RU2072855C1 - Method of preparing dna sodium salt from fish milt - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves homogenization of the matured fish milts and addition of enzymes complex isolated from Kamchatka crab hepatopancreas (complex has proteolytic, lipase and nuclease activities) to homogenate. Also, ethylenediaminetetraacetic acid sodium salt is added up to its final concentration 3-30 mM in reaction mixture. Reaction mixture is incubated, then sodium chloride is added up to concentration 20-100 g/l in reaction mixture, heated to 56-60 C, kept at this temperature, cooled to 2-8 C and precipitate is separated by centrifugation or Nutsch-filtration. Method ensures to obtain DNA of different molecular mass at protein content 0.7% or 0.1%. Method can be used for preparing immunomodulating agents based on nucleic acids in pharmaceutical industry. EFFECT: improved method of DNA preparing. 5 cl, 3 dwg

Description

 The invention relates to a method for producing sodium salt of DNA from mature fish milk and may find application in the preparation of nucleic acid immunomodulators in the pharmaceutical industry.

 Currently, in addition to the widespread use of deoxyribonucleic acid preparations for scientific purposes, to study the structural and functional organization of the cell’s genetic apparatus, schemes for their use as therapeutic agents for cancer, radiation sickness, diabetes, and other pathological conditions of the body that require immunocorrection or immunomodulation.

 A well-established source of the isolation of deoxyribonucleic acids (DNA) for industrial purposes is mature fish milk, which has a number of advantages over raw materials of other origin, such as, for example, organs and tissues of animals or plants, biomass of microorganisms, etc. The main advantages of fish milk as a raw material can be attributed to: the highest DNA content compared to other sources, the relatively low content of ribonucleic acids (RNA) and other impurities, as well as the low cost and availability of milk, which are waste products from the fishing industry.

 Known methods for producing DNA involve the isolation of a deoxynucleoprotein (DNP) in a first step, from which a protein is removed in a second step with detergents, cation exchangers or proteases.

A known method of producing DNA (ed. Certificate. USSR N 1373709, 1988, MKI C 07 H 21/04), according to which animal tissue is homogenized and then centrifuged. The precipitated chromatin is washed from the ribonucleoproteins at least 6 times, each time by centrifugation and the whole process is carried out at 4 ^° C. The washed DNPs are suspended in saline buffer solution with 0.15 M sodium chloride, the salt concentration is adjusted to 2 M, after which cation exchange resin is added. and the DNP obtained is dialysed suspension cation against saline under constant stirring for 12 hours at 4 ^o C. The dialyzed DNA preparation was centrifuged, the supernatant remains in the DNA solution concentration of not more 0.2 mg / ml and containing protein it is less than 1%
The disadvantages of this method are the need for work at low temperatures (the complexity of the technology), the ability to obtain only dilute solutions of DNA and small amounts. The method is suitable only for use in laboratory practice.

A known method (ed. Certificate of the USSR N 496279, 1975, MKI C 07 H 21/04), in which proteins are removed from DNP by extraction with chloroform in the presence of bentonite at 15-20 ^o C with vigorous stirring. After centrifugation, a suspension of DNA from the supernatant was precipitated with ethyl alcohol. In this case, the authors demonstrated the possibility of obtaining several tens of grams of DNA with a content of the main substance in the final product of 80-85%
The disadvantages of the method are the use of environmentally harmful compounds of chloroform, the high cost of the process, as well as its complexity.

 A known method (ed. Certificate of the USSR N 644484, 1979, MKI C 07 H 21/04), according to which the deproteinization of washed DNP is also carried out by extraction with organic solvents with a mixture of chloroform with isoamyl alcohol. After separation of the precipitate by centrifugation, DNA is precipitated from the supernatant with 2 volumes of chilled ethyl alcohol. Unfortunately, the authors do not indicate the degree of purity of the DNA they receive.

 The disadvantage of this method is the use of environmentally harmful chloroform compounds.

