Complex of biologically active recombinant protein with polysialic acid
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, in particular to obtaining protein complexes and can be used in medicine. A complex of biologically active recombinant protein is obtained, which is non-covalently bonded to polysialic acid which is inactive in aqueous solution at pH 4.4-4.7 which has prolonged therapeutic effect and has general formula:
where m=20-110; n=1-4; P is biologically active recombinant protein.
EFFECT: invention enables to obtain a complex of biologically active recombinant protein with polysialic acid, having stability and prolonged effect.
6 dwg, 2 tbl, 15 ex
The invention relates to the field of biochemistry, and can find application in medicine for the production of biologically active recombinant proteins, stable and prolonged action, which has therapeutic value, such as human insulin, alpha - and beta-interferons, granulocyte colony-stimulating factor and other
To improve the stability of recombinant proteins in the finished dosage forms use different techniques: from covalent modification of proteins of low molecular weight and high molecular weight agents to create complexes with biopolymers.
There is a method of preparation of long-acting insulin in the form of a suspension, resulting from the interaction of insulin with Protamine-sulfate. These develop crystals in the solution, containing in its composition the insulin, Protamine sulfate, ions of divalent zinc and phenol. The drug has a prolonged action due to slow dissolution of the crystals in the subcutaneous tissue and prolonged intake of free insulin in the blood. It maintains the level of blood glucose during 12-16 hours after subcutaneous injection of Protamine-insulin (RF Patent No. 2281756, IPC AC 9/10, publ. 2006).
Disadvantages obtained a prolonged form of the drug is insulin are:
a) the need to obtain a crystalline suspension containing alien to the human body protein Protamine sulfate;
b) the complexity of the dosing of the drug in the introduction, due to the inhomogeneity of the phase state (suspension of crystals in solution).
There is a method of stabilization of biologically active proteins by complexation with natural polysaccharides and amino acids (R.S.Brody, S.Alavattam, W.M.Fountain, R.L.Jones. Stabilization of biologically active proteins with mixtures of polysaccharides and amino acid based compounds. US 2008/0219951 A1). The combination of therapeutic protein with a mixture of polysaccharides obtained from vegetable raw materials, such as gum Arabic or starch, can improve the stability of the pharmaceutical composition in solution and in gel form. Gum Arabic is a highly branched polymer, the main chain of which is composed of residues of D-galactopyranose connected by a linkage (1→3)and the side chain of oligosacharides links attached link (1→6). The molecular structure of this polysaccharide are also other monosaccharide residues joined to the main and side chains. Gum Arabic also contains about 1% silnoagressivnyh proteins, such as oxidase and peroxidase activity that must be deactivated before you can connect with squirrels, to prevent unwanted pobo the different reactions with the functional groups of the protein.
Disadvantages obtained by this method compounds are: a) the content of impurities heavily glycosylated proteins through the use of a mixture of natural polysaccharides, which are almost impossible to separate from polysaccharides; b) possible adverse immune reactions in patients on impurity proteins and immunogenic polysaccharides; b) the impossibility of standardization ready protein preparation because of the difficulty of controlling the composition of complex natural mixtures. Known closest to the claimed complex of engineered proteins with sulfated polysaccharides (Y.Uemura, Neglige, .Morise, S.Funakoshi, .Suyama. Process for recovering interferon. US 4314935). The complex is produced by the interaction of a solution of α-interferon produced by human cells, with a polysaccharide containing negatively charged sulfopropyl. As polysacharide use bentonite or SP-Sephadex. The resulting complex is insoluble in water. When this protein remains of native and can be separated in the form of this complex from other impurities.
However, this method is not suitable for the preparation of the water-soluble polymer complexes of negatively charged carbohydrates with recombinant proteins.
The invention solves the problem of expanding the range of stable, water-soluble biologically active recombinant proteins prolon the new action.
