RU2561467C1 - Method of production of preparation to increase meat and milk productivity of farm animals (versions) and preparation produced by method - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: method comprises cultivating the recombinant strain Escherichia coli RNCIM B-6519 on a medium containing a source of amino nitrogen, a source of vitamins, salt components and glucose, with stirring and aeration. The control of pH level and the optical density or dissolved oxygen level and the optical density is carried out through stagewise glucose inflow based on 2-3 g/dm³ of culture fluid and 10-12% yeast extract solution at the rate of 3-4 g/dm³ of culture fluid. Upon reaching the optical density of 3 to 3.5 units, the alcohol solution of isopropyl-beta-D-thiogalactoside is added in a concentration of 8 to 12 mg/dm³. Extraction, purification, drying of the protein solution and making the oil suspension of the preparation is carried out. The resulting preparation additionally comprises bee wax, vitamins A and E, and has a stimulating effect on milk and meat productivity of farm animals.

EFFECT: increase in meat and milk productivity of farm animals.

11 cl, 9 tbl, 3 ex, 3 dwg

Description

The invention relates to biotechnology and agricultural microbiology and consists in intensifying the process of biosynthesis by cells of a recombinant strain of Escherichia coli somatostatin-containing protein, which regulates the formation and concentration of growth hormone in farm animals.

In the mid-90s of the XX century, Russian researchers proposed an original solution to increase the growth rate of animals, based on the possibility of regulating the levels of biologically active peptides and the activity of enzymes in the gastrointestinal tract of mammals. Somatostatin, a biologically active polypeptide produced in the hypothalamus and gastrointestinal tract of animals, attracted attention. Somatostatin, having a strong inhibitory effect on a number of hormones (somatotropin, TSH, insulin, glucagon, etc.), initiates inhibition of the secretion of gastric enzymes, pancreas, small intestine, slows down the motility of the gastrointestinal tract and the evacuation of its contents. As a result, a decrease in the concentration of somatostatin in the body initiates an increase in the content of endogenous somatotropic hormone, which leads to an intensification of anabolic processes.

A promising approach for the production of growth hormone in an industrial environment is the use of microorganisms as producers of this drug [1, 2].

A known method of producing somatotropin, including its expression in Escherichia coli cells, which consists in a sufficiently rapid biosynthesis of recombinant somatotropin by microbial cells in the form of insoluble “inclusion bodies” [3].

In RF patent No. 2233879, the authors describe the construction of plasmid DNA pES1-6, which provides the synthesis of recombinant growth hormone in Escherichia coli cells [4].

Known strain of Escherichia coli - producer of porcine somatotropin (RF Patent No. 2072393) [5].

In the patent of the Russian Federation No. 2031121, the authors proposed a method for producing a recombinant somatostatin-containing protein used as a substance of a veterinary medicinal product to increase the meat and milk productivity of farm animals. The dosage form of somatostatin-containing protein used to increase the productivity of farm animals contains a carrier protein in the form of an enzymatically inactive chloramphenicol acetyltransferase without 10 C-terminal amino acids, an immunogenic active part of somatostatin and an oil adjuvant [6].

However, the yield of the target protein produced by these methods is low and is about 0.2 g per 1 liter of producer fluid.

With the intensification of methods for the production of biological products, the optimization of biosynthesis conditions is usually carried out according to the following scheme: search for the optimal values of physicochemical parameters of cultivation (pH, temperature, etc.), as well as the selection of the composition of the nutrient medium. Also, the intensification of the biosynthesis process is achieved by dosed introduction of additional sources of carbon and nitrogen nutrition.

In some cases, the intensification of biosynthesis processes can be achieved due to changes in substrate concentrations and / or physico-chemical factors. The formation of the desired target peptides occurs at the beginning of the stationary phase of the growth of microbial cells when certain specific substrates are exhausted, in particular, when the inhibition of some enzymes is removed.

A similar induction of regulation of gene transcription factors has been studied with continuous cultivation of Saccharomyces cerevisiae yeast in a chemostat. A steady-state culture in a state limited by galactose was pulsed by various glucose concentrations. The inclusion of two different transcription systems into disturbing transition states has been shown [6].

In the patent of the Russian Federation No. 2451070 describes a method of fermentation with water to obtain a high yield of recombinant protein. It includes the continuous addition of a carbon source to a culture medium with a culture of recombinant bacterial cells and the subsequent continuous continuous addition of an inducer to a culture medium after the threshold value of one of the parameters is reached in the culture [7].

An object of the present invention is to develop a method for intensifying the process of biosynthesis of a somatostatin-containing protein in the deep cultivation of cells of the producer strain Escherichia coli, based on the use of a new algorithm for introducing carbon nutrition sources into the culture medium. The next objective of the present invention is to increase the biological activity of somatostatin-containing protein when used in farm animals by adding new ingredients that potentiate the action of the chimeric protein in the composition of the dosage form.

