Recombinant dna pa3, recombinant dna pqe 30-pa3 providing producing polypeptide a3, strain e. coli m 15-a3 transformed by recombinant plasmid dna pqe 30-pa3 and expressing recombinant polypeptide a3, recombinant polypeptide a3 possessing ability to bind human serum albumin, and rfa test system for qualitative detection of microalbuminuria, test system for quantitative determination of microalbuminuria

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions concerns recombinant DNA pA3, recombinant plasmid DNA pQE 30-pA3, strain E.coli M 15-A3, recombinant polypeptide A3, test systems for the qualitative detection and quantitative determination of microalbuminuria. The characterised recombinant DNA pA3 is produced by a polymerase chain reaction with the use of chromosomal DNA of the strain DG 13 of group B streptococci and structured primers. An amplification fragment has been cloned by a system of expression vectors pQE-pQE 30 in E.coli M 15 to produce recombinant DNA pQE 30-pA3, which codes an amino acid sequence of the recombinant polypeptide A3 having an ability to bind human serum albumin (HSA) selectively.

EFFECT: presented inventions can be used in the medical practice and industry for producing the test systems for the detection and quantitative determination of microalbuminuria - one of the earliest signs of renal irritation in the patients suffering from diabetes mellitus and essential arterial hypertension.

6 cl, 9 dwg, 1 tbl, 8 ex

 

Recombinant DNA will get RA3, recombinant DNA pQE 30-will get RA3 generating polypeptide A3, the strain E. coli M 15-A3, transformed recombinant plasmid DNA pQE 30-will get RA3 and expressing the recombinant polypeptide A3, recombinant polypeptide A3 having the ability to selectively bind NAV, test-XRF system for the qualitative detection of microalbuminuria, a test system x-ray fluorescence analysis for the quantification of microalbuminuria.

The invention relates to the field of genetic engineering and biotechnology and can be used in medical practice and industry for the production of test systems for the detection and quantitative determination of microalbuminuria is one of the early signs of kidney damage in patients with diabetes mellitus (DM) and essential hypertension (AH).

In recent years all over the world is steadily increasing mortality from diseases leading to chronic renal failure (CRF). The most common causes of kidney failure are diabetes and essential hypertension. Given the significant prevalence of diabetes and hypertension, the importance of search early signs of kidney damage in patients with this pathology.

The leading cause of mortality in the world ESRD is due to the progression of diabetic nephropathy (L�) - specific renal disease in DM. In the United States and Japan NAM ranks first in prevalence among all diseases of the kidneys (35-40%), pushing the second or third position such the renal disease, such as glomerulonephritis, pyelonephritis, polycystic, etc. In Europe NAM is less threatening, but kept at the level of 20-25% needs replacement therapy (chronic dialysis, kidney transplantation). In Russia, the mortality from ESRD with DM type I according to the State register does not exceed 18%, which is three times below the level recorded in the world for the past 30 years. In DM type II mortality from ESRD in Russia is 1.5%, which is 2 times lower world. This paradox can only be explained by the lack of a unified methodology of registration of mortality in Russia.

Currently a limiting factor for the effective treatment of diabetes are:

- the lack of widespread implementation of screening programmes DN in diabetes-related institutions in Russia;

- the lack of a unified methodology of screening days;

- unavailability of dialysis ESRD treatment methods for many patients with diabetes.

One of the earliest signs of kidney damage in diabetic patients is microalbuminuria. Therefore, early and reliable method of diagnosis of primary stages of kidney failure is the test for m�realmoney. The term "microalbuminuria" understand the excretion of albumin in the urine in relatively low concentrations ranging from 30 to 300 mg/day. This amount of protein could not be detected by conventional routine examination of the urine, and in connection with the early (preclinical) stage of development of kidney failure can be diagnosed and ignored. However, since this stage is only reversible with timely pathogenetic drug therapy, its identification is a prerequisite for a successful fight against DN and ESRD in diabetes of both types.

