RU2613420C1 - RECOMBINANT PROTEIN Mio-HSP, METHOD OF ITS PRODUCTION, INJECTION PREPARATION FOR MUSCLE MASS INCREASE IN FARM ANIMALS, BIRDS AND ANIMALS OF CANIDS, AS WELL AS METHOD OF USING PREPARATION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology and can be used for increasing muscle mass of farm animals, livestock and canines. Fused protein is obtained, consisting of myostatin, Gli-Ser spacer and glucan binding domain of alpha-glucan binding domain of Streptococcus mutans (Mio-HSP), that is used to produce injection preparation for subcutaneous or intramuscular injections in a dose of 50–200 mcg of said protein per one kg of animal body weight or poultry. For producing recombinant protein is grown cell strain E. coli M15 [rMio-HSP] - producer of recombinant protein myocardial HSP, which is obtained by transformation of cells of E. coli M15 plasmid rMio-HSP M15 containing a nucleotide sequence gene coding myocardial HSP, then bind protein myocardial HSP in paragraph 1 in the cell extracts strain E. coli M15 [rMio-HSP] with alpha-glucan binding sorbent by affine interaction incubation procedure, with further washing from unbound bacterial proteins and separating end product.

EFFECT: invention allows to effectively induce synthesis of specific antibodies to myostatin, inhibit and, consequently, stimulate growth of muscular tissue.

4 cl, 7 tbl, 6 ex

Description

Application area

The invention relates to genetic engineering, biotechnology and veterinary medicine, and in particular to a recombinant protein Myostatin-glucagon binding domain (GDM), a method for its production, an immunogenic composition containing myostatin as an antigen, an injection preparation for increasing muscle mass of farm animals, poultry and canine animals , as well as the method of using the specified injection drug to increase muscle mass of animals.

Known level

Myostatin (Myo) is a protein that inhibits the growth and differentiation of muscle tissue. Animal studies show that blocking the activity of myostatin leads to a significant increase in muscle mass of the body.

A known drug consisting of peptides that bind myostatin and inhibit its activity (WO 2004/058988 A2, IPC A61K 38/08, 2004). The binding agent includes at least one myostatin binding protein attached directly or indirectly to at least one carrier, such as a polymer or Fc domain. Drugs of this kind are not associated with the inhibition of myostatin through the synthesis of anti-myostatin antibodies and require regular administration into the animal's body to maintain the effect.

The pharmaceutical company Wyeth Pharmaceuticals has developed the drug MYO-029 (Stomalumab; Stamulumab), a combination of recombinant humanized antibodies that bind to myostatin and block its effects. At the beginning of 2005, clinical studies of the safety and tolerability of the drug were conducted in 116 patients (Wagner et al., A phase I-II trial of MYO-029 in adult subjects with muscular dystrophy, Annals of Neurology, 2008 Vol 63, Is 5, p . 561-571).

Clinical trial data were published in January 2008, and in March 2008, after the completion of clinical trials, Viet Pharmaceuticals announced the suspension of this project, since none of the examined patients showed a statistically significant improvement in muscle strength or growth muscle tissue.

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a recombinant immunologically active myostatin-containing protein that is easy to purify and has sufficient immunogenicity for myostatin as an antigen that can be used to increase the meat productivity of farm animals (cattle, pigs, horses, rabbits), poultry, and also to increase the muscle mass of canine animals by inducing the synthesis of specific autoantibodies to myostatin, blocking its action and as a result, stimulation of muscle growth.

Another objective of the invention is to implement a preparation based on the indicated protein and to create a method for using this preparation, which solves the problem of increasing the meat productivity of farm animals, poultry and increasing the muscle mass of animals of the canine family under the condition of unsystematic use of the drug. It should be noted that the amino acid sequence of myostatin is identical in all farm animals and canine animals. As a result of this, the developed preparation is a universal tool for increasing the meat productivity of farm animals, poultry and increasing the muscle mass of canine animals.

The solution to the first problem is provided by the following objects of the present invention.

Recombinant Mio-GDM protein for increasing muscle mass of farm animals, poultry and canine animals with a molecular weight of 35.9 kDa, encoded by the nucleotide sequence of the Mio-GDM gene SEQ ID NO 1.