A known method of producing DNA (ed. Certificate of the USSR N 487897, 1975, MKI C 07 G 7/026), according to which fish milk is homogenized in a cold citrate-salt solution, centrifuged and the residue is again homogenized in a citrate-salt solution, then with stirring add a 6% solution of sodium dodecyl sulfate (DNS) to a final concentration of SDS of 1% and, without interrupting stirring, heat the solution to 60 ^° C. and hold at this temperature for 1.5 hours. Then add the citrate-salt mixture again to a final concentration sodium chloride 16% and concentration of tre substituted sodium citrate 2.6% and the mixture was stirred for 1.5 hours at 60 ^o C, at the end of which the mixture is cooled to 2-4 ^o C, mixed with a diatomaceous earth and eluted the DNA with it citrate saline. The DNA solution is separated from kieselguhr by filtration through a layer of balting and filter paper on a suction filter. DNA from the solution is precipitated with cold 96% ethanol. DNA obtained by this method contains no more than 1.5% protein, has a significant hyperchromic effect (41%).

 The disadvantage of this method is the consumption of significant amounts of solutions: to obtain 1 g of DNA requires about 12 l of citrate-saline solution, which requires the use of large capacities of the reaction vessels.

A known method of producing DNA (US Pat. RF N 2017496, 1994, MKI C 07 H 21/04), according to which the milk is homogenized in a citrate-salt solution, the precipitate after centrifugation is washed three times with the same solution, the washed precipitate is suspended in a citrate-salt solution with 6% sodium dodecyl sulfate in 45% ethanol, heated to 60 ^o C, after which an equal volume of 5 M sodium chloride was added, stirred and cooled to 12-16 ^o C. The resulting reaction mass was sonicated and the precipitate was separated by filtration of the suspension through kieselguhr . DNA from the filtrate is separated on a membrane with a pore size of 0.45 μm and washed on the same membrane to a negative sample for protein in washing solutions.

 The disadvantage of this method is the use of ultrasound during the production of DNA, which inevitably entails the use of powerful ultrasonic machines in the technology, which makes obtaining a substance more expensive and complicates the technology.

A known method (ed. Certificate of the USSR N 652187, 1979, MKI C 07 H 21/04), in which the homogenate of fish milk in 0.15 M sodium chloride is centrifuged and the resulting precipitate is washed repeatedly with salt buffer until a negative protein sample in the washing liquid whereupon the precipitate is resuspended in a solution of a proteolytic enzyme (usually trypsin or pronase) and incubated at 370 ^° C for 18 hours. Then, the cells are lysed with a 0.5% sodium dodecyl sulfate solution and proteins are removed from the resulting viscous mass by phenol extraction. After centrifugation, sodium chloride was added to the supernatant to a final concentration of 3 M and extracted with an equal volume of a mixture of chloroform with isoamyl alcohol. After centrifugation, DNA is precipitated from the supernatant with an equal volume of concentrated ethyl alcohol. To obtain high purity DNA, it is treated with RNAase and then trypsin to remove RNA impurities and protein. Then, the obtained DNA is reprecipitated several times and one or two deproteinizations are carried out with chloroform, after which it is precipitated with concentrated ethanol, washed several times with 70% ethanol.

 In this way, it is possible to obtain DNA of a high degree of purity (protein content of about 1% lack of polysaccharide impurities, RNA impurities). However, the disadvantages of the method include multistage and the use of organic solvents for the deproteinization of DNPs, which are environmentally harmful and quite expensive, which makes the process more expensive and complicated.

The closest technical solution is a method for producing DNA, in which protein is removed from DNP using protosubtilin protease isolated from Bacillus Subtilis (ed. Certificate of the USSR N 780444, 1985, MKI C 07 D 13/06). The homogenate of salmon milk is diluted with an equal volume of 0.15 M NaCl, centrifuged, the precipitate is washed three times with 0.15 M NaCl, then with ethanol. The resulting DNP is suspended in saline buffer, the suspension is heated to 35 ^° C., protosubtilin is added to 1.2 units. act / ml and incubated for 30 minutes with gentle stirring. After centrifugation of the reaction mixture, the supernatant is mixed with an equal volume of ethyl alcohol and strands of DNA are recovered.

 However, a significant disadvantage of the known method is the production of protosubtilin DNA with a high protein content (2.4%), which cannot be used without further purification to obtain injectable or other drugs in which the protein content should not exceed 1.5%. Another disadvantage is the fact that according to the described method it is possible to obtain only high molecular weight DNA, which slowly dissolves, forms very viscous solutions and is also not suitable without additional fragmentation for obtaining medications based on it.