One of the aspects of the invention is that to intensify the biosynthesis of the recombinant somatostatin-containing protein, cells of the producer strain Escherichia coli are grown on a medium containing sources of amine nitrogen, vitamins, salt components and glucose with stirring and aeration, and isopropyl- is used as an inductor beta-D-thiogalactoside. At the same time, the dynamics of the biosynthesis process is controlled by the pH of the medium. With a sharp increase in the pH level by 0.2-0.4 units, the medium is fed with carbon sources, with the first feed, glucose in the amount of 2-3 g / dm ^{3 of} culture fluid is used as the carbon source, the second feed is carried out with a 10-12% yeast solution extract at the rate of 3-4 g / dm ^{3 of the} culture fluid, and the third feed is carried out by the introduction of glucose in an amount of 2-3 g / dm ^{3 of the} culture fluid.

Another aspect of the invention is that in order to intensify the biosynthesis of recombinant somatostatin containing protein, cells of the producer strain Escherichia coli are grown on a medium containing sources of amine nitrogen, vitamins, salt components and glucose with stirring and aeration, and isopropyl is used as an inductor. beta-D-thiogalactoside. At the same time, the dynamics of the biosynthesis process is controlled by the level of dissolved oxygen.

With an increase in the level of dissolved oxygen by 20–40% of the initial indices, they feed the cultivation medium with carbon sources. At the first recharge, glucose in the amount of 2-3 g / dm ^{3 of} culture fluid is used as a carbon source. With a secondary increase in the level of dissolved oxygen, a second feed is made in the form of a 10-12% solution of yeast extract at the rate of 3-4 g / dm ^{3 of the} culture fluid. With the next increase in the level of dissolved oxygen by 20-40%, glucose is introduced in an amount of 2-3 g / dm ^{3 of the} culture fluid, while the aeration is increased to 1 rpm / min.

Another aspect of the invention is that the inductor is introduced when the suspension density is from 3 to 3.5 units, and the isopropyl-beta-D-thiogalactoside alcohol solution is used as the inductor at a concentration of from 8 to 12 mg / dm ³ , preferably at a concentration 10.5 mg / dm ³ .

Another aspect of the invention is that a veterinary medicinal product for increasing the meat and milk productivity of farm animals consists of a somatostatin-containing protein obtained by deep growing cells of a recombinant strain producer Escherichia coli VKPM B-6519, an adjuvant based on refined vegetable oils and additionally contains bee wax, as well as medicinal compounds (vitamins A and E) that potentiate the action of the chimeric protein. In this case, the main components of the drug per 100 ml of oil suspension contain 0.9 g of beeswax, 250 mg of chimeric protein, 300,000 IU of vitamin A and 200 mg of vitamin E.

List of figures

FIG. 1 Control of cultivation by optical density, where I is the time of introduction of the IPTG inducer, and A, B. C are the periods of introduction of additional carbon sources during pH control.

FIG. 2 Dynamics of pH changes during the biosynthesis of recombinant somatostatin-containing protein.

FIG. 3 Dynamics of changes in the concentration of dissolved oxygen during the biosynthesis of recombinant somatostatin-containing protein.

In the process of developing a method for intensifying the biosynthesis of somatostatin-containing protein using a new algorithm for feeding carbon substrates during the deep cultivation of Escherichia coli cells, it was found that a combination of different carbon sources at the cultivation stages improves the efficiency of the synthesis of recombinant somatostatin-containing protein by microbial cells. So the pulsed introduction at the second stage of deep cultivation of cells of the producer strain of the yeast extract containing several types of carbon sources, allows to increase the yield of the target product.

The biosynthesis of the target protein is carried out under conditions of deep cultivation of the cells of the producer of Escherichia coli (strain VKPM B-6519) on a nutrient medium based on tryptone, yeast extract, as well as sources of nitrogen, phosphorus, mineral salts and containing glucose as a source of carbon and energy. Isopropyl-beta-D-thiogalactoside (IPTG) is used as an inductor at the rate of 10.5 mg / dm ^{3 of the} growth medium.

The cells of the strain VKPM B-6519 are gram-negative, sedentary sticks that form filaments under adverse conditions. The cells of the strain grow well in the temperature range (30-42) ° C on rich media of the LB type, as well as on synthetic media with additives that compensate for auxotrophic mutations. On rich media, the cells of the strain form smooth colonies with smooth edges, which mucilize over time, which is explained by the lon mutation. At an incubation temperature of 40-42 ° C, colonization of colonies does not occur. When grown on synthetic nutrient media with additives, the cell colonies of this strain are always mucous.

The cells of the strain Escherichia coli VKPM B-6519 (the strain was deposited in the All-Russian collection of industrial microorganisms) have the following genetic markers: F-, lacY, supE, gal 6, xyl4, mal A1, arcH, his ′, lon-, apr24, rpl. The cells of the strain are resistant to streptomycin, do not ferment lactose, galactose, xylose and maltose. The cells of the strain grow on a synthetic medium supplemented with glucose, arginine and histidine. The cells of the VKPM B-6519 strain, due to the presence of the plasmid pC (Sp) nS, acquire resistance to ampicillin. A culture for a working cell bank is grown on a semi-synthetic liquid nutrient medium of the following composition (g / dm ³ ):

Glucose 10.0 Tripton 15.0 Yeast extract 7.5 K ₂ HPO ₄ 1,0 KH ₂ PO ₄ 0.5 NaCl 10.0 Ampicillin sodium salt 0.05 Distilled water up to 1 l pH 7.0-7.1

A sign of the resistance of strain cells to streptomycin provides a simple selection of the strain, and the absence of lon protease determines a high level of synthesis of recombinant proteins.