The problem is solved at the expense of a stable complex of biologically active recombinant protein with polisialovoi acid with prolonged therapeutic effect, the General formula:
where: m=20-110; n=1-4; R is a biologically active recombinant protein.
Polivanova acid is a natural biopolymer that is part of the adhesion molecules in the nervous mammalian cells and is a component of the cell wall of some bacteria (Gregoriadis G., McCormack C., Wang Z. et al. // FEBS Lett. 1993. V.315. P.271). She has no immunogenicity, recognized by sialidase body, splitting her to residues neuraminic acid, and excreted from the body. Policylevel acid used for covalent modification of proteins (S.Jain, .Laing, G.Gregoriadis, D.H.Hreczuk-Hrist, Y.P.apaoannou. Sialic acid derivatives for protein derivatisation and conjugation. WO 2005016974), but non-covalent complexes of this acid protein, which is the subject of this invention, is not known.
Complex polisialovoi acid protein produced by mixing solutions of each component at a certain pH value, usually below 6.0, stirring for some time and then bringing the pH to specific for this dosage form values, typically 7.4. Policylevel acid before the introduction of the complex triggers, acidification its water is of astora with subsequent separation on Sephadex G25. In some cases, activation polisialovoi acid is conducted immediately before preparation of the complex without the stage filtration through a column of Sephadex. The resulting complexes polisialovoi acid and protein can be stored in solution or in lyophilized crystalline drug. The formation of the complex Polivanova acid-protein can be recorded by direct observation using an atomic force microscope. Prolonged action of recombinant proteins in complex with polisialovoi acid confirmed by biological tests in vivo. The preservation of the specific biological activity of the protein in complex with polisialovoi acid follows from the data of experiments on cell cultures that are sensitive to these proteins. Improved stability of a protein molecule to the action of natural substances, inducing aggregation, as demonstrated by a special experiment.
Thus, the invention solves the problem of increasing the stability and duration of pharmacological action of genetically engineered proteins by the introduction of the non-covalent complex with natural polisialovoi acid.
The invention is illustrated graphics.
Figure 1. AFM image of complexes of insulin with polisialovoi acid. The scale of 60 nm. A - Molek is s insulin.
Figure 2. Electrophoregram separation in polyacrylamide gel. Polyacrylamide gel, adenocarinoma conditions, without concentrating gel, pH 8.8, 8%T, staining EZBlue (Sigma, USA). 1 - native interferon-β, 2 - complex of example 7, 3 - native granulocyte colony-stimulating factor 4 complex from example 6, 5-7 - molecular weight markers, 5 - carbohydrate, 6 - α-lactoglobulin, 7 - BSA.
Figure 3. Electrophoregram insulin and its complex with polisialovoi acid after incubation with arachidonoylglycerol (AA-DAQ). Polyacrylamide gel denaturing conditions without recovery, Trichinosis buffer. 1 - complex with insulin polisialovoi acid of example 2, 2 - complex with insulin polisialovoi acid from example 2+AA-DAQ, 3 - insulin + AA-DAQ, M - token.
Figure 4. Effect of interferon-alpha 26 on the proliferation of the cell line Daudi. 1 - set of interferon and polisialovoi acid 22 kDa, obtained as described in example 5,2, standard sample interferon alpha 2B.
Figure 5. The influence of drugs granulocyte colony-stimulating factor human (g-CSF) on the proliferation of HL60 cells differentiated dimethylsulfoxide. 1 - comparison drug filgrastim, 2 - complex of g-CSF and polisialovoi acid 22 kDa, obtained in example 6.
6. Effect of interferon-beta 1B on the proliferation of the cell line Daudi. 1 - set of interferon and polisialovoi acid 22 kDa, obtained as described in example 7, 2, a standard sample of interferon beta 1B.
The invention is illustrated by the examples.
Example 1. Getting activated polisialovoi acid.