 The objective of the invention is to develop such an enzymatic method for producing DNA from the milk of mature fish, which would make it possible to obtain DNA with a protein content of not more than 1.5% without additional purification and complication of the technological scheme and at the same time would allow to obtain a given nucleic acid size without resorting to additional technical means, for example the use of ultrasonic devices.

 The problem is solved in that a method is proposed for producing sodium salt of DNA from fish milk by homogenizing them and removing proteins from the complex with DNA by treatment with an enzyme preparation isolated from the hepatopancreas of the Kamchatka crab, which has proteolytic, lipase and nuclease activities. Another important technique is the addition of EDTA sodium salt to the reaction mixture, followed by incubation.

 The molecular weight of the resulting sodium salt of DNA can be controlled by varying the amount of EDTA added. To obtain low molecular weight DNA add EDTA to a concentration of 3-10 mm. To obtain medium-sized DNA, EDTA is added to a concentration of 10-20 mM. To obtain high molecular weight DNA add EDTA to a concentration of 20-30 mm.

 In addition, it was found that re-incubation of the reaction mixture in the presence of sodium chloride in a final concentration of 20-100 g / l allows you to get a DNA preparation with a lower protein content.

 To obtain highly purified DNA with a protein content of less than 0.1%, the DNA solution is passed through a column with an modified silica gel amino exchanger (polysil CA-500, 20-30 μm), the column is washed with a 0.5-1 M sodium chloride solution and a 2 M high-purity DNA is eluted NaCl solution.

In comparison with known methods, a higher degree of DNA purity is achieved by adding instead of a specific protease of protosubtilin a mixture of proteases from king crab hepatopancreas and an additional stage
heating the reaction mixture at 56-60 ^o C after hydrolysis in saline. After these treatments, the protein content in the DNA preparation does not exceed 0.7%
It should also be added that in order to obtain DNA as a drug substance, it must be fragmented, in particular, ultrasound is used for these purposes and to increase the yield of DNA to 3%. In the inventive method, the use of ultrasound is not required, since DNase and RNase are also present in the complex of enzymes from king crab, so only by regulating the amount of EDTA added can we obtain a DNA preparation with a given distribution of molecular weights of the fragments, and the yield of DNA from fish milk also increases from 3% up to 4-7.5% in the proposed method.

 Figure 1 shows a diagram of the preparation of a DNA preparation according to the claimed method, where (A) additional steps necessary to obtain highly purified DNA with a protein content of less than 0.1% in figure 2, the absorption spectrum of DNA samples obtained by the present method in the UV region; figure 3 electrophoretogram of DNA samples isolated according to the claimed method at different levels of EDTA in the reaction mixture, 1-22 molecular weight markers. The arrows indicate the position of the largest fragment (1300 base pairs) and the smallest (50 base pairs).

 1, 22 molecular weight markers, Bspl plasmid DNA hydrolyzate pQPR, arrows indicate the position of the largest fragment (1300 base pairs) and the smallest (50 base pair).

 2, 7, 12, 17 DNA obtained according to the proposed method at a concentration of EDTA of 5 mm.

 3, 8, 13, 18 DNA obtained at an EDTA concentration of 10 mM.

 4, 9, 14, 19 DNA obtained at an EDTA concentration of 15 mM.

 5, 10, 15, 20 DNA obtained with an EDTA content of 20 mM in the reaction mixture.

6, 11, 16, 21 DNA samples obtained according to [5]
Example 1. The scheme for obtaining DNA is presented in figure 1. 1 kg of frozen salmon milk is washed with drinking tap water and then rinsed with distilled water, homogenized in 900 ml of 0.15 M sodium chloride solution for 10-15 minutes at maximum revolutions of the homogenizer knife. 100 ml of a 0.40 M solution of EDTA disodium salt (pH 8.0) was added to the homogenate to a final concentration of 20 mM and homogenized for an additional 5 minutes. To the resulting viscous mass is added 1 g of an enzyme complex obtained from hepatopancreas of the Kamchatka crab, and the mass is mixed by homogenization for 2 minutes. Then the mixture is incubated at 37 ^o C for 16 hours. To the resulting hydrolytic mass, 20-100 g / l of sodium chloride are sprinkled, heated with stirring to 56 ^o C and kept at this temperature. Next, the reaction mass is cooled and the DNA solution is separated from the precipitate by centrifugation or filtration on a suction filter. Two volumes of chilled ethyl alcohol are added to the cooled solution with stirring and a DNA precipitate is formed for at least 2 hours. The resulting precipitate is separated on a suction filter, washed with 300 ml of 70% ethyl alcohol, dried in air and dissolved in 800-1000 ml of 0. 5 M sodium chloride solution. The resulting solution was reheated and precipitated DEC with ethyl alcohol. The precipitate formed is filtered off on a suction filter, washed successively with 300 ml of 70% and then 96% ethanol, and dried at 30-50 ^° C. in a vacuum of a water-jet pump. The yield of sodium salt of DNA, depending on its content in the feedstock 40-75 g.