The culture for the working cell bank is grown in a semi-synthetic liquid nutrient medium of the above composition.

Cultivation is carried out in rocking flasks with a volume of 750 cm ³ from 100 cm ^{3 of} nutrient medium. The flasks are seeded with a suspension of cells grown on the surface of the agar medium in Petri dishes and incubated on a shaker at a temperature of (37 ± 1) ° C and a stirring speed of 200 rpm for 7-10 hours.

The microbial culture of the producer of the recombinant somatostatin-containing protein in a production fermenter is grown on a semi-synthetic liquid nutrient medium of the above composition.

Maintain the following values of the technological parameters of the cultivation process:

temperature - (37 ± 1) ° C; pH value (initial) - 7.0 ± 0.1; mixing speed - 350 rpm aeration - 0.5 rpm growing time - 8-12 hours.

The control of the cultivation process is carried out according to the readings of the optical density of the medium. Optical density samples are taken every hour. Upon reaching a density of 3-3.5 units (the middle of the exponential growth phase), an alcohol solution of the isopropyl-beta-D-thiogalactoside inducer (IPTG) is added to the fermenter at the rate of 10.5 mg / dm ³ .

In the process of biosynthesis using Escherichia coli cells as a producer and using glucose as the main carbon source, “acidic” metabolic products are formed that lower the pH of the medium. During biosynthesis, the optimal pH value of the medium (about 7.0) is maintained using a controller that automatically adds a dose of alkali to the fermenter to maintain a given pH value. The rate of alkali supply during the biosynthesis of the target beige correlates with the rate of consumption of the carbon substrate. At the time of complete utilization of the dose of the substrate, the pH increases sharply, which is a very informative indicator for feeding the next dose of the carbon substrate.

When the pH value is increased above 7.2, the first feeding of the substrate with glucose is made at the rate of 2-3 g / dm ^{3 of the} culture fluid, the aeration is increased to 1 rpm / min. With a repeated increase in the pH value above 7.1-7.2, a second feed of the substrate is introduced in the form of a 10-12% solution of yeast extract at the rate of 3-4 g / dm ^{3 of} culture fluid. Then, when the decrease in pH is stopped and its value exceeds 7.1 -7.2 make a third feed of the carbon substrate with glucose at the rate of 2-3g / dm ^{3 of the} culture fluid.

The cultivation process is continued for another 3-5 hours until the cessation of the decrease in pH and its increase to values equal to 7.3-7.5.

According to another embodiment of the present invention, the level of dissolved oxygen is controlled to control the level of consumption of the carbon substrate. When the substrate is exhausted, the rate of oxygen consumption by producer cells decreases sharply and the level of dissolved oxygen increases. To correct the level of the carbon substrate, the substrate is fed with an increase in the level of dissolved oxygen by 20-40% of the initial values. At the first recharge, glucose in the amount of 2-3 g / dm ^{3 of} culture fluid is used as a carbon source. With a secondary increase in the level of dissolved oxygen by 20-40%, a 10-12% solution of yeast extract is added at the rate of 3-4 g / dm ^{3 of the} culture fluid. With the next increase in the level of dissolved oxygen by 20–40%, a third feed is introduced with a carbon source — glucose in an amount of 2-3 g / dm ^{3 of the} culture fluid, while aeration is increased to 1 rpm.

After completion of the synthesis process, the culture fluid is cooled to a temperature of 10-12 ° C and transferred to concentration.

An overpressure of 0.05 MPa is established in the fermenter with compressed air and the culture fluid is fed through a sterile line to the nozzle of the S-Z-61 flow-through centrifuge at a rotor speed of 17,000 rpm. The speed of separation of bacteria is 15-20 DM ³ h ^-1 . The microbial paste is resuspended in a lysis buffer solution and fed to the input of a Braun & Luebbe homogenizer, model SHL 05. The microbial suspension is homogenized once at a feed rate of 40-50 dm ³ h ^-1 .

Purification of the target recombinant protein consists in the denaturation of Taurus inclusions in a solution of guanidine chloride and the subsequent renaturation of the native conformation of somatostatin in the composition of the chimeric protein.

Insoluble components of the preparation are removed in a flow centrifuge at a rotation speed of 17,000 rpm.

The filtered supernatant is transferred to dialysis bags and sealed. Renaturation is carried out using a 3-5 step centrifuge at a speed of rotation of 17,000 rpm.

The filtered supernatant is transferred to dialysis bags and dried in KS-30 brand dryers or similar models. Upon reaching a working vacuum not exceeding 100 microns, the preparation should be cooled to minus (30 ± 35) ° С and kept at the indicated temperature for 45-60 minutes.