To 110 mg polisialovoi acid 22 kDa (m=75) was added 1.72 ml of water and stirred at room temperature (23°C) for 15 minutes, measure the pH. A value of 7.0. Add 70 ál of 0.5 M HCl, the resulting solution pH 4.5. Column PD-10, filled with Sephadex G25 (GE Healthcare, UK), washed with water, detektywa the eluate at 240 nm. The solution is applied polisialovoi acid and continue eluted with water. Collect fractions by absorption at 240 nm. The main substance is released in the first fraction, pH 4.9. This fraction (3.9 ml) is transferred into a round bottom flask and lyophilized. Obtain 86 mg of dry crystalline substances. Store at temperatures below -18°C.
Example 2. Obtaining complex polisialovoi acid (m=50) with insulin.
In a vessel with a magnetic stir bar is placed 250 mg polisialovoi acid with a mass of 15 kDa (m=50) and dissolved in 5 ml of water under stirring. Measure the pH value of the obtained solution (usually located within a 6.5-5.5) and DataRowView solution to a value of 4.5 0.5 M HCl. At the same time prepare for a solution of the substance of genetically engineered human insulin of 96 mg of the polypeptide and 4 ml of water at pH 2.0. To the resulting clear solution polisialovoi acid added PR is stirring in portions a solution of insulin in the water. Immediately formed a voluminous white precipitate. Stirred at room temperature (23°C) for 30 minutes and bring the solution pH to a value of 6.75-7.0. The resulting solution was sterile filtered through a filter with a pore diameter of 0.22 μm and lyophilized. Receive 300 mg of a white crystalline substance. The ratio Polivanova acid - protein is 1:1 (n=1).
Example 3. Obtaining complex polisialovoi acid (m=20) with insulin.
In a vessel with a magnetic stir bar is placed 250 mg polisialovoi acid with a mass of 6 kDa (m=20) and dissolved in 5 ml of water under stirring. Measure the pH value of the obtained solution (usually located within a 6.5-5.5) and DataRowView solution to a value of 4.5 0.5 M HCl. At the same time prepare for a solution of the substance of genetically engineered human insulin of 60 mg of the polypeptide and 3 ml of water at pH 2.0. To the resulting clear solution polisialovoi acid is added with constant stirring portions solution of insulin in the water. Stirred at room temperature (23°C) for 30 minutes and bring the solution pH to a value of 6.75-7.0. The resulting solution was sterile filtered through a filter with a pore diameter of 0.22 μm and lyophilized. Obtain 280 mg of a white crystalline substance. The ratio Polivanova acid protein 4:1 (n=4).
Example 4. Obtaining complex polisialovoi acid (m=110) with insulin.
In a vessel with a magnetic stir bar is placed 456 mg policy is gross acids with a mass of 32 kDa (m=110) and dissolved in 9 ml of water with stirring. Measure the pH value of the obtained solution (usually located within a 7.3-6.5) and DataRowView solution to a value of 4.5±0.1 0.5 M HCl. At the same time prepare for a solution of the substance of genetically engineered human insulin of 73 mg of the polypeptide and 3.2 ml of water at pH 2.0. To the resulting clear solution polisialovoi acid is added with constant stirring portions solution of insulin in the water. Immediately formed a voluminous white precipitate. Stirred at room temperature (23°C) for 30 minutes and bring the solution pH to a value of 7.15±0.1. The resulting solution was sterile filtered through a Millex filter 0.22 μm and lyophilized. Obtain 495 mg of a white crystalline substance. The ratio Polivanova acid - protein is 1:1 (n=1).
Example 5. Obtaining complex polisialovoi acid with interferon alpha 2B.
457 mg activated polisialovoi acid 22 kDa, obtained as described in example 1, is dissolved in 75 ml of purified water. Stirred at room temperature (23-25°C) until complete dissolution. Check the pH value. If necessary dotiagivaut 0.5 M solution of hydrochloric acid to a value of 4.6±0.1 under the control of the pH meter. To the obtained solution under stirring at a speed of 300±50 rpm poured 100 ml of protein solution with a concentration of interferon 2±0.1 mg per ml, obtained at the stage was stirred at room temperature is 30 minutes Bring the pH to a value of 7.0 with 0.1 N NaOH and sterile filtered. The ratio Polivanova acid - protein 2:1.
