RU2422532C2 - Method of recovery low-molecular nuclear ribonucleoproteids (rnp) from tissues and organs of mammals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: neutral detergent Cevtalone is used for recovery and purification of a low-molecular ribonucleoproteid fraction from DNA and high-molecular RNA. Cevtalone forms from water-insoluble salts with DNA and RNA thereby enables consistent precipitation of DNA and high-molecular RNA from the solution. As a result, the solution contains the low-molecular ribonucleoproteid fraction which then precipitated with Cevtalone. The produced precipitate is dissolved in ethanol for pure ribonucleoproteid precipitation. Cevtalone salts of nucleic acids are resistant to ribonuclease enzymes thereby the given method can be staged with great time intervals (up to several days) without loss of biological properties of recovered ribonucleoproteids. The given method of recovery allows to producing high-purity low-molecular ribonucleoproteids (the ribonucleoproteid contents in the prepared samples is 90 %) in preparative amounts.

EFFECT: recovery of low-molecular nuclear ribonucleoproteid from tissues of mammals with using non-toxic and low-cost reagents.

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Description

The invention relates to the field of biochemistry, molecular biology, veterinary medicine and.

A new method is proposed for the isolation of preparative amounts of ribonucleoproteins (RNPs) using the neutral detergent cetavlon, previously used only for the purification of analytical amounts of RNA and DNA.

Tissues and organs of young (1-2 years old) animals (cattle) are used as sources of RNP, which are subjected to homogenization with subsequent isolation of cell nuclei. Then, the nuclei are lysed with a detergent and the preparation is purified from proteins, DNA and high molecular weight RNA using concentrated solutions of NaCl and the neutral detergent cetavlon, which forms water-insoluble salts with DNA and high molecular weight RNA. The final product is reprecipitated with ethanol, lyophilized and stored in sealed ampoules at 4 ° C.

Low molecular weight nuclear ribonucleoproteins (RNPs) are genetic regulators, the set of which is specific for the cells of each tissue and for each stage of the ontogenetic development of the body. RNPs are found in the cells of normal healthy organisms; therefore, they do not have a toxic effect and differ slightly from one animal species to another. Currently, they are actively being investigated for use as drugs and adaptogens for the treatment of various human diseases.

Presently known methods for isolating ribonucleoproteins (Kirkby KS A new method for the isolation of nucleic acids from mammalian tissues, 1956, Biochem. J., 64, 405 (1); Feramisco JR, Helfman DM, Smart JE, Burridge K., Thomas GP, 1982, Coexistence of vinculin-like protein of higher molecular weight in smooth muscle, J. Biol. Chem. (2); T. Maniatis, E. Fritsch, J. Sambrook, 1984, Methods of Genetic Engineering, “The World” , Moscow, pp. 191-192 (3)) have the following disadvantages:

1. The use of highly toxic reagents: requires special equipment and personal protective equipment.

2. Expensive operations for the disposal of phenol and chloroform, which leads to a significant increase in the cost of the resulting product.

3. The final cleaning of the resulting preparations from toxic impurities greatly complicates the procedure and increases the time of allocation.

The aim of the present invention is to develop an easily reproducible, non-toxic method for the isolation of preparative quantities of low molecular weight ribonucleoproteins (RNP) of high purity, stably retaining activity, suitable for administration to mammals.

As a prototype of the proposed method for the isolation of low molecular weight ribonucleoproteins, we can consider methods for producing low molecular weight ribonucleopeptides from the nuclei of tissue cells and organs of cattle described by V. Vitvitsky. and co-authors in the patent RU 2238756, published October 27, 2004 (application for the grant of a patent of the Russian Federation No. 2003101855 dated January 24, 2003). To obtain low molecular weight nuclear ribonucleoproteins in the known method, the tissue (for example, liver) of cattle (young healthy animals) is separated from the connective tissue membranes, blood vessels, fatty layers and washed from blood cells in physiological saline. The tissue thus treated is homogenized at a low temperature in a solution containing Tris buffer, calcium chloride, EDTA and Triton X-100. The homogenate is centrifuged, the precipitate is washed in the same solution, but without Triton X-100, suspended in a solution of sodium acetate containing Tris-HCl buffer (pH 5.0), EDTA and sodium dodecyl sulfate. Then pour a mixture of phenol: chloroform (1: 1) and heated in a water bath. After cooling, the system is centrifuged, the upper phase containing RNA is selected, a mixture of phenol: chloroform (1: 1) is again poured onto it, heated, cooled and centrifuged. This procedure is repeated several times with a successively increasing heating temperature. The obtained centrifugate phases are again deproteinized, removing most of the proteins, DNA and high molecular weight RNA, which are removed by centrifugation from solutions after adding sodium chloride to 1-2 M. Chilled ethanol is added to the obtained phases of the centrifugate to precipitate the desired product, the precipitate is separated, washed with ethanol . After sterilizing filtration, the target product is poured into ampoules and freeze-dried. Ampoules are sealed under vacuum and stored in the refrigerator.

The essence of the applicant’s proposal aimed at isolating preparative amounts of low molecular weight nuclear ribonucleoproteins using available simple technological methods without the use of toxic reagents with a high degree of purification, which makes it possible to introduce human and animals into the body, is as follows.