When the preparation is dried, the temperature on the shelves is regulated in such a way as to reach 0 ° C in the preparation by 16-18 hours of drying, then, gradually increasing the temperature, reach + (27-30) ° C in the material, the preparation is kept at this temperature for 8 -10 hours. The duration of the drying process is 24-28 hours.

The dosage form of the preparation is prepared according to the scheme using vegetable oils included in the group of refined sunflower, corn, cottonseed and rapeseed oils and their combinations as adjuvant, with the addition of 0.9% by weight of purified beeswax. The physiological effect of somatostatin-containing protein on the body of farm animals can be enhanced, bearing in mind the following.

The action of growth hormone on the body of mammals is carried out indirectly, namely through the formation of hepatocytes somatomedin C (insulin-like growth factor-1). On this basis, the additional use of drugs that enhance the synthesis of somatomedins, in addition to growth hormone, will lead to a summation of the final effect. One of these compounds is vitamin A. The use of vitamin A oil solution in the preparation will enhance the synthesis of somatomedins A1, A2, B, and C in the animal’s body, as well as the formation of enzymes necessary for activating phosphoadenosine phosphosulfate (FAFS), which is involved in the synthesis of mucopolysaccharides: chondroitin sulfur acids and sulfoglycans, components of connective tissue, cartilage, bones of farm animals [8].

In practice, vitamin A is used in conjunction with vitamin E. Their synergism is so high that vitamin A in the absence of tocopherol loses its properties and is rapidly destroyed [9]. Vitamin E, in addition, improves the use of protein by the cells of the body, affects the function of the genital and endocrine glands [10]. From this point of view, an additional introduction of vitamin E in the form of an oil solution into the finished form is justified. Thus, when preparing a new dosage form of a preparation based on a somatostatin-containing protein, it contains oily solutions of vitamin A at a concentration of 3000 IU / ml of adjuvant and vitamin E at a concentration of 2 mg / ml of adjuvant. The use of such a prescription of the drug allows to achieve more pronounced clinical effects when using a veterinary drug based on somatostatin-containing protein in farm animals.

Example 1. The biosynthesis of somatostatin-containing protein was carried out as follows. A strain VKPM B-6519 was used as a producer.

The strain Escherichia coli VKPM B-6519 was stored in cryovials at minus 40 ° C.

The seed for sowing the fermenter was grown in flasks on a semi-synthetic liquid nutrient medium of the following composition (g / dm ³ ):

Glucose 10.0 Tripton 15.0 Yeast extract 7.5 K ₂ HPO ₄ 1,0 KH ₂ PO ₄ 0.5 NaCl 10.0 Ampicillin sodium salt 0.05 Distilled water  up to 1 l pH 7.0-7.1

Contents from a cryovial were transferred to a flask with 100 cm ^{3 of} medium in compliance with aseptic rules. The flask was placed on a rocking chair and incubated at (180-200) rpm at (37 ± 1) ° C for 8 hours.

The process of biosynthesis of recombinant somatostatin-containing protein was carried out in a manufacturing fermenter BIOR-025 (OKB TBM, Kirishi) with a volume of 250 dm ³ on a nutrient medium of the above composition.

The following values of the technological parameters of the cultivation process were supported:

temperature - (37 ± 1) ° C; pH value (initial) - 7.0 ± 0.1; mixing speed - 350 rpm aeration - 0-4 hours of growth 0.5 rpm / min, then 1 rpm

During the cultivation process, culture fluid samples were taken at intervals of 60 minutes to determine the optical density. Where And - the time of introduction of the IPTG inductor, A, B, C - the time periods for the introduction of additional carbon sources. Upon reaching an optical density of 3.5 units (4 hours of growth), an isopropyl-beta-D-thiogalactoside inducer (IPTG) alcohol solution was added to the fermenter at the rate of 10.5 mg / dm ³ .

Then, when the decrease in pH was stopped and its values increased to 7.2–7.4 (4.5 hours of growth), the first glucose feed was added at the rate of 2 g / dm ^{3 of the} culture fluid, and aeration was increased to 1 rpm.

With a repeated increase in pH to 7.2 (6 hours of growth), a second feed was made with a 10% solution of yeast extract at the rate of 4 g / dm ^{3 of the} culture fluid.

Then, when the decrease in pH was stopped and increased to 7.2 (7 hours of growth), a third glucose feed was introduced at the rate of 3 g / dm ^{3 of the} culture fluid.

The cultivation process was continued for another 3 hours until the cessation of the decrease in the pH level and its increase to 7.3-7.5, then the culture fluid was cooled to a temperature of 10-12 ° C and transferred to concentration.

In FIG. Figure 2 shows a diagram characterizing the dynamics of changes in pH values during the biosynthesis of somatostatin-containing protein and the moments of additions.

The culture fluid from the fermenter was supplied with compressed air through a sterile line to the nozzle of the S-Z-61 flow-through centrifuge at a rotor speed of 17,000 rpm. The speed of separation of bacteria was 15-20 DM ³ h ^-1 . Received 2.7 kg of microbial paste.