Example 6. Obtaining complex granulocyte colony-stimulating factor human (g-CSF) and polisialovoi acid.
Get the solution of the complex of g-CSF and activated polisialovoi acid 22 kDa, as described in example 4, 745 mg polisialovoi acid and 28 ml of protein solution with a concentration of g-CSF 9 mg per ml of the resulting solution of the complex quantitatively injected into the chromatographic column PD-50, filled with Sephadex G50 (GE Healthcare, UK). The column was washed with at least three volumes of phosphate-saline buffer, pH 7.4. Control of the eluate are carried out by the increase of absorption at 240 nm using UV detector. Fractions corresponding to the front, top and main descending part of the peak is collected in plastic tubes in 15 ml. Fractions preceding the peak of the matter, and consistent with its tail drop. Measure the volume of solution complex with g-CSF polisialovoi acid after gel chromatography. Select a sample of 500 μl for analysis of protein content. According to the analysis, calculate the quantity of phosphate-saline buffer (pH 7.4), which is required to obtain the concentration of 0.54-0.66 mg/ml and added to a solution of the complex. The resulting solution of the substance is sterilized by passing through a sterilizing filter in the septic conditions. The ratio Polivanova acid - protein 2:1.
Example 7. Obtaining complex polisialovoi acid with interferon beta-1B.
Bottle of dark glass on 4.5 ml was placed 2 ml of the prepared solution of the interferon containing 0.89 mg/ml protein in 10 mm phosphate buffer (pH 7.3)containing 2% mannitol. To this solution under stirring at room temperature (20°C) was added in one portion a solution of polisialovoi acid 22 kDa with a concentration of 27 mg/ml (pH 4.6). Stirred at this temperature for 35 minutes DataRowView to a value of 7.2 0.1 M NaOH. The resulting solution was stirred for 30 min at room temperature. Determine the protein concentration. If necessary, diluted with 10 mm phosphate buffer (pH 7.3) to a concentration of 0.25 mg/ml Sterile filtered. The ratio Polivanova acid protein 3:1.
Example 8. Visualization of complex polisialovoi acid and human insulin using atomic force microscope (AFM).
On the surface sizescolors high-oriented pyrolytic graphite (HOPG) put 40 μl of a solution of a modifier graphite "GM" company "Nantong" (Russia). After 10 min incubation drop blow off with compressed argon. On the thus prepared surface of HOPG put a drop of aqueous complexes of insulin-Polivanova acid obtained in example 2 with a concentration of 0.1 μg/ml After incubation during the 5 min drop blow off with compressed argon. AFM izobrazheniya obtained in intermittent-contact mode using surgestrip cantilever high-resolution production "Nantong" (Russia) on the device Solver P-47 bio company NT-MDT (Russia).
Figure 1 shows the typical AFM images of complexes of insulin with polisialovoi acid. Molecules polisialovoi acid look on AFM images well straightened. The arrows in figure 1 indicate the insulin molecule, having a greater height compared to the molecules polisialovoi acid. Measured in AFM height molecules polisialovoi acid is 0.3 - 0.5 nm, the average length of the molecules is about 10-20 nm, which corresponds to a molecular weight of 23 KD, given the reduction in the length of the molecule due to the formation of spiral sections and bends. The interaction is specific because of the insulin molecule are distributed unevenly along the length of the molecule polisialovoi acid and are mainly associated with the one end of the molecule. The height of the molecules of insulin, measured using AFM corresponds to 0.7 to 1 nm.
Example 9. Characterization of protein complexes with polisialovoi acid by electrophoresis in polyacrylamide gel adenocarinoma conditions.
The division performed without concentrating gel, pH 8.8, 8%T, staining EZBlue (Sigma, USA).
