SU1055732A1 - Method of recovering growth factor of nerve tissue from snake poison - Google Patents

Abstract

THE METHOD OF INCREASING THE GROWTH FACTOR OF NERVOUS TISSUE FROM SNAIL POISON, providing for the dissolution and in a buffer solution, followed by centrifugation of the gel filter; of the resulting adiposic liquid on Sephadex in the presence of a buffer solution and ion-exchange chromatography using eluent with increasing ionic strength, about tl and -. Since, in order to simplify the process and increase the yield of the target product, dyurgy is used as the starting material, gel filtration is carried out on Sephadex G-100, ion-exchange chromatography - on a column with carboxymethylcellulose, as a buffer solution in the process 0.19-0.21 M ammonium acetate solution with a pH of 6.4-6.6 is used, and the indicated radar is used as an eluent in a concentration gradient from 0.19-0.21 to 0.95-1.05 m;

Description

The invention relates to the industry of biologically active substances and is a method of obtaining a growth factor of the nervous tissue (NRTF) from yes, which enhances the growth and differentiation of sympathetic and embryonic sensory nerve cells, the introduction of the NRTT to an animal, starting from the first day of the day, causes hypertrophy of the sympathetic nervous system, and sensory cells are sensitive to the PHRNT only in the embryonic period, the NTF is used for investigative work, and can also be used in medicine. Known methods are vyy-gpeni NGNTs from animal tissues (such as placenta 1, heart and other 2) and animal biological fluids, such as snake venoms, Z. The production of NDTs from tissues of the crotch is very laborious and does not ensure the purity of the product, since the sources of the preparation are a multicomponent mixture of various protein and protein components of the divisions of which is a difficult task .. The most effective source of FRTT are serpentine dyes, which contain only protein components and have a stable compound. , The NTDF is engraved in dahs of representatives of all the families of snakes 4J. In some dah, the initial concentration of the TNNT is impossible to determine because of their high toxic components (for example, decobras. Usually, elapid and .viperides have more powerful Crotalid dyns. - The composition and variety of snakes varies, this is due to the species specificity of the species, as well as to the habitat and the conditions of existence, and therefore the production of NEFT by methods developed on dyes of snakes alone is not suitable for working with dy other . The method of isolating the NTTF from the veneer of Central Asian snakes is not known. The closest to the invention to the technical essence is a method for isolating a factor, the growth of nerve tissue from a sand viper. (Uipera ammodytes ammodytes), providing for dissolution and in a buffer solution - 0.1 M solution of ammonium ammonium pH 4.7}, followed by centrifugation, gel filtration of the obtained supernatant liquid on Sephadex Q-50 in the presence of the same buffer solution, desalting , drying and dissolving in .0.08 M tris-citrate buffer, pH 7.3 and ion-exchange chromatography on a column with SE-Sephadex C-25 using eluent with increasing ionic strength — linear gradient 0.5 M NaCl + 0.5 M tris-citrate buffer (pH 7.3). arativnuyu isoelectric focusing in the range of pH 7-10. An NTDF preparation with a biological activity of 5-10 mg / ml and a yield of 0.05% is obtained. However, the use of different buffer systems per k; each purification step necessitates labor-intensive operations — desalting between stages, the use of buffer systems from nonvolatile components during lyophilization causes the need for desalivanide, the use of the preparative isoelectric focusing stage requires the availability of expensive imported equipment and expensive I-type ampholytes (pH 7-10). In addition, the disadvantage of this method is the low yield of the drug (o, 05%). . The purpose of the invention is to simplify the method and increase the yield of the preparation of the NTF. This aim .dostigaets fact that according to the method vscheleni nerve growth factor from snake venom, and comprising dissolving in a buffer solution, followed by centrifugation, gel filtration hdadkosti resulting supernatant on Sephadex in the presence of buffer and ion exchange chromatography using elyuen .ta with ionic led ayuscheys - force, g Sigurza is used as the source, gel filtration is carried out on G-100 Sephadex, ion exchange chromatography - on a column with carboxymethyl cellulose y, as a buffer solution in the process using 0,19-0,21 M ammonium acetate solution with a pH of 6.4-6.6, and as an eluent use the specified solution in a concentration gradient from 0.19 0, 21 to 0, 95 - 1.05 M. The invention simplifies the process by eliminating a number of stages (isoelectrofocusing, desalting) and carrying out the process in one buffer. At the same time, the yield of the desired product is up to 30 times. Ammonium acetate solution with a molar content of 0.19–0.21 M, pH 6.4–6.6 and specific electrical conductivity (14.5–15f5) is used as a buffer. 10 pC "1-, and a linear gradient from ammonium acetate solutions 0.19-0.21 and 0.95-1.05 M with specific electrical conductivities of solutions (14.4 - 15.5) is used as eluent. 10- (63-65). 