The microbial paste was resuspended in a lysis buffer solution and the cells were destroyed using a Braun & Luebbe homogenizer, model SHL 05. The microbial suspension was homogenized once at a feed rate of 40-50 dm ³ h ^-1 . After destruction, insoluble components of the drug were separated on a S-Z flow-through centrifuge. 1.15 kg of washed inclusion bodies were obtained.

Further purification of the obtained chimeric protein was the denaturation of Taurus inclusions in a solution of guanidine chloride.

The renaturation of the native conformation of somatostatin in the composition of the chimeric protein was carried out by dialysis.

The dissolved intermediate was transferred to dialysis bags and sealed. Renaturation was performed using stepwise 3-5-fold dialysis (dialysate: buffer ratio of approximately 1:10).

The protein solution was dried in a KS-30 freeze dryer. Drying temperature reached + (27-30) ° С. The duration of the drying process was about 24 hours.

Received 193 g of dry somatostatin-containing protein with a molecular weight of 29.5 kDa.

Example 2. The biosynthesis of somatostatin-containing protein was carried out as follows. As a producer, a strain of Escherichia coli VKPM B-6519 was used.

Strain VKPM B-6519 was stored in cryovials at minus 40 ° C.

The seed for sowing the fermenter was grown in flasks on a semi-synthetic liquid nutrient medium of the following composition (g / dm ³ ):

Glucose 10.0 Tripton 15.0 Yeast extract 7.5

K ₂ HPO ₄ 1.0

KH ₂ PO ₄ 0.5 NaCl 10.0 Ampicillin sodium salt 0.05 Distilled water up to 1 l pH 7.0-7.1

Contents from a cryovial were transferred to a flask with 100 cm ^{3 of} medium in compliance with aseptic rules. The flask was placed on a rocking chair and incubated at (180-200) rpm at (37 ± 1) ° C for 8 hours.

The process of biosynthesis of recombinant somatostatin-containing protein was carried out in a manufacturing fermenter BIOR-025 (OKB TBM, Kirishi) with a volume of 250 dm ³ on a nutrient medium of the above composition.

The following values of the technological parameters of the cultivation process were supported:

temperature - (37 ± 1) ° C; pH value (initial) - 7.0 ± 0.1; mixing speed - 350 rpm aeration - 0-4 hours of growth 0.5 rpm / min, then 1 rpm

During the cultivation process, culture fluid samples were taken at intervals of 60 minutes to determine the optical density. Upon reaching an optical density of 3.8 units (4 hours of growth), an alcohol solution of the isopropyl-beta-D-thiogalactoside inducer (IPTG) was added to the fermenter at the rate of 10.5 mg / dm ³ .

Carbon substrates were fed by monitoring the level of dissolved oxygen. With an increase in the level of dissolved oxygen by 25-30% (5 hours of growth), the first glucose supplement was added at the rate of 2 g / dm ^{3 of the} culture fluid, and aeration was increased to 1 rpm.

With a repeated increase in the level of dissolved oxygen by 25-30% (6.5 hours of growth), a second feed was made with a 10% solution of yeast extract at the rate of 4 g / dm ^{3 of the} culture fluid.

Then, with an increase in the level of dissolved oxygen by 25-30% (8.5 hours of growth), a third glucose supplement was introduced at the rate of 3 g / dm ^{3 of the} culture fluid.

In FIG. Figure 3 shows a diagram characterizing the dynamics of changes in the concentration of dissolved oxygen during the biosynthesis of somatostatin-containing protein and the moments of introduction of additives.

The cultivation process was continued for another 3 hours until the cessation of the decrease in pH and its increase to 7.3-7.5, then the culture fluid was cooled to a temperature of 10-12 ° C.

The culture fluid was separated on a Sulfur Z-61 flow centrifuge at a rotor speed of 17,000 rpm. The speed of separation of bacteria was 15-20 DM ³ h ^-1 . Received 3.0 kg of microbial paste.

The microbial paste was resuspended in a lysis buffer solution and the cells were destroyed using a Braun & Luebbe homogenizer, model SHL 05. The microbial suspension was homogenized once at a feed rate of 40-50 dm ³ h ^-1 . After destruction, insoluble components of the drug were separated on a S-Z flow-through centrifuge. Received 1.0 kg of washed inclusion bodies.

Further purification of the obtained chimeric protein was the denaturation of Taurus inclusions in a solution of guanidine chloride.

The renaturation of the native conformation of somatostatin in the composition of the chimeric protein was carried out by dialysis.

The dissolved intermediate was transferred to dialysis bags and sealed. Renaturation was performed using stepwise 3-5-fold dialysis (dialysate: buffer ratio of approximately 1:10).

The protein solution was dried in a KS-30 freeze dryer. Drying temperature reached + (27-30) ° С. The duration of the drying process was 24 hours.

245 g of dry somatostatin-containing protein was obtained.

As follows from the results given in examples 1 and 2, the yield of the target protein with 1 liter of culture fluid is 3.5–5 times higher than previously known [6].

The possibility of carrying out the invention is confirmed by examples of the practical use of the drug to increase the meat and milk productivity of farm animals.