A method of obtaining a recombinant protein Mio-GDM on glucan includes:

- growing cells of E. coli strain expressing the Mio-GSD gene;

- the binding of the Mio-GDS protein in the composition of cell extracts of E. coli M15 strain with a glucan-containing sorbent due to affinity interaction during the incubation procedure;

- subsequent washing from unbound bacterial proteins and the selection of the target product.

The recombinant Mio-GSD protein includes the protein sequence of the glucan binding domain, which determines the ability of this protein to bind to the glucan-containing sorbent, which allows concentration, purification and immobilization of the protein product on glucan in one step. Immobilization on glucan is ensured by the presence in the recombinant protein of a glucan binding domain from the alpha glucan binding domain of a gene from Streptococcus mutans, which has a high affinity for alpha glucans (pullulan, glycogen, dextran, starch) and provides an irreversible broad binding range with pH 6.0-9.0 and salt concentrations of 0-3 M NaCl.

Since there are no proteins binding to alpha-glucan in E. coli cells, the recombinant Myo-GDS protein synthesized in E. coli cells is the only protein of the producer strain cells that binds strongly to alpha-glucan. This provides the possibility of a single-stage production of a highly purified recombinant protein preparation immobilized on a glucan-containing sorbent.

The solution to the second problem is provided by the following objects of the present invention.

The injection preparation for increasing the muscle mass of farm animals, poultry and canine animals contains the recombinant Mio-GDS protein, described above, suspended in a medium from a glucan-containing sorbent in an acceptable liquid carrier for injection. The method of increasing the muscle mass of farm animals, poultry, and canine animals includes: administering subcutaneous or intramuscular injections of a preparation containing recombinant Myo-GDS protein twice in a medium from a glucan-containing sorbent in a liquid carrier acceptable for injection use at a dose of two times with an interval of 14 days in a dose 5-200 μg of the indicated protein per kilogram of body weight of an animal or bird.

Thus, a bifunctional recombinant Mio-GDS protein was created, which has the ability to spontaneously bind to a glucan-containing sorbent, forming a highly immunogenic composition in the form of a polyantigen, induce the synthesis of specific autoantibodies to myostatin when introduced into animals and, as a result, stimulate muscle tissue growth.

Obtaining a recombinant Mio-GDS fusion protein

At the first stage, the myostatin gene is produced, followed by its cloning.

The myostatin gene was obtained by the chemical-enzymatic method. An oligonucleotide duplex encoding an appropriate gene optimized for expression in E. coli was planned. Then, the plasmid rMio-HSD was obtained, containing the sequences encoding myostatin, the Gly-Ser spacer and the glucan binding domain (HSD).

Obtaining a strain of E. coli, a producer of the recombinant myostatin antigen connected to the cellulose-binding domain

To obtain a strain of E. coli, a producer of the recombinant protein Myo-GDS, E. coli M15 cells were transformed with the plasmid rMio-HSD. 3 μl of a 0.1 M solution of isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the culture and grown for 3 hours at 37 ° C. When comparing the spectrum of proteins synthesized by cells of the strain E. coli M15 [rMio-GDM], the appearance of an additional protein band was detected. The molecular weight of the additional band corresponded to the expected mass of 35.9 kDa for the recombinant Myo-GDS protein. The level of protein synthesis in E. coli was determined by comparing the staining intensity of the band of the recombinant protein with the band of the corresponding protein, the molecular weight standard. It was shown that the recombinant Mio-HSD protein is synthesized in E. coli cells in an insoluble form in the form of Taurus inclusion.

Obtaining recombinant protein Mio-GDM immobilized on alpha-glucan

To obtain a recombinant protein, the cell culture of E. coli M15 strain [rMio-GDM] was grown in 1000 ml of LB medium with ampicillin (100 μg / ml) at 37 ° C to an optical density corresponding to 1 unit. absorption at a wavelength of 550 nm. 15 μl of a 0.1 M IPTG solution was added to the medium and grown for 3 hours. Cells were besieged by centrifugation at 5500 g for 15 minutes.

