RU2585494C2 - Recombinant protein rec-mp, containing sequence of myelopeptides for treating secondary immunodeficiency conditions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to production of recombinant protein containing sequences of myelo-peptides, and can be used for treatment of secondary immunodeficiencies. Recombinant protein (REC-MP) is constructed by alternation of short active myelopeptides MP-1 (FLGFPT), MP-2 (LVVYPW), MP-3 (LVCYPQ) and MP-4 (FRPRMTP), alternating with thrombin and urokinase recognition sites.

EFFECT: invention allows to increase clinical effectiveness of treatment of secondary immunodeficiencies by more evident stimulation of antibodies formation, by increasing the number of B-lymphocytes expressing CD25 and CD69 on their surface.

5 cl, 4 dwg, 2 tbl, 6 ex

Description

The invention relates to the field of biotechnology, in particular to the production of a recombinant protein containing myelopeptide sequences (rec-MR) for use in the treatment of secondary immunodeficiencies.

One of the important medical problems of our time is the spread of secondary immunodeficiency states (VIDS), caused by exposure to adverse environmental factors. Secondary IDS can provoke: injuries of a physical, chemical and biological nature, poor ecology, endocrinopathies (diabetes mellitus, hypothyroidism, etc.), surgery (anesthesia + stress + trauma), drugs (glucocorticoids, antibiotics, cytostatics and other immunosuppressants), defects nutrition, stress syndrome, etc. For the treatment of secondary IDS, more than 30 different drugs are currently used [Diagnostic and treatment standards "Immunology and Allergology", 2001]. However, every year the expanding spectrum of various lesions of the immune system by rapidly changing adverse environmental factors requires the development of new drugs to restore it.

The methods of drug treatment of secondary immunodeficiencies are based on the idea that changes in the absolute or relative number of cells involved in immune reactions, or their functional activity, can be restored using appropriate immunotropic drugs. One of the sources for the development of drugs that restore the normal functioning of the immune system are immunologically active peptides isolated from lymphoid tissues or their synthetic analogues.

Based on the myelopeptides produced by bone marrow cells, myelopid was developed [Petrov RV. Immunomodulating activity of myelopeptides: clinical trials, Ann N Y Acad Sci. 1993 Jun 23; 685: 351-61. Review], representing a complex of bioregulatory mediators. This is a group of low molecular weight peptides with a molecular weight of 500-3000 daltons. These myelopeptides are obtained by extraction of pre-cultured in vitro pig or calf bone marrow cells. Obviously, obtaining short peptides from animal tissues and cells is very costly and poorly reproducible. Current requirements for the composition and purity of drugs limit this method of production.

In patent RF 2507212 a method for producing recombinant peptides using producer strains of E. coli is proposed. The disadvantage of the proposed method is the expression of each peptide separately. First, short peptides can only be expressed as part of a fusion protein with a total mass of at least 10 kD. With an average weight of the target peptide of less than 1 kD, this sharply reduces the yield of the product and increases its cost. Secondly, as already noted, the preparations contain several peptides. Therefore, it is necessary to obtain several separate producer strains producing the desired number of peptides. This still significantly increases the cost of production. As a result, in the case of the implementation of the proposed invention, the price of the product will be significantly higher than the mixture of peptides obtained by the traditional method from animal tissues.

The prototype of the claimed invention is the patent document of the Russian Federation 2198178. This document discloses the four myelopeptides that are part of the claimed REC-MP, but their individual biological activity is shown. It was shown that these peptides separately do not have the same characteristics (description of page 2), which will determine their suitability for any particular application. So myelopeptide (MP-3) protects against bacterial infection by stimulating macrophage phagocytosis, while MP-4 induces terminal differentiation in leukemia cells. In addition, the increase in serum interferon induced by different MPs was also different. So, with a single injection of 0.1 μg MP-4 in serum after 48 hours, the level of interferon reached 80 units / ml, while a single injection of 0.01 μg MP-5 led to a very strong increase in late (48 h) serum interferon - 320 units / ml. Moreover, MP-3 and MP-6 weakly induced late interferon at various concentrations. Thus, the different activity of MP in relation to the production of interferon will manifest different efficacy in the treatment of secondary immunodeficiencies.

Therefore, the object of the invention is to obtain a recombinant fusion polypeptide with higher immunological activity for the treatment of IDA.