10p-CIV (pH 6.5-6.6), respectively. Ammonium acetate is a very hygroscopic substance and in order to prepare well-reproducible buffer solutions, control of the ionic strength of the solution is necessary, and the most convenient method is the conductometric method. Here, the electrical conductivity is calculated by the formula V3 pip.c "), where K is the constant of the conductor meter cell,; P - solution resistance, Ohms. Using ammonium acetate as a buffer at the dissolution stage and ensures the creation of a medium for the subsequent separation. The gel filtration is subjected to a solution and a filter, the separation takes place on a gel, and the hyperfine Cz-100 ephadex, which separates proteins within a molecular weight of 4000-150000 daltons, is used as a gel. The gurzy-containing venom containing a multi-component mixture is divided into 9 protein peaks. 1-1 And and V-1X. By picking up proteases, nucleases, C-aminooxidase, and other substances, are excluded. The degree of cleaning - 5-6 times. Subsequent ion exchange chromatography is subjected to a fraction of 1V gelfiltration peak. A selectively acting carboxymethyl cellulose is used as the ion exchanger and selective separation is carried out. A solution of TNT with a specific electrical conductivity is applied onto conit (14.5 15.5). |} cm- (pH 6.4-6.6). In this case, the main part of the protein passes through the ion exchanger, and the TNTF is bound to a cation exchanger, which is eluted by a linear gradient of ionic strength. Thus, the proposed method consists in the following: the dye is dissolved in an ammonium acetate solution (0.19-0.21 m) with a specific electrical conductivity (14.5 15.5). cm- (pH 6.4-6.6), centrifuged, the precipitate is discarded, the supernatant is used for Gelfiltration, gelfiltration is carried out on an ultrathin Sephadex Q-.100 with a solution of 0.19-0.21 M ammonium acetate with a specific electrical conductivity . (14,5-15,5) -. cm (pH 6.4-6.6). The resulting fraction of PDHT with approximately the same molecular weight is applied directly to the carboxymethylcellulose column. The main part of the protein passes through cation exchanger, and the NDFH is linked to carboxymethylcellulose at pH 6.4-6.6 and the specific electrical conductivity of the solution (l4.5 - 15.5} -Cd cm; eluted by Linear gradient ionic strength of ammonium acetate solution (0,19-0,21) - (0,95-1,05 M) with a specific electrical conductivity (14.5 15, 5 105- (bz-65; "10-b. Cm- (pH 6.4 6 , b). The fractions of NBHT are collected and dried by means of lyophilization. The yield is 1.5% biological activity 5 10 mg / ml. Example 1. To 4.9 g and 35 ml of 0 ml are added. 19 M solution of ammonium acetate with specific electrical conductivity of 14, R. cm (RP 6.4) / s The wood is slowly stirred for 4 hours. The solution is diluted and centrifuged at 5000 rpm at 4 ° C for 30 minutes. The precipitate is dispensed, the supernatant is used for gel filtration. For ammonium acetate balanced with solution (specific electrical conductivity of 14.5 to 10 cm) Column with ultrathin Sephadex Q-100 (column size 4.2–140 cm) is applied with solution yes. The column is eluted with acetic acid solution with specific electrical conductivity of 14.5-10 () (pH 6.4) at a rate of “13 ml / h and collect fractions of 19 ml .. The optical density of the eluate R. segist Surgery continuously with Uwikord-11. Get 9 protein peaks. In the IV peak, the fractions that have the biological activity of the TNTF are combined (volume 193 ml). A solution of NBHT is applied to a column of KM-.52 carboxymethyl cellulose column equilibrated with a solution of 0.19 M ammonium acetate with a specific electrical conductivity of 14 / 5-10 c. (PH 6.4). Through the column, 0.5 l of a 0.19 M solution of agfuni acetate is passed through a specific electrical conductivity of 14.510 r. cm (pH 6.4); Elution rate 45 ml / h; 15 ml fractions are collected. The optical content of the eluate is recorded continuously with Uvikord. The main part of the protein passes through cation exchanger under these conditions, and the NTDF, the isoelectric point above 8.5, is bound to KM-52 cation exchanger and eluted with a linear gradient of ionic strength of ammonium acetate from 0.19 m) specific conductivity of 14.5 p. cm (500 ml) to. . 0.95 m). 63. (500 mp) (rI 6.4). The fractions containing the biological activity of the NEF, are combined (yield 605 w) and freeze-dried. Drug characteristics: Yield, 76.56 mg mg (1.56% for protein). Activity yield,% 98.0 Biological activity, 5 mg / ml U, Purification degree, 64.0 times. Example 2. 35 ml of 0.2 M ammonium acetate solution with a specific conductivity of 15.0. pH 6.5). The mixture is slowly stirring for 4 hours. The dissolved d is centrifuged at 5000 rpm for 30 minutes at 4 ° C. The precipitate is discarded, the supernatant is used for gel filtration. An equilibrated 0.2 M ammonium acetate solution (conductivity of a 15.0-10 p-cm column with ultrathin Sephadex Q-100 with a column size of 4.2140 cm was applied to a solution of yes. The column was eluted with a solution of ammonium acetate with a specific conductivity of 15.0 , cm (pH 6, 5) at a rate of 13 ml / h and collecting 19 ml fractions. The optical density of the eluate is recorded continuously using Uwikorb-li. 9 protein peaks are obtained, B the IV peak fraction; they are put (190 ml).

