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Preparation method of high-purity mixed sodium deoxyribonucleotide

A high-purity sodium deoxynucleotide technology, which is applied in the preparation of sugar derivatives, chemical instruments and methods, sugar derivatives, etc., can solve the problems that deoxynucleosides cannot be effectively removed, and the content of finished products can only reach 75%.

Active Publication Date: 2015-05-20
NANTONG QIUZHIYOU BIOSCI & BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defect of this method is that deoxynucleoside and deoxynucleotide impurities, proteins and pigments cannot be effectively removed, so that the content of the final product can only reach 75%.

Method used

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  • Preparation method of high-purity mixed sodium deoxyribonucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Dissolve 500g of DNA in 50L of distilled water, adjust the pH to about 5.4 with hydrochloric acid, heat up to 90°C for 10 minutes, cool to 71°C, add 10g of 5'-phosphodiesterase (8000 units / g enzyme activity, first use distilled water Make into a paste and keep warm at 72°C for 8 minutes. React for 3 hours, then heat up to 90°C and keep warm for 10 minutes (to inactivate 5'-phosphodiesterase) to stop the reaction. Cool the solution to below 40°C and adjust the pH to 10, filter Impurities such as protein are removed to obtain an enzymolysis solution for subsequent use. Wherein: the weight content of mixed deoxynucleotide sodium is 50% to 60%;

[0037] Prepare 3 chromatographic columns, No. 1 is equipped with 4L HZ-201 anion resin, No. 2 is equipped with 4LHZ-201 anion resin, and No. 3 is equipped with 5L769 activated carbon. The method reported by "Animal Biochemical Pharmacy" People's Health Publishing House in February, 1981 carried out regeneration treatment to No. 1 a...

Embodiment 2

[0044] The same method as in Example 1 was used to obtain the enzymatic hydrolyzate for subsequent use. Wherein: the weight content of mixed deoxynucleotide sodium is 50-60%;

[0045] Prepare 3 chromatographic columns, No. 1 is equipped with 4L 711 anion resin, No. 2 is equipped with 4L711 anion resin, and No. 3 is equipped with 5L G15 activated carbon; the regeneration treatment is the same as in Example 1.

[0046] Load the reaction filtrate through columns No. 1 and No. 2 in series; after loading the sample, wash the column with 2 times the column volume of distilled water until the pH of the effluent from the outlet of No. Pass the hydrochloric acid solution into No. 1 column to wash the column until the pH of the effluent at the outlet of No. 2 column is 3.0. At this time, various deoxynucleoside impurities on the column can be washed away;

[0047] Then pass into No. 1 column elution with the mixed solution of sodium chloride and acid, and No. 2 column effluent is all p...

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Abstract

The invention provides a preparation method of high-purity mixed sodium deoxyribonucleotide. The method comprises the following steps of: (1) loading an enzymolysis liquid onto a No.1 column and a No.2 column which are filled with regenerated anion exchange resin and are connected in series; (2) introducing water into the No.1 column, washing till the pH of an effluent liquid of the No.2 column is 7.0-9.0, introducing an acid into the No.1 column, and washing till the pH of an effluent liquid of the No.2 column is 3.0-3.5; (3) introducing a mixed solution of sodium chloride and an acid into the No.1 column, and introducing the effluent liquid of the No.2 column into a No.3 column filled with active carbon; and (4) washing the No.3 column with water, eluting with a cholamine solution, collecting a part of deoxyribonucleotide of which the concentration is higher than 20 g / L and the HPLC (High Performance Liquid Chromatography) is over 98 percent from an eluent, concentrating, and performing ultrafiltration and freeze drying to obtain a finished product. By adopting measures such as serial combination, stepwise elution and the like, a high-purity product which only contains four types of sodium deoxyribonucleotides and has a stable ratio can be obtained.

Description

technical field [0001] The invention relates to a preparation method of sodium deoxynucleotide. Background technique [0002] Enzymatic hydrolysis of DNA can obtain sodium deoxyadenylate (dAMP, 2Na), sodium deoxyguanylate (dGMP, 2Na), sodium deoxycytidylate (dCMP, 2Na), sodium deoxythymidylate (dTMP, 2Na) ) and other four sodium deoxynucleotide products (hereinafter referred to as mixed sodium deoxynucleotide). DNA can be extracted from calf thymus or fish testis. Mixed sodium deoxynucleotides can enhance the hematopoietic function of bone marrow, have a good effect on leukopenia caused by radiotherapy and chemotherapy, and improve the body's immunity. [0003] In industrial production, the 5'-phosphodiesterase (or nuclease) used to enzymatically digest DNA often contains a small amount of hybrid enzymes such as phosphomonoesterase and deaminase, so a small amount of deoxygenated enzymes will be contained in the DNA enzymatic hydrolysis solution. Impurities such as dIMP (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H1/06C07H19/20C07H19/10
Inventor 邱蔚然曹静邱志云
Owner NANTONG QIUZHIYOU BIOSCI & BIOTECH
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