Figure 2 shows a typical electropherogram of protein complexes with alicelove acid 22 kDa, obtained as described in examples 6 and 7. It is seen that the mobility of the complexes is different from the mobility of the native protein.
Example 10. The stability of the complexes of recombinant proteins with polisialovoi acid by the action of the modifying agents.
The oxidized form of the neurotransmitter dopamine (n-ethylamino-o-benzoquinone) is assumed as a damaging agent in Parkinson's disease, and it is known that it is able to form covalent adducts with cysteine residues and lysine different proteins. To check the stability of genetically engineered proteins in complex with polisialovoi acid to the action of diaminononane use its fatty acid derivative (arachidonoylglycerol, AA-DAQ), with greater stability in aqueous solutions compared to the unmodified diningroom. Recombinant proteins (CSF, interferon alpha 2B and beta 1B and insulin) and their complexes with polisialovoi acid incubated with a five-fold molar excess (relative to the total concentration of lysine and residues) AA-DAQ at pH 7.4 or 6.5 for one hour at 37°C and analyzed by the method of denaturing electrophoresis in polyacrylamide gel in non conditions. In the absence of polisialovoi acid incubation of the proteins in AA-DAQ lead to the formation of protein oligomers. The dominant product is the dimer, followed by a trimer and a small number of tetramer. Complexation with polisialovoi acid significantly reduces the formation of oligomers. Figure 3 presents electrophoregram typical experiment with insulin and its complex with polisialovoi acid. Electrophoretic separation was carried out under denaturing conditions without recovery using Technologo buffer according to published methods (Judd R.C. Electrophoresis of Peptides // Methods in Molecular Biology. V. 32 Basic Protein and Peptide Protocols, Humana Press, 1994). You can see the almost complete absence of oligomeric forms of the protein in the sample containing the complex with polisialovoi acid.
Example 11. Test the biological activity of the drug insulin in rabbits.
Biological activity and prolonged action of the drug is determined by reducing the concentration of blood glucose in rabbits.
Use rabbits of the same sex weighing 2.5÷3.5 kg contained on a standard diet of the vivarium. In the experiment take at least 18 animals, which by the way is randomly assigned to two equal groups. One group subcutaneously injected undiluted FE (test preparation), another group - the same dose of the basic solution WITH (a standard form of soluble insulin), corresponding to species specificity. The recommended dose range is 0.5÷0.8 IU/kg of Taking shelter is from the ear vein of the rabbit carried out immediately before injection and after 1.5; 3.0; 4.5 and 6.0 h after it. Determination of glucose in blood serum is carried out glucoseoxidase method using a set of "Glucose" (Human, Germany).
For the control time point take the time, when the average concentration of glucose in the blood of animals that received a basic solution, returned to the original level (100%). At this point, for each rabbit received FROM or PI, calculate the individual relative concentration of glucose in the blood in percent from baseline from this animal. If for 6 h average concentration of glucose in the blood of rabbits that received FROM, not returned to the original level, as a reference time point take the point in time at which the average level of glucose in this group became as close as possible to the original.
IE consider having passed the test if the control time point, the average relative concentration of glucose in the blood of animals that received FE, calculated from the individual relative concentrations, significantly lower than in the blood of animals that received a basic solution. The reliability of differences test using student's criterion with p<0,05. The results of the tests are presented in table 1.
|Test the biological activity of a complex of insulin with polisialovoi acid in rabbits|
|Description sample||% reduction of glucose over a period of time, h|
|WITH the Standard sample of insulin), 40 IU/ml||37||21||0||0|
|FE (Complex insulin with polisialovoi acid from example 2), 40 IU/ml||43||30||18||21|
Example 12. Test the biological activity of the drug insulin in mice.
Biological activity and prolonged action of the drug is determined by reducing the concentration of blood glucose in mice.