A solution of NGTHs is applied to a balanced ammonium acetate solution with a specific electrical conductivity of 15, (pH 6.5 column of KM-52. 0.5 l of ammonium acetate solution is passed through the column (pH 6.51 with a specific electrical content; conductivity 15 , 0 CT. The elution rate is 45 ml / h, 15 MP fractions are collected. The optical density of the eluate is recorded continuously using Wickord. Proteins that bind to KM-52 under these conditions are eluted with a linear gradient of 0.2-1 ionic strength , 0 M ammonium acetate from the conductivity of the solution .15,0 .SM- (500 ml) to 64.0 10; r. Cm (500 ml) at pH 6.5. The fractions containing the biological activity of TNT, are combined (yield 588 ml) and lyophilized.

Characteristics of the drug: Output, 77.52 ml (1.55% by

squirrel).

The output of the activity,% 98,2 Biological

activity, mg / ml 510 Degree of purification, 64.5 times

Example 3. To 5.1 g and yes of a bag, 36 ml of a 0.21 M ammonium acetate solution with an electrical conductivity of 15.5 are added. see pH 6.6), the mixture is slowly stirred for 4 hours. Dissolved

The mixture is centrifuged at BOOO rpm for 30 minutes, at 4 ° C. The precipitate is discarded, the supernatant is used for gel filtration. On balanced. 0.21 M ammonium acetate solution with a specific electrical conductivity of 15.5 cm column with an ultrathin Sephadex G-100 with a column size4, with a solution applied yes. The column was eluted with a 0.21 M ammonium acetic acid solution with a specific electrical conductivity of 15.5 rpm (pH 6.6) at a speed of 13 MP / h and collecting 19 ml fractions; The optical density of the eluate is recorded continuously using Wicord-I. Get 9 protein peaks. At the IV peak, the fractions that have the biological activity of the TNTF are combined (volume 196 m

A solution of NBHT is applied to a balanced 0.21 M ammonium acetate solution with a specific electrical conductivity of 15.510 / 5 cm (pH 6.6} column of KM-52. 0.5 l of a 0.21 M solution of ammonium acetate is passed through the column (pH 6, b1 with a specific electrical conductivity of 15,) - s The elution rate is 45 ml / h, fractions of 15 ml are collected. The optical density of the eluate is recorded continuously using Wicord. Proteins binding Pa KM-52 under these conditions are eluted with a linear gradient of acetic acid ionic strength ammonium (1.05 M) from the specific electrical conductivity of a solution of 15, 5 10V-cav | (500 ml) to 65.0–10 ppm of 550 ml at pH 6.6. The fractions containing the biological activity of NGFH are combined (yield 593) and dried lyophilized. The PDNT is eluted under these conditions at the first peak of the gradient and It comes from a proteolytic enzyme having a higher pH than the NEF.

Characteristics of the drug: Output, mg 79.9 (1.57% by

squirrel)

Activity yield,% 98.0 Biological activity, mg / mp. 6-10 Degree of purification, 63.8 times. The lyophilized form NGNT retains its biological activity for 24 months. when stored at 4.

The determination of the biological activity of the NEFT is carried out according to the well-known method D.

The biological activity of NGNTs was determined using a culture of sensory ganglia of an 8-day-old chicken embryo, and radial growth of axons from the ganglia was observed.

The sensory ganglia of the chicken embryo are placed in 3 ml of a solution consisting of 10% serum in the culture medium W 199, 300 units of penicillin and streptomycin and 0.01-0.05 ml of the test sample dissolved in the same solution. The sample is incubated for 18-24 h at. In the presence of a NTPT solution, one can observe radial growth of axons from the ganglion. Depending on the density and the width of the crown formed, a positive response to

the presence of NGNT is estimated on a 5-point scale.

The minimal concentration of the drug in the culture medium, which causes the +3 - response of the sensory ganglia during the 18 h incubation time on a 5-point scale, is taken for the biological activity of the NEFT.

The technical and economic effect of the invention is to save the serpentine and by increasing the yield of the preparation FRNT. This simplifies the process of cleaning FRG from yes gurzy.

Claims (1)

METHOD FOR INTRODUCING NERVOUS FABRIC FACTOR FROM SNAKE POISON, which involves dissolving the poison in a buffer solution followed by centrifugation? gel filtration of the obtained supernatant on Sephadex in the presence of a buffer solution and ion-exchange chromatography using an eluent with increasing ionic strength, about t and -. I want to note that, in order to simplify the process and increase / yield the target product, gyurza venom is used as a source, gel filtration is performed on Sephadex C-100, ion-exchange chromatography on a column with carboxymethyl cellulose, and buffer is used in the process 0.19-0.21 M solution of ammonium acetic acid with a pH of 6.4-6.6, and as the eluent use the specified solution in a concentration gradient from 0.19-0.21 to 0.95 1.05 M. '>