To prepare the finished form of the drug, refined vegetable oil and beeswax were used. Beeswax is added to the refined vegetable oil at a rate of 0.9% by weight, vitamins A and E in the form of an oil solution in concentrations of 3000 IU / ml of vitamin A adjuvant and 2 mg / ml of vitamin E adjuvant. The oil is heated to a temperature of 45-50 ° C. By mixing, they achieve complete dissolution of the wax. Then the wax oil is cooled to a temperature of 35-37 ° C and a portion of dry protein is added at the rate of 250 mg per 100 ml of adjuvant. The suspension is homogenized for 1-2 minutes and transferred to the filling equipment. The finished emulsion of the drug is packaged in containers (glass bottles and disposable syringes). At a different concentration of components, the drug is prepared similarly. The following component contents are preferred: per 100 ml of refined vegetable oil: 250 mg of chimeric protein, 0.9 g of beeswax; Vitamin A - 300,000 ME, Vitamin E - 200 mg.

Example 3. In the conditions of an industrial pig-breeding complex, pigs of large white breed aged 18-21 days and weighing 5.5-6.2 kg were introduced preparations at the rate of 50-200 μg of recombinant protein per 1 kg of live body weight according to the following scheme: the first two injections, with an interval of 14 days, the next two injections of the drug - with an interval of 60 days between injections. As a positive control, we used the dosage form of the drug without adding vitamins A and E to the adjuvant. The meat productivity of animals when using drugs at a dose of 50 μg per 1 kg of body weight are shown in table 1.

Table 1 Indicators of meat productivity of animals when using drugs at a dose of 50 μg per 1 kg of live body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The body weight of the animals during the first injection, kg 6.2 ± 0.3 5.9 ± 0.2 6.0 ± 0.3 The body weight of the animals during the second injection, kg 10.7 ± 0.2 10.9 ± 0.2 11.2 ± 0.1 The body weight of the animals during the third injection, kg 34.7 ± 0.4 35.5 ± 0.4 37.1 ± 0.2 The body weight of the animals during the fourth injection, kg 71.8 ± 0.3 75.4 ± 0.2 81.4 ± 0.2

As follows from the data presented in table 1, the dosage form of the drug with the addition of vitamins A and E had a more stimulating effect on the meat productivity of animals. So, after the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 4.50%, and by the end of the experiment this indicator was higher by 7.95%.

Indicators of meat productivity of animals when using drugs at a dose of 100 μg per 1 kg of live body weight are shown in table 2.

table 2 Indicators of meat productivity of animals when using drugs at a dose of 100 μg per 1 kg of live body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The body weight of the animals during the first injection, kg 6.2 ± 0.3 6.0, ± 0.2 6.1 ± 0.4 The body weight of the animals during the second injection, kg 10.7 ± 0.2 10.8 ± 0.1 11.1 ± 0.2 The body weight of the animals during the third injection, kg 34.7 ± 0.4 35.2 ± 0.3 36.9 ± 0.2 The body weight of the animals during the fourth injection, kg 71.8 ± 0.3 74.9 ± 0.2 81.2 ± 0.3

As follows from the data presented in table 2, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the meat productivity of animals. So, after the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 4.82%, and by the end of the experiment this indicator was higher by 8.41%.

Indicators of meat productivity of animals when using drugs at a dose of 200 μg per 1 kg of live body weight are shown in table 3.

Table 3 Indicators of meat productivity of animals when using drugs at a dose of 200 μg per 1 kg of live body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The body weight of the animals during the first injection, kg 6.2 ± 0.3 5.7 ± 0.1 5.9 ± 0.2 The body weight of the animals during the second injection, kg 10.7 ± 0.2 10.7 ± 0.2 11.0 ± 0.3 The body weight of the animals during the third injection, kg 34.7 ± 0.4 35.0 ± 0.3 36.3 ± 0.2 The body weight of the animals during the fourth injection, kg 71.8 ± 0.3 74.1 ± 0.2 80.2 ± 0.2

As follows from the results presented in table 3, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the meat productivity of animals. So, after the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 3.71%, and by the end of the experiment this indicator was higher by 8.23%.

The study of the effect of the drug on the meat productivity of bulls during fattening was carried out in livestock feeding organizations.

An oily suspension of preparations was administered to animals subcutaneously at a dose of 50-200 μg of protein per 1 kg of body weight twice with an interval of 14 days between injections. The third and fourth injections of the preparations were carried out with an interval of two months between injections. As a positive control, we used the dosage form of the drug without adding oil solutions of vitamins A and E to the adjuvant. The meat productivity of animals when using drugs at a dose of 50 μg of recombinant protein per 1 kg of body weight are shown in table 4.