In the experiment take at least 30 mice deviation of body mass is not more than ±1.0, Mice distribute 3 equal groups by way of random selection and mortality in order each individual within the group.
Animals of the first group every 15 seconds subcutaneously injected into the withers working standard solution sample, prepared from the primary rest the RA, in a volume of 0.1 ml/10 g of body weight of the animal. The working solution WITH prepared immediately before the experiment at a concentration of 0.1 IU/ml
Animals of the second group enter the working solution of the test drug (PI), in a volume of 0.1 ml/10 g body weight. The working solution of FE is prepared in a similar manner, immediately before the experiment, with a concentration of 0.1 IU/ml
The time interval between the introduction of each working solution should not be less than two minutes.
The third (control) group injected solution, not containing insulin (placebo), in the same amount and in the same pattern as experienced by the animal.
Blood samples (50 μl) is conducted from the orbital venous sinus using sterile heparinised capillary or Pasteur pipettes 40±1 min, 90±2 min and 120±3 min after injection of insulin in accordance with the number of each animal within a group, respecting the same time intervals as in the infusion. Determination of glucose in blood serum is carried out glucoseoxidase method using a set of "Glucose" (Human, Germany). Calculate the average concentration of glucose in the blood of mice from each of the three groups at each time point. Determine the difference between the average concentration of glucose in the blood of animals third, the control group and the average concentration of glucose in the blood first (poluchivshey the solution FROM a) and the second (which solution PI) groups. The test is considered satisfactory if the values obtained are statistically significant (p<0,05). IE consider having passed the test if the control point 120 min average concentration of glucose in the blood of the animals of the second group (who received a solution of FE) at least 10% lower than in the blood of the animals of the first group (who received the solution FROM a). The results are presented in table 2.
|Test the biological activity of a complex of insulin with polisialovoi acid in mice|
|Description sample||% reduction of glucose through time, min|
|The control solution (placebo)||0||0||0||0|
|WITH the standard sample of insulin), 40 IU/ml||62||31||6||0|
|FE (Complex insulin with polisialovoi acid of the example 2), 40 IU/ml||68||38||32||27|
Example 13. Test the biological activity of a complex of interferon alpha 2B human and polisialovoi acids in cell culture.
Biological activity of recombinant interferon alpha 2B men in non-covalent nanocomplex with polisialovoi acid determined by the ability of this drug to inhibit the proliferation of the cell line Daudi obtained from the lymphoma Bernita and a standard model for studying the antiproliferative effect of drugs group of interferons.
Aliquots of solutions of complex interferon alpha 2B human and polisialovoi acid 22 kDa, obtained as described in example 5, and the comparison drug in phosphate-buffered saline was added to the culture medium ARPMI 1640 with cells Daudi so that the final concentration of protein ranged from 0.064 to 250 ng per ml of medium. Cells incubated with the substances within 72 hours. Upon expiration of the specified term determine the number of viable cells in each sample compared to the intact cell line. Each test replicate at least five times.
After incubation to determine the number of viable cells using a colorimetric MTT-assay. Records of the results is the ATA 3-4 measurements for each concentration of each drug produced by the relative number of cells line Daudi MTT test after 72 h after application of the samples.
As can be seen from figure 4, the complex interferon and polisialovoi acid have persistent antiproliferative effect observed at concentrations above 1 ng/ml and fully comparable with those of the standard drug interferon, which is statistically no significant differences between the results of the tests.
Example. 14. Test the biological activity of complex granulocyte colony-stimulating factor human (g-CSF) human and polisialovoi acids in cell culture.
The test of the drug complex of recombinant human g-CSF and polisialovoi acid 22 kDa carried out analogously to example 13, with the difference that instead of the cell line Daudi use of cell lines of myeloid leukemia human HL60, differentiated dimethylsulfoxide. Drug comparison in this case acted as an official drug on the basis of recombinant g-CSF - Filgrastim. The effect of each sample drug study with 3-fold when determining that reproduce the experiment three times. Results proliferative actions of the tested drugs are presented in figure 5.