According to the present invention, a recombinant rec-MP protein is constructed by alternating short active myelopeptides alternating with specific protease recognition sites having the amino acid sequence of SEQ ID NO: 1. The recombinant protein contains four myelopeptides: FLGFPT, LVVYPW, LVCYPQ and FRPRMTP. As protease recognition sites, sequences for hydrolysis by thrombin and urokinase were used.

Recombinant rec-MP protein was obtained by expression in E. coli cells; methods for purification and refolding of this product were developed. To obtain a recombinant protein, a genetic construct was created based on the pET30a plasmid, which contains the gene encoding this recombinant protein. The gene sequence is represented by SEQ ID NO: 2. The primary structure and homogeneity are confirmed by mass spectrometry and HPLC. The recombinant protein rec-MR after introduction into the body is able to hydrolyze with the release of its myelopeptides.

The technical result achieved by the implementation of this invention is to increase the effectiveness of the prevention and treatment of secondary IDS when using REC-MP containing a myelopeptide complex and represented by the sequence SEQ ID NO: 1, by more pronounced stimulation of antibody formation, an increase in the number of B-lymphocytes expressing on their surfaces of CD25 (receptor for interleukin 2) and CD69 (lymphocyte activation marker).

A brief description of the drawings.

FIG. 1. The chromatographic profile of the passage of the recombinant rec-MP protein through SP-Sepharose. The major peak corresponds to the yield of the recombinant protein.

FIG. 2. Chromatographic profile of elution of recombinant rec-MP protein from a Kromasil 300-5C18 analytical column.

FIG. 3. The effect of recombinant protein rec-MP on the content in the lymph nodes of experimental animal cells carrying receptors CD25 and CD69.

FIG. 4 Analysis of lymph node cells of animals immunized with recombinant rec-MP protein.

The invention is illustrated by examples.

Example 1. Obtaining a producer strain of the recombinant protein REC-MP.

To obtain a recombinant protein, a genetic construct was created based on the pET30a plasmid, which contains the gene encoding this recombinant protein. The gene sequence is represented by SEQ ID NO: 2.

Plasmid transfected cells of E. coli strain BL21 (DE3). Transformed cells were scattered on a Petri dish with LB agar containing 25 mg / ml kanamycin and incubated for 16 hours at 37 ° C. 24 single colonies were incubated for 10 hours in 1 ml LB containing 25 mg / ml kanamycin. 50 μl of cell cultures were introduced into 0.5 ml LB containing 25 mg / ml kanamycin, incubated for 2 hours and expression of rec-MP protein was induced by adding IPTG to 1 μM. Matrix cultures were stored at -80 ° C until the end of clone analysis. The presence of rec-MR protein was analyzed by SDS page electrophoresis. One clone producing the highest amount of rec-MP protein was used as the producer strain of the recombinant rec-MP protein.

Example 2. Obtaining and chromatographic purification of recombinant protein REC-MP.

The cells of the producer strain were subcultured into a culture flask with 200 ml LB containing 25 mg / ml kanamycin. The culture was grown on a shaker at 280 rpm at a temperature of 37C until the optical density reached OD585 = 0.8E. Protein expression was induced by adding IPTG to a concentration of 1 mM. The culture was additionally cultured for 4 hours, then the cells were precipitated by centrifugation at 5000 g. Cell pellet was lysed in 8M urea and processed on an ultrasonic disintegrator. The cell lysate was centrifuged at 20,000 g for 2 hours. The supernatant was filtered through a Q-Sepharose column. The fraction that did not bind to this sorbent was chromatographed on SP-Sepharose in a NaCl gradient (start buffer: 20 mM TrisHCl pH6.8, 20 mM NaCl, final buffer 20 mM TrisHCl pH6.8, 400 mM NaCl). The chromatographic profile is shown in FIG. 1. Fractions containing the target product were combined and dialyzed in 0.9% NaCl. The purity of the product was analyzed by HPLC (Fig. 2). Amino acid sequence of recombinant protein rec-MP:

. Подчеркнуты сайты узнавания протеаз: GlyArgGly - сайт распознавания тромбином, ThrGlyArg - сайт распознавания урокиназой

. Underlined protease recognition sites: GlyArgGly - thrombin recognition site, ThrGlyArg - urokinase recognition site

Example 3. The effect of recombinant protein on the number of cells producing antibodies in the lymph nodes of immunized animals.