Table 4 Indicators of meat productivity of animals when using drugs at a dose of 50 μg of recombinant protein per 1 kg of live body weight Indicators Groups of animals Animals without drug treatment Animals treated with the drug obtained Animals treated with the drug obtained (the control) according to old technology (first experimental group) on new technology (second experimental group) The body weight of the animals during the first injection, kg 147.2 ± 3.3 148.3 ± 3.4 147.1 ± 3.0 The body weight of the animals during the second injection, kg 158.7 ± 3.9 161.8 ± 4.9 164.1 ± 4.2 The body weight of the animals during the third injection, kg 206.1 ± 4.4 221.2 ± 3.8 227.9 ± 4.0 The body weight of the animals during the fourth injection, kg 271.8 ± 5.3 281.9 ± 3.1 295.2 ± 3.3

As follows from the results presented in table 4, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the meat productivity of animals. After the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 3.03%, and by the end of the experiment this indicator was higher by 4.71%.

Indicators of meat productivity of animals when using drugs at a dose of 100 μg of recombinant protein per 1 kg of live body weight are shown in table 5.

Table 5 Indicators of meat productivity of animals when using drugs at a dose of 100 μg of recombinant protein per 1 kg of live body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The body weight of the animals during the first injection, kg 146.2 ± 3.3 147.3 ± 3.4 147.1 ± 3.1 The body weight of the animals during the second injection, kg 157.7 ± 3.7 162.4 ± 4.6 164.9 ± 4.0 Body mass 207.2 ± 4.6 223.4 ± 3.6 229.6 ± 3.0 animals at the third injection, kg The body weight of the animals during the fourth injection, kg 273.5 ± 5.1 283.6 ± 2.9 297.4 ± 3.6

As follows from the results presented in table 5, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the meat productivity of animals. So, after the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 2.77%, and by the end of the experiment this indicator was higher by 4.87%.

Indicators of meat productivity of animals when using drugs at a dose of 200 μg of recombinant protein per 1 kg of live body weight are shown in table 6.

Table 6 Indicators of meat productivity of animals when using drugs at a dose of 200 μg of recombinant protein per 1 kg of live body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The body weight of the animals during the first injection, kg 143.4 ± 3.3 144.1 ± 2.9 143.9 ± 3.7 The body weight of the animals during the second injection, kg 158.5 ± 3.2 160.9 ± 4.4 165.4 ± 3.8 The body weight of the animals during the third injection, kg 203.8 ± 4.0 222.2 ± 5.8 228.2 ± 3.9 The body weight of the animals during the fourth injection, kg 269.8 ± 5.5 282.2 ± 3.6 295.4 ± 3.8

As follows from the results presented in table 6, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the meat productivity of animals. So, after the second injection of the drug, the live weight of animals in the second experimental group exceeded the same indicator in the first experimental group by 2.70%, and by the end of the experiment this indicator was higher by 4.67%.

To study the effect of the drug on the milk productivity of cattle in livestock farms, control and experimental groups of Holstein cows were formed, clinically healthy at the age of 4-5 years, with an approximately equal number of calving. An oily suspension of preparations was administered to animals subcutaneously at a dose of 50-200 μg of protein per 1 kg of body weight twice with an interval of 14 days between injections. The first injection of drugs was carried out on 75-80 days after calving, the second - after 14 days. As a positive control, the dosage form of the drug was used without adding oil forms of vitamins A and E to the adjuvant. Indicators of animal milk productivity when using drugs at a dose of 50 μg of recombinant protein per 1 kg of body weight are shown in table 7.

Table 7 Indicators of animal milk productivity when using drugs at a dose of 50 μg / kg body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The initial milk productivity of animals, kg / day 11,2 10.9 11.0 The milk productivity of animals 30 days after the second injection of the drug, kg / day 10.9 11.7 12.0 The milk productivity of animals 60 days after the second injection of the drug, kg / day 11.0 11.8 12,2 The milk productivity of animals 90 days after the second injection of the drug, kg / day 10.9 11,4 12.0

As follows from the results presented in table 7, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the milk productivity of animals. So, 30 days after the second injection of the drug, the milk production of animals in the second experimental group exceeded the same indicator in the first experimental group by 2.56%, and 90 days after the second injection of the drug this indicator was higher by 5.26%.

Indicators of milk productivity of animals when using drugs at a dose of 100 μg of recombinant protein per 1 kg of live body weight are shown in table 8.

Table 8 Indicators of milk productivity of animals when using drugs at a dose of 100 μg / kg body weight. Indicators Groups of animals Animals without drug treatment Animals treated with the drug Animals treated with the drug (the control) obtained by the old technology (first experimental group) obtained by new technology (second experimental group) The initial milk productivity of animals, kg / day 11,2 10.7 11.1 The milk productivity of animals 30 days after the second injection of the drug, kg / day 10.9 11.8 12.3 The milk productivity of animals 60 days after the second injection of the drug, kg / day 11.0 11.7 12,2 The milk productivity of animals 90 days after the second injection of the drug, kg / day 10.9 11.3 12.0

As follows from the results presented in table 8, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the milk productivity of animals. So, 30 days after the second injection of the drug, the milk production of animals in the second experimental group exceeded the same indicator in the first experimental group by 4.24%, and 90 days after the second injection of the drug this indicator was higher by 6.19%.

Indicators of milk productivity of animals when using drugs at a dose of 200 μg of recombinant protein per 1 kg of live body weight are shown in table 9.