The absorption spectrum obtained by this DNA scheme is presented in figure 2, the electrophoregram in figure 3. Protein was determined according to the standard Bradford method using a human serum albumin solution as a standard. DNA yield was calculated by absorbance at 260 nm using a conversion factor of 1 p.u. 50 μg. DNA hyperchromism (c) was determined by comparing the optical density at 260 nm of a DNA solution before and after hydrolysis with 5% perchloric acid at 100 ^° C for 20 minutes.

 The protein content in DNA samples obtained by the present method is significantly lower than that of the known methods. In addition, the DNA yield is also significantly higher (4-7.5% instead of 3, as in the known methods). The DNA obtained by our method has a double-stranded structure, as evidenced by the high value of the hyperchromic effect (33-42%). The presence of nuclease activities in the enzyme preparation results in fragmented DNA, with fragment sizes ranging from 50 to 1300 base pairs. The maximum distribution of DNA across the fragments is shifted towards longer fragments with increasing concentration of EDTA in the reaction mixture (Fig. 3).

 Example 2. 1 kg of frozen salmon milk is washed with drinking tap water and then rinsed with distilled water, homogenized in 950 ml of 0.15 M sodium chloride solution for 10-15 minutes at maximum revolutions of the homogenizer knife. 50 ml of a 0.40 M solution of EDTA disodium salt (pH 8.0) was added to the homogenate to a final concentration of 10 mM and homogenized for an additional 5 minutes. Next, from the obtained homogenate isolated low molecular weight native DNA according to example 1.

 Example 3. 1 kg of frozen salmon milk is washed with drinking tap water and then rinsed with distilled water, homogenized in 850 ml of 0.15 M sodium chloride solution for 10-15 minutes at maximum revolutions of the homogenizer knife. To the homogenate, add 150 ml of a 0.40 M solution of EDTA disodium salt (pH 8.0) to a final concentration of 30 mM and homogenize for an additional 5 minutes. Next, from the obtained homogenate isolated high molecular weight native DNA according to example 1.

 Example 4. The DNA obtained according to examples 1-3 is dissolved in physiological saline (concentration 10 mg / ml) and passed through a column with modified silica gel (Polysil CA-500, 20-30 μm). After application, the column is washed and eluted with 2 M NaCl DNA. Analysis of the relative protein content in various fractions showed that high-purity DNA (protein content less than 0.1%) is eluted with 2 M NaCl.

 Industrial applicability. The invention can be used in medicine and the pharmaceutical industry, as well as in the preparation of cosmetic preparations.

Claims (5)

1. The method of obtaining sodium salt of DNA from fish milk by homogenizing raw materials in a solution of sodium chloride, incubating the homogenate in the presence of a proteolytic enzyme, followed by separation and purification of the target product, characterized in that the homogenate is treated with a solution of sodium salt of EDTA to a final concentration in the reaction mixture 3 30 mm, incubated in the presence of a complex of proteolytic enzymes at 20 40 ^o C, then add sodium chloride to a final concentration of 20 to 100 g / l in the reaction mixture, and before separation of the target product the reaction mixture is cooled to 2 - 8 ^o C.  2. The method according to p. 1, characterized in that to obtain low molecular weight DNA add EDTA to a final concentration in the reaction mixture of 3 to 10 mm.  3. The method according to p. 1, characterized in that to obtain medium-sized DNA add EDTA to a final concentration of 10 20 mm in the reaction mixture.  4. The method according to claim 1, characterized in that in order to obtain high molecular weight DNA, EDTA is added to a final concentration of 20-30 mM in the reaction mixture.  5. The method according to PP. 1 to 4, characterized in that to obtain highly purified DNA with a protein content of less than 0.1 percent, the DNA solution is passed through a column with an anion exchanger.

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