The pellet was resuspended in phosphate buffer with lysozyme. Additionally, the suspension was treated with ultrasound. After centrifugation at 6000 g, the insoluble Mio-HSD protein remained in the sediment. The precipitate was suspended in 8M urea, centrifuged at 12,000 g for 30 minutes, and the supernatant was collected. To immobilize the recombinant Mio-HSD protein on a sorbent, the supernatant was diluted four times with phosphate buffer, 1/10 of the volume of alpha-glucan suspension was added, incubated at 25 ° C for 2 hours. Centrifuged at a speed of 8000 rpm, the precipitate was resuspended in phosphate buffer; Alpha-glucan was washed 3 times. The Mio-GSD antigen immobilized on alpha-glucan is a suspension of a sorbent with a protein adsorbed on it. The purity of the drug was at least 95%. Preservation of the drug was carried out by adding benzyl alcohol to a concentration of 0.1%.

The biological effect of the recombinant protein Mio-GSD

In a preferred embodiment of the invention, the preparation contains a recombinant Mio-GDS protein suspended in a solution of glycan (90% by weight), a water-oil suspension (10% by weight) or aluminum hydroxide (equal volume); used by subcutaneous or intramuscular injection of the drug twice with an inter-injection interval of 14 days at a dose of 50-200 μg of recombinant protein per kilogram of animal or bird body weight. The mechanism of action of the drug is based on the temporary blocking of the activity of endogenous myostatin using autoantibodies.

The effectiveness of the drug to increase muscle mass in farm animals is illustrated by the following examples.

Example 1. The effect of the drug with the recombinant Mio-GDM protein on the weight gain of piglets.

Under the conditions of an industrial pig-breeding complex, piglets aged 100-110 days with a body weight of 50-52 kg were injected with a preparation containing the recombinant Mio-GDS protein suspended in medium from alpha-glucan (90% by weight), a water-oil suspension (10 % by weight) twice with an interval of 14 days at the rate of 50-200 μg of recombinant protein per 1 kg of live body weight of animals. The drug was administered subcutaneously. The results are presented in table 1.

As can be seen from the data presented, a twofold (with an interval of 14 days) administration of the preparation containing the recombinant Mio-GSD protein led to an increase in body weight of piglets 90 days after the second injection of the drug when 12.2 mg of the recombinant protein was used at a dose of 50 μg / kg % in relation to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 12.6%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 13.4%.

Table 2 shows the titers of autoantibodies to myostatin after immunization with a preparation containing the recombinant Myo-GDS protein (ELISA method).

From the data in table 2 it follows that the optimal dose of the drug containing the recombinant Mio-GDM protein for the induction of autoantibodies in animals is a dose of 50 mg / kg of animal body weight.

Example 2. The effect of the drug with the recombinant protein Mio-GDM on the increase in body weight of meat bulls.

Under the conditions of the feeding complex, gobies of Kalmyk breed at the age of 10 months were injected with a preparation containing the recombinant Mio-GSD protein suspended in alpha-glycan medium (90% by weight), a water-oil suspension (10% by weight) twice with an interval of 14 days at the rate of 50-200 μg of recombinant protein per 1 kg of animal body weight. The drug was administered subcutaneously or intramuscularly. The results are presented in table 3.

As can be seen from the data presented, a double (with an interval of 14 days) administration to the calves of the Kalmyk breed of the preparation containing the recombinant Mio-GSD protein led to an increase in the body weight of the animals 90 days after the second injection of the drug when used at a dose of 50 μg / kg of recombinant protein per 9.0% in relation to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 9.8%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 9.9%.

Example 3. The effect of the drug with the recombinant protein Mio-GSD on the increase in body weight of lambs.

Romanov lambs at the age of 3.5-4 months were injected with a preparation containing the recombinant Mio-GDS protein suspended in alpha-glycan medium (50% by weight), aluminum hydroxide suspension (50% by weight), twice with an interval of 14 days at the rate of 50-200 μg of recombinant protein per 1 kg of animal body weight. The drug was administered subcutaneously or intramuscularly. The results are presented in table 4.