To isolate low molecular weight fractions of ribonucleoproteins, organ tissues of young (1-2 years) animals (cattle) were used. Tissue was freed from adipose tissue and connective tissue layers, homogenized and centrifuged to obtain single cells. Cells were lysed with the neutral detergent Triton X-100 to isolate cell nuclei. The resulting nuclei were homogenized in the presence of Na dodecyl sulfate (SDS), and then the resulting homogenant was freed from proteins and DNA using NaCl solutions and cetavlon neutral detergent. The developed technique can be divided into stages, between which breaks are possible for one or more days.

Since the solubility of cetavlon salts of single-stranded and double-stranded nucleic acid molecules in aqueous solutions is different and depends on the concentration of NaCl, cetavlon was previously used mainly for cleaning DNA preparations from impurities of RNA and other high molecular weight compounds (Sulimova, 1978, Sulimova et al., 1976, 1978 ; Dohim, 1977; Hvoika et al., 1978). In this method of isolation, cetavlon is used as the main component that allows you to separate low molecular weight ribonucleoproteins from DNA impurities and high molecular weight RNA; previously, cetavlon was not used for these purposes.

Detailed description of the implementation of the method

The following conditions (temperature, concentration of reagents, centrifugation parameters) are described that are optimal for obtaining the maximum yield of low molecular weight nuclear ribonucleoproteins with a high degree of purification from liver and kidney tissue.

The purified tissue is resuspended in 0.01 M Tris buffer (pH = 7.0) containing 0.35 M sucrose and 0.01 M calcium chloride. Then add Triton X-100 to a final concentration of 1% and homogenize the resulting mixture. The resulting suspension is centrifuged for 7 minutes at 7000 rpm, 4 ° C.

The supernatant is discarded, and the resulting nuclear pellet is resuspended in 0.1 M Tris-HCl buffer (pH = 7.6) containing EDTA (0.05 M) with SDS added to it to a final concentration of 1% and 5 M NaCl to a final concentration 1 M, after which this mixture is left to incubate for 30 min, 60 ° C.

To the resulting suspension was added 5 M potassium acetate to a final concentration of 1 M, after which the mixture was incubated in the cold for 1.5 hours to precipitate SDS and the SDS-protein complex.

The resulting suspension is centrifuged for 25 minutes at 6000 rpm, 10 ° C.

To the supernatant obtained by centrifugation, add an equal volume of a 1% solution of cetavlon and incubate for 30 minutes at room temperature.

The resulting suspension is centrifuged for 10 minutes at 4000 rpm, 15 ° C.

An equal volume of a 0.5% solution of cetavlon is added to the supernatant obtained by centrifugation and incubated for 2.5-3 hours at room temperature.

The resulting suspension is centrifuged for 20 minutes at 10,000 rpm, 15 ° C.

The precipitate is dissolved in 1 M NaCl, after which the target product is precipitated - low molecular weight nuclear RNP with 2.5 volumes of chilled ethanol.

The resulting precipitate was dissolved in distilled water and lyophilized.

The lyophilized dried preparation is an odorless and tasteless hygroscopic white powder. It remains active for one year when stored at a temperature not exceeding 4 ° C, soluble in water.

Yield: usually about 1.2 mg of the final preparation is obtained from 1 g of tissue.

The total release time takes about 6-7 hours.

The use of nucleic acid electrophoresis in an agarose gel, spectrophotometric analysis and a specific reaction for the determination of nucleic acids (according to Schmidt-Townhauser) showed that the characteristics of the drug obtained by this method and the drug obtained using the phenolic method are similar and both drugs have similar purity and the ratio of RNA / DNA. The drug contains 90% of low molecular weight ribonucleoproteins, as well as impurities of DNA and protein.

The method can be divided into stages, between which breaks for 24 hours or more are possible, which ensures the manufacturability of the process.

In this way, it is possible to isolate low molecular weight ribonucleoproteins from other organs and tissues, according to the same principle. Moreover, such parameters of separation as centrifugation modes, the concentration of individual reagents (Triton X-100, SDS), etc., can be varied to obtain the maximum yield of the target product from a specific type of tissue. The main process parameters, such as the sequence of stages, the concentrations and ratios of cetavlon and sodium chloride used, are common for the isolation of low molecular weight ribonucleoproteins from various tissues and organs of mammals.

This method eliminates the use of toxic reagents for the isolation of highly purified RNP preparations suitable for administration to humans and animals, while allowing for the preparation of preparative amounts of RNP (more than 100 mg) for one selection.

Claims (1)

A method for producing low molecular weight nuclear ribonucleoproteins (RNPs) from mammalian tissues and organs, comprising isolating a fraction of cell nuclei using a neutral detergent Triton-XI00 followed by lysis using Na dodecyl sulfate (SDS), releasing the obtained nuclear homogenate from proteins and DNA with the isolation of the target product - low molecular weight nuclear RNP and reprecipitation with ethanol, characterized in that the liberation of the obtained nuclear homogenate from proteins and DNA is carried out using concentrated solutions of NaCl and neutral detergent cetavlon (cetyltrimethylammonium bromide), while 5M potassium acetate is added to the homogenate of cell nuclei to a final concentration of 1M, the mixture is incubated in the cold for 1.5 hours, then centrifuged, an equal volume of a 1% solution is added to the obtained supernatant cetavlon and incubated for 30 min, the resulting suspension is centrifuged, the supernatant is separated, an equal volume of a 0.5% solution of cetavlon is added to it and incubated for 2.5-3 hours, the resulting suspension is centrifuged, the obtained the precipitate is dissolved in 1 M NaCl, after which the desired product was reprecipitated with cold ethanol.