The drawing shows that g-CSF in the composition of non-covalent nanocomplex with polisialovoi acid fully proliferative effect on cells of the myeloid Rostock Trovatore the Oia and at a concentration in the culture medium above 0.4 ng/ml is not inferior in this respect, the official drug.
Example. 15. Test the biological activity of a complex of interferon beta 1B and polisialovoi acids in cell culture.
The test of the drug complex of recombinant interferon beta 1B human and polisialovoi acid 22 kDa carried out analogously to example 13. Drug comparisons were native interferon beta 1B. The effect of each sample drug study with 3-fold definition, that reproduce the experiment three times. Results antiproliferative action of the tested drugs presented on Fig.6.
As can be seen from Fig.6, the complex interferon and polisialovoi acid has a persistent antiproliferative effect observed at concentrations above 10 ng/ml and fully comparable with those of the standard drug interferon, which is statistically no significant differences between the results of the tests.
A stable complex of biologically active recombinant protein, ecovalence associated with activated in aqueous solution at pH of 4.4 to 4.7 polisialovoi acid with prolonged therapeutic effect, the General formula:
where m=20-110; n=1-4; R is a biologically active recombinant protein.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.
EFFECT: as a result of expression of the nucleotide sequence coding the said tandem, GH-linker-GH polypeptides are obtained, which exhibit growth hormone receptor agonist properties, which determine the possibility of their use in medicinal agents for treating diseases related to the need to administer the growth hormone.
10 cl, 31 dwg, 5 ex
SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPR∧GS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.
EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.
3 cl, 4 dwg, 4 tbl, 6 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.
EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.
16 cl, 4 dwg, 1 tbl, 13 ex
SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp4Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.
EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.
4 cl, 4 dwg, 5 ex
SUBSTANCE: protein is constructed, which includes DNA-binding domain SSBTne from thermophile microorganism Termatoga neapolitana, connected to C-end of domain VirD2 from Agrobacterium tumefaciens, which is a signal of nuclear localisation.
EFFECT: efficient transport of transgene into cell nucleus.
6 dwg, 1 tbl
SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.
EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.
7 cl, 13 dwg, 4 tbl, 7 ex
SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.
EFFECT: application of the invention provides low-immunogenicity antibody.
2 cl, 8 dwg, 1 tbl, 2 ex
SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.
EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.
4 cl, 6 dwg, 22 tbl, 19 ex
FIELD: chemistry, medicine.
SUBSTANCE: claimed is novel hybrid protein CFP10-ESAT6 from M. tuberculosis, inducing reaction of hypersensitivity of delayed type with respect to M. tuberculosis. Chimeric NA, coding claimed protein is described. Described is method of obtaining claimed protein by cultivating cells of strain BL21(DE3) E.Coli, transformed with constructed recombinant expression vector on the basis of plasmid pET22b(+). Claimed is dosed medicinal form, containing claimed protein, for intracutaneous injection for diagnostics of tuberculosis infection.
EFFECT: high output of protein CFP10-ESAT6, which possesses specific immunogenicity.
8 cl, 2 dwg, 7 tbl, 3 ex
FIELD: genetic engineering.
SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.
EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.
9 cl, 6 ex
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex
FIELD: medicine, oncology, biochemistry.
SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.
EFFECT: valuable medicinal properties of protein complexes.
13 cl, 40 dwg, 18 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: genetic engineering, pharmaceutical and medical-biological industry.
SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.
EFFECT: improved preparing method, valuable biological properties of polypeptide.
23 cl, 67 dwg, 1 tbl, 35 ex
FIELD: biotechnology, gene engineering, microbiology.