15 female mice, first-generation hybrids (CBACCHC57BL), weighing 20-22 g, were immunized with 5% suspension of sheep erythrocytes in physiological saline, 0.1 ml subcutaneously in the hind limb pads. 14 days after the first immunization was re-immunized with the same dose and in the same way. On the 4th day after the second immunization, mice were divided into 3 groups of 5 animals per group. The first group (control) was injected subcutaneously in the pads of the hind legs in 0.1 ml of physiological saline; the second group was administered 0.1 ml of physiological saline containing 1.5 μg of the commercial preparation myelopid and the third group - 0.1 ml of physiological saline containing 1.5 μg of recombinant protein. After 24 hours, all the mice were blocked and their popliteal lymph nodes were removed. Lymph nodes were homogenized and the number of cells producing an IgG antibody against sheep erythrocytes was determined in the obtained cell suspension by the method of Erne. The stimulation coefficient (K) of antibody formation was calculated as the ratio of the number of hemolysis zones in the test cups to the number of hemolysis zones in the control. The significance of the difference between the experimental groups was evaluated by student t-test. The results obtained are presented in Table 1. From the obtained data it is seen that the stimulation coefficient - myelopid / control is (36.4 / 25.2) = 1.44, and the stimulation coefficient - recombinant protein / control was (92.4 / 25, 2) = 3.67.

Thus, the introduction of the recombinant rec-MP protein into experimental animals leads to a significantly more pronounced stimulation of antibody formation (K = 3.3), i.e. providing a synergistic effect of the effects of its myelopeptides than after the introduction of the myelopeptide (K = 1.45), which confirms the increased effectiveness of rec-MR for the prevention and treatment of secondary IDs by activating a humoral immune response.

Example 4. The effect of recombinant protein rec-MP on the content in the lymph nodes of experimental animal cells carrying receptors for interleukin 2.

Fifteen mice of the first generation hybrids (CBA × C57BL) were immunized with a 10% suspension of EB in physiological saline subcutaneous solution of 0.05 ml in the pads of the hind legs. 14 days after the first immunization was re-immunized with the same dose and in the same way. On the 3rd day after the second immunization, one group of mice (5 animals) was injected with 0.15 μg of rec-MR in 0.1 ml of saline in the pads of the hind legs. The other 5 immunized mice were injected with rec-MR in a dose of 1.5 μg, Control animals were injected with 0.1 ml of physiological saline into the pads of the hind legs. On the 4th day after immunization, animals were killed, popliteal lymph nodes were taken, homogenized, cell suspensions were obtained, which were stained with fluorescein-labeled dyes with BD Bioscience antibodies (Anti-Mouse CD4 FITC, cat. 11-0042-82 and Anti-Mouse CD25 APC cat. 17-0251-82) according to the manufacturer's instructions. Stained cells were analyzed on a Gallios flow cytometer (Beckman Coulter). The results were processed using the FlowJo v.7.6 program. The cells of the lymphocytic gate, constructed according to the parameters of direct and lateral light scattering, were analyzed. The results are presented in figure 4.

In FIG. Figure 4 shows that the introduction of rec-MR into the productive phase of the humoral immune response leads to an increase in cells carrying receptors for Interleukin 2 (CD 25). This effect has a dose-dependent nature (control - 1.47%, rec-MR at a dose of 0.15 μg - 4.09% and rec-MR at a dose of 1.5 μg - 7.33%). These cells do not belong to a subpopulation of T-lymphocytes expressing the CD4 marker.

Example 5. The effect of recombinant protein rec-MP on the content in the lymph nodes of experimental animal cells carrying a marker of activation of lymphocytes (CD69).

10 mice of the first generation hybrids (CBA × C57BL) were immunized with a 10% suspension of EB in physiological saline 0.05 ml subcutaneously in the pads of the hind legs and at the base of the tail. 14 days after the first immunization was re-immunized with the same dose and in the same way. On the next day after the second immunization, mice were injected with RB at a dose of 1 μg or saline into the paw pads and the base of the tail. On the 3rd day after the second immunization, animals were killed, popliteal and inguinal lymph nodes were taken, homogenized, cell suspensions were obtained, which were stained with Lymphocyte Activation Antibody Cocktail, with Isotype Control (PE-CY ™ 7 CD25, PE CD69, & APC CD 19 ) (BD Biosciences), according to the manufacturer's instructions. Stained cells were analyzed on a Gallios flow cytometer (Beckman Coulter). The results were processed using the FlowJo v.7.6 program. The cells of the lymphocytic gate, constructed according to the parameters of direct and lateral light scattering, were analyzed. The results are presented in FIG. 3.