As can be seen from the data presented, a two-fold (with an interval of 14 days) administration to the lambs of the Romanov breed of the preparation containing the recombinant Mio-GSD protein led to an increase in the body weight of the animals 90 days after the second injection of the drug when used at a dose of 50 μg / kg of recombinant protein per 12.9% in relation to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 14.1%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 16.0%.

Example 4. The effect of the drug with the recombinant protein Mio-GSD on the increase in body weight of rabbits.

A preparation containing recombinant protein was used on rabbits of the Soviet chinchilla breed at the age of 60 days twice with an interval between injections of 14 days. The drug was used at a dose of 50, 100, 200 μg / kg of recombinant protein. The drug was administered subcutaneously. The results are presented in table 5.

As can be seen from the data presented, a two-fold (with an interval of 14 days) administration to the rabbits of the Soviet chinchilla breed of the preparation containing the recombinant Mio-GSD protein led to an increase in the body weight of animals 60 days after the second injection of the drug when used at a dose of 50 μg / kg of recombinant protein by 10.6% in relation to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 12.0%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 11.5%.

Example 5. The effect of the drug with the recombinant protein Mio-GDM on the increase in body weight of turkeys.

The preparation containing the recombinant protein was used on turkeys (males) of the breed white broad-chested (heavy cross) at the age of 50 days twice with an interval between injections of 14 days. The drug was used at a dose of 50, 100, 200 μg / kg of recombinant protein. The drug was administered subcutaneously or intramuscularly. The results are presented in table. 6

As can be seen from the data presented, a double (with an interval of 14 days) administration of a white broad-breasted (heavy cross) turkey (male) breed preparation containing the recombinant Mio-GSD protein led to an increase in animal body weight 90 days after the second injection of the drug when used in a dose of 50 μg / kg of recombinant protein by 12.1% with respect to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 12.6%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 12.0%.

Example 6. The effect of the drug with the recombinant protein Mio-GDM on weight gain in dogs.

The drug was used on Caucasian shepherd dogs (males) at the age of 50-60 days twice with an interval between injections of 14 days. The drug was used at a dose of 50, 100, 200 μg / kg of recombinant protein. The drug was administered subcutaneously. The results are presented in table 7.

As can be seen from the data presented, a two-fold (with an interval of 14 days) administration to males of a Caucasian shepherd dog a preparation containing the recombinant Mio-GSD protein led to an increase in animal body weight 90 days after the second injection of the drug when a dose of 50 μg / kg of recombinant protein was used per 10.1% in relation to this indicator in the control group; when using the drug in a dose of 100 μg / kg of recombinant protein - by 6.7%; when using the drug in a dose of 200 μg / kg of recombinant protein - by 13.1%.

The examples of the invention are not exhaustive. Other examples of implementation are possible, corresponding to the scope of patent claims.

Claims (4)

1. Recombinant protein myostatin-glucan binding domain (Mio-GDM) with a molecular weight of 35.9 kDa to increase the muscle mass of farm animals, poultry and canine animals, encoded by the nucleotide sequence of the Mio-GDM gene SEQ ID NO 1. 2. A method for producing a recombinant Mio-GDS protein on glucan, comprising binding the Mio-GDS protein according to claim 1 as part of cell extracts of E. coli strain M15 [rMio-GDM] with an alpha-glycan-containing sorbent due to affinity interaction during the incubation procedure, followed by washing off unbound bacterial proteins and isolating the desired product. 3. An injection preparation for increasing the muscle mass of farm animals, poultry and canines, containing the recombinant Mio-GSD protein according to claim 1, suspended in an environment with an alpha-glycan-containing sorbent in a liquid carrier acceptable for injection, for subcutaneous or intramuscular injections in a dose of 50-200 μg of the specified protein per kilogram of body weight of an animal or bird. 4. A method of increasing the muscle mass of farm animals, poultry, and canines, including double, with an interval of 14 days, subcutaneous or intramuscular injections of a preparation containing the recombinant Myo-GDS protein according to claim 1, suspended in a medium from an alpha-glycan sorbent in acceptable for injection use in a liquid carrier, at a dose of 50-200 μg of the indicated protein per kilogram of animal or bird body weight.