Table 9 Indicators of animal milk productivity when using drugs at a dose of 200 μg / kg body weight. Indicators Groups of animals Animals without drug treatment (control) Animals treated with a preparation obtained according to old technology (first experimental group) Animals treated with a drug obtained by new technology (second experimental group) The initial milk productivity of animals, kg / day 11,2 11.0 11.1 The milk productivity of animals 30 days after the second injection of the drug, kg / day 10.9 11.7 12,2 The milk productivity of animals 60 days after the second injection of the drug, kg / day 11.0 11.7 12,2 The milk productivity of animals 90 days after the second injection of the drug, kg / day 10.9 11,4 11.9

As follows from the results presented in table 9, the dosage form of the drug with the addition of vitamins A and E had a more active stimulating effect on the milk productivity of animals. So, 30 days after the second injection of the drug, the milk production of animals in the second experimental group exceeded the same indicator in the first experimental group by 4.27%, and 90 days after the second injection of the drug this indicator was higher by 4.38%.

Bibliography

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Claims (11)

1. A method of obtaining a preparation for increasing the meat and milk productivity of farm animals, consisting in the fact that the recombinant producer strain Escherichia coli is grown on a medium containing an amine nitrogen source, a vitamin source, salt components and glucose with stirring and aeration, control the pH level and optical density, and isopropyl-beta-D-thiogalactoside is used as an inductor, isolation, purification, drying of a protein solution and preparation of an oil suspension of the preparation are carried out, characterized in that in quality The producer uses the recombinant producer strain Escherichia coli VKPM B-6519, and in the process of biosynthesis when stopping the decrease in pH and increasing its values to 7.2-7.3, glucose is fed at the rate of 2-3 g / dm ^{3 of the} culture fluid, this increases aeration to 1 rpm / min, with a repeated increase in pH values to 7.2-7.3, they are fed with a 10-12% solution of yeast extract at the rate of 3-4 g / dm ^{3 of} culture fluid, then when the decrease is stopped pH and increasing its values to 7.2-7.3 carry out a feed of glucose at the rate of 2-3 g / d ³ culture broth, the inductor is introduced when the suspension density of 3 to 3.5 units as an inducer of an alcoholic solution of isopropyl-beta-D-thiogalactoside in a concentration of from 8 to 12 mg / dm ^3. 2. The method according to p. 1, characterized in that tryptone is used as the source of amine nitrogen. 3. The method according to p. 1, characterized in that the yeast extract is used as a source of vitamins. 4. The method according to p. 1, characterized in that the fermentation is carried out under sterile conditions at a temperature of 36-38 ° C with stirring and aeration for 9-12 hours. 5. The method according to p. 1, characterized in that as an inductor use an alcohol solution of isopropyl-beta-D-thiogalactoside, preferably at a concentration of 10.5 mg / DM ³ . 6. A method of obtaining a preparation for increasing the meat and milk productivity of farm animals, consisting in the fact that the recombinant producer strain Escherichia coli is grown on a medium containing a source of amine nitrogen, a source of vitamins, salt components and glucose with stirring and aeration, control the level of dissolved oxygen and optical density, and isopropyl-beta-D-thiogalactoside is used as an inducer, the protein solution is isolated, purified, dried, and an oil suspension of the preparation is prepared, ex sistent with the fact that as the producer strain, recombinant-producing strain Escherichia coli VKPM B-6519, and during biosynthesis with increasing level of dissolved oxygen at 20-40% of glucose feed is performed at the rate of 2-3 g / ^dm3 culture liquid, while increasing aeration to 1 rpm / min; with a repeated increase in the level of dissolved oxygen by 20-40%, they are fed with a 10-12% solution of yeast extract at the rate of 3-4 g / dm ^{3 of the} culture fluid; then, with an increase in the level of dissolved oxygen by 20-40%, glucose is fed at the rate of 2-3 g / dm ^{3 of the} culture fluid, the inductor is introduced when the suspension density is from 3 to 3.5 units, an isopropyl-beta alcohol solution is used as an inductor D-thiogalactoside in a concentration of from 8 to 12 mg / DM ³ . 7. The method according to p. 6, characterized in that tryptone is used as the source of amine nitrogen. 8. The method according to p. 6, characterized in that the yeast extract is used as a source of vitamins. 9. The method according to p. 6, characterized in that the fermentation is carried out under sterile conditions at a temperature of 36-38 ° C with stirring and aeration for 9-12 hours. 10. The method according to p. 6, characterized in that as the inductor use an alcohol solution of isopropyl-beta-D-thiogalactoside, preferably at a concentration of 10.5 mg / DM ³ . 11. A preparation for increasing the meat and milk productivity of farm animals obtained by the method according to any one of paragraphs. 1-14 together with an adjuvant based on refined vegetable oils, characterized in that the oil suspension additionally contains beeswax, vitamins A and E, while the content of the components in 100 ml of the drug is as follows:
beeswax 0.9 g omatostatin-containing protein 250 mg vitamin a 300,000 ME vitamin e 200 mg vegetable oil up to 100 ml

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