From the presented results in FIG. 3 it follows that in the lymph nodes of mice draining the site of introduction of antigen (EB), under the influence of rec-MR, the relative number of cd25 + cd19 + cells increases by 2.5, i.e. B-lymphocytes carrying the receptor for IL2, and approximately 2 times the cd69 + cd19 + cells, i.e. cells carrying a marker of activation of b-lymphocytes. It should also be noted that along with an increase in B lymphocytes carrying the cd25 marker, the number of other lymphocytes carrying the IL2 receptor also increased by 1.6, which indicates the possible participation of various types of immune system cells in the implementation of the antibody-stimulating activity of the claimed rec-MR.

Thus, REC-MP, containing a myelopeptide complex and represented by the sequence SEQ ID NO: 1, increases the number of B-lymphocytes expressing CD25 (receptor for interleukin 2) and CD69 (lymphocyte activation marker) on their surface.

Example 6. The study of the ability of a recombinant protein to restore the humoral immune response in a secondary immunodeficiency state.

Ten female mice, first-generation hybrids (CBA4C57BL) weighing 18-20 g were injected intraperitoneally with cyclophosphamide at a dose of 200 mg / kg in physiological saline. 5 mice were injected with saline. On the next day, all 15 animals were immunized with a 5% suspension of sheep erythrocytes in physiological saline, 0.1 ml subcutaneously in the hind limb pads. On the 2nd and 3rd day after immunization, 5 animals from the group that received cyclophosphamide were intramuscularly injected with 1.5 μg of recombinant protein in 0.1 ml of physiological saline. On the 4th day after immunization with sheep erythrocytes, all mice were clogged and popliteal lymph nodes were removed. Lymph nodes were homogenized and the number of cells producing an IgM antibody against sheep erythrocytes was determined in the resulting cell suspension by the method of Erne. The significance of the difference between the experimental groups was evaluated by student t-test. The results obtained are presented in Table 2.

From the presented data it is seen that the introduction of experimental recombinant protein rec-MP to experimental animals with secondary immunodeficiencies leads to the restoration of the humoral immune response.

Thus, the creation of REC-MP containing a complex of myelopeptides and represented by the sequence SEQ ID NO 1 allows not only to preserve the functional activity of its myelopeptides, but also to increase the effectiveness of the treatment of secondary immunodeficiencies. Since, after introduction into the body, REC-MP is able to hydrolyze at the recognition sites of specific proteases with the release of short myelopeptides entering it, providing a synergistic effect during immunization (as noted in the above examples).

Sequence listing

<110> FSBI "SSC Institute of Immunology" FMBA of Russia

<120> Recombinant rec-MP protein containing myelopeptide sequences for the treatment of secondary immunodeficiency conditions.

<160> NUMBER OF SEQ ID NOS: 6

<210> SEQ ID NO 1

<211> 73

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 1 :

MFLGFPTGRGLVVYPWGRGLVCYPQGRGFRPRIMTPGRGFLGFPTTGRLVVYPWTGRLVCYPQTGRFRPRIMT

<210> SEQ ID NO 2

<211> 228

<212> DNA

<213> artificial

<400> SEQUENCE 2:

ATGTTCTTAGGCTTTCCAACTGGCCGTGGTCTGGTGGTGTATCCATGGGGTCGTGGTCTGGTGTGCTATCCGCAAGGTCGTGGCTTCCGTCCACGCATCATGACTCCAGGCCGCGGCTTTCTGGGCTTCCCGACCACTGGTCGTCTCGTTGTGTACCCGTGGACCGGCCGCCTGGTTTGTTACCCACAGACGGGTCGCTTTCGCCCGCGTATTATGACGCCGTAATGA

<210> SEQ ID NO 3

<211> 6

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 3:

FLGFPT

<210> SEQ ID NO 4

<211> 6

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 4:

LVVYPW

<210> SEQ ID NO 5

<211> 6

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 5:

LVCYPQ

<210> SEQ ID NO 6

<211> 7

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 6:

FRPRMTP

Claims (5)

1. A recombinant polypeptide for the treatment of secondary immunodeficiency conditions, comprising myelopeptides interspersed with protease recognition sites having the amino acid sequence of SEQ ID NO: 1. 2. The recombinant polypeptide according to claim 1, characterized in that it consists of myelopeptides: FLGFPT, LVVYPW, LVCYPQ and FRPRMTP. 3. The recombinant polypeptide according to claim 1, characterized in that its composition includes a urokinase recognition site. 4. The recombinant polypeptide according to claim 1, characterized in that the thrombin recognition site is included in its composition. 5. The recombinant polypeptide according to claim 1, characterized in that its composition includes recognition sites with thrombin and urokinase.

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