SUBSTANCE: invention relates to TUL4spCBD recombinant protein comprising TUL4 Francisella tulergenis protein, Gly-Ser spacer and cellulose-binding domain of CelD endoglucanase gen from Anaerocellum thermophilus. Said protein has TUL protein antigen properties and is capable of spontaneous binding to cellulose-containing sorbents. Described is recombinant plasmid pTUL4spCBD DNA of 4388 n.p. length, encoding TUL4spCBD recombinant protein. Abovementioned plasmid contains: artificial bacterial operon of TUL4spCBD recombinant protein, including promoter region of N5 bacteriophage earlier promoter (7-87 n.p.); TUL4spCBD recombinant protein gene (115-1113 n.p.); transcription terminator (1134-1230 n.p.); beta-lactamase bacterial operon (4183-3323 n.p. of complementary chain)), providing ampicillin resistance; ColE1-type bacterial site of replication initiation, providing plasmid replication in E.coli strain (4388 n.p.). Also discoised is M15[pREP4, pTUL4spCBD] Escherichia coli strain as producer of TUL4spCBD recombinant protein and method for production of purified, concentrated and immobilized TUL4spCBD recombinant protein from said strain by treatment of cell hydrolyzate supernatant from M15[pREP4, pTUL4spCBD] Escherichia coli with cellulose-containing sorbent. Method for production of specific antibodies against TUL4spCBD protein also is described. Said method includes animal immunization with TUL4spCBD recombinant protein immobilized onto cellulose.
EFFECT: method for high yield production of TUL4spCBD recombinant protein; simplified method for purification, concentration and immobilization of said protein.
5 cl, 1 dwg, 4 ex
FIELD: biotechnology, molecular biology, medicine.
SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.
EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.
23 cl, 71 dwg, 12 tbl, 17 ex
FIELD: biotechnology, medicine.
SUBSTANCE: the present innovation deals with CFP10-ESAT6 hybrid protein that is able to induce Mycobacterium tuberculosis-specific hypersensitivity reaction of delayed type. This hybrid protein contains complete protein CFP10 out of M.tuberculosis combined with complete protein ESAT6 out of M.tuberculosis through linker amino acid sequence. It is, also, [presented a recombinant plasmid DNA pTBD16 for expression of hybrid protein CFP10-ESAT6. The latter should be obtained due to applying DLT1270 E.coli strain transformed with the obtained recombinant plasmid DNA pTBD16. The innovation suggests to apply the above-mentioned protein as a diagnostic kit of tuberculous infection both in mammalians and human beings. The method for predicting tuberculous infection has been revealed. Tuberculous infection should be predicted due to induction of hypersensitivity reaction of delayed type in case of subcutaneous injection of hybrid protein. Application of the present innovation enables to obtain a Mycabacterium tuberculosis-referring diagnostic kit, at specificity being 100%, sensitivity - 80%, not less.
EFFECT: higher accuracy and efficiency of diagnostics.
6 cl, 4 dwg, 4 ex
FIELD: medicine, biotechnology, pharmacy.
SUBSTANCE: invention relates to exchangers of ligand/receptor specificity delivering antibodies to receptors on pathogen. In particular, invention describes variants related to manufacturing and using exchangers of ligand/receptor specificity. Exchangers comprise at least one specificity domain containing ligand for receptor wherein ligand is not antibody or its part, and at least one antigenic domain combined with abovementioned specificity domain wherein antigenic domain comprises epitope of pathogen or toxin. Advantage of the invention involves enhanced specificity in delivery of drug.
EFFECT: improved and valuable properties of exchangers.
30 cl, 5 tbl, 6 ex
FIELD: biotechnology, immunology.
SUBSTANCE: invention reports about preparing and characterizing two forms of Nogo protein bound with the natural myelin with respect to the presence of immunogenic properties in their. These two forms correspond to natural products of alternative splicing, their fragments and derivatives, in particular, derivatives comprising deletions in amino acid residues, and chimeric protein also and comprising novel immunogenic polypeptides and their fragments. Invention can be used in medicine for diagnostic and curative aims.
EFFECT: valuable medicinal properties of protein.
18 cl, 79 dwg, 3 tbl, 8 ex