro is centrifuged at 6000 rpm Adosados used for selection of cryoprotectant. In adosados add ethanol to a final concentration of 36% (V/V) and the mixture is cooled at a temperature of 4-6°C for 3 hours the Mixture was centrifuged at 3000 rpm for 15 min, the precipitate discarded, and adosados with stirring to 0.6 N solution of chloric acid to the ratio of 1:1 rpm and about 15 min, centrifuged at 6000 rpm for 10 min. the supernatant add cooled to 0°With acetone in the ratio 1:2 rpm rpm and centrifuged at 6000 rpm for 10 minutes and the process of obtaining glikoproteid takes place as in example 1. The output in this case is in average 35 mg glikoproteid of 1 kg is taken of the mass of the ctenophore Beroe ovata.

As can be seen from the examples, the highest output glikoproteid obtained in the second and third options.

The efficiency obtained by the proposed method of glycoproteins has been proven. As the object of cryopreservation used donor sperm. The control was the same sperm sample in crossride without adding glycoproteins of the ctenophore Beroe ovata. The comparison of the two environments showed that in the standard environment, the added after thawing kept pryamolineinym Beroe ovata is available and popular views of the seas, therefore, the use of the proposed method will allow you to get the required number of cryoprotective glycoproteins with significant simplification and cheapening of the process for their preparation.

Sources used:

1. The Japan patent 3025931, MKI C12N 5/06.

2. Tsvetkova L. I. and other Methodological manual on cryopreservation of sperm of common carp, salmon and sturgeon. - M.: IFF, 1997. - 11 S.

A method of obtaining a cryoprotectant for crossed, including the allocation of glikoproteid of hydrobionts, characterized in that as the source of glikoproteid use the jellyfish, for example Vague ovata, and glycoprotein emit from his body.



 

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