In addition, microalbuminuria is an important early sign of kidney damage, reflecting the initial stage of the vascular pathology and consistently correlated with increased cardiovascular morbidity and mortality. As clinical studies show, even the small levels of increasing albumin excretion with urine clearly associated with a significant increased risk of cardiovascular events, including fatal and progressive over time, the increase in the level of microalbuminuria clearly indicates the deterioration of blood vessels and, accordingly, causes an additional increase in risk. In this regard, microalbuminuria is recognized as an independent cardiovascular risk factor and the earliest (up to�clinical) a symptom of the defeat of such vulnerable target organ, as the kidneys.

Microalbuminuria is not determined by conventional methods, e.g. by precipitation of protein sulfosalicylic acid.

The currently used diagnostic test for the detection of microalbuminuria are mainly foreign and are designed for semi-quantitative and quantitative immunological determination of the concentration of albumin in the urine. Semi-quantitative - test strips or latex-system, in which various reagents are applied to albumin. The General principle is to register a "positive" result only when identifying the sample of urine the albumin concentration corresponding to the range of microalbuminuria (20-200 mg/ml).

Known diagnostic kit "Micro-Bumin test" (Mile's Ins Ekkhart, FIN). It is based on the use of albumin blue Bromphenol, fixed on the alkaline matrix. Staining occurs when the concentration of albumin in the sample is greater than 40 mg/L. the Low specificity of this test (<80%) due to the ability of the reagent to contact with other plasma proteins.

Known diagnostic kits "Albu Screen" and "Albusure" ("Cambridge Life Siences, Cambridge, UK), which are latex-agglutinating inhibition test based on the use of antiserum to human albumin and latex-particles with albumin. If conc�tion of albumin in the urine sample is less than 30 mg/l, there is a latex-agglutination. The specificity and sensitivity of this test is 90%.

Known diagnostic kit "Micral-test (Boehringer Mannheim, Indiana-polis, Germany), which is based on the use of monoclonal antialbumin antibodies labeled with a galactosidase, and fixed on the test strip. Assessment of response is made after 5 minutes by visual comparison of staining reaction zone of the test strip with the attached colour scale division which correspond to the albumin concentration of 0, 10, 20, 50, 100 µg/L.

Using test strips is quite acceptable as a screening test for the semiquantitative detection of microalbuminuria. However, the test needs to measure albumin excretion of urine.

For the quantitative determination of the level of albumin excretion with urine used radioimmunoassay, enzyme immunoassay and immunoturbidimetric methods based on the interaction of the "antigen-antibody". However, the relative specificity of antibodies, they can interact with a number of other antigens and to give so-called "non-specific" response.

1. Direct immunoturbidimetry. It is based on the reaction of albumin with an antibody and the precipitation of immune complexes in the presence of ethylene glycol. In conditions of excess antibodies precipitate causes turbinate (absorption of light), the degree of which depends� on the concentration of albumin in the sample. Turbinate determined photometrically at the wavelength of light of 340 nm.

2. An immunochemical method using the system HemoCue ® Urine Albumin. The system is based on the reaction with anti-human antibodies. The complex antigen-antibody forms a precipitate, which is detected photometrically in the range of 610 nm.

Thus, currently used for the detection of microalbuminuria methods are:

- expensive for large-scale screening programmes and monitoring of microalbuminuria in large groups of patients with DM and hypertension;

based on the immunological determination of the concentration of albumin in the urine;

- time consuming, since the complex antigen-antibody forms a precipitate, so the research is preliminary centrifugation of the samples, since any contamination of the urine, i.e. any level of turbidity leads to false-positive results;

Known group of inventions on the application number 2011125752 on which the decision to grant a patent, which is a recombinant polypeptide A2, which is characterized by the amino acid sequence of 333 amino acids, covalently linked to 13 amino acid residues encoded by the plasmid pQE 32, having the ability to selectively bind human serum albumin (NAV), recombinant DNA re encoding the recombinant polypeptide A2 and which of CSA-binding fragment of chromosomal DNA of strain DG 13 streptococci group G-CTG, recombinant plasmid DNA pQE 32-RA representing plasmid DNA pQE 32, carrying the recombinant DNA, E. coli strain M 15 transformed with recombinant plasmid DNA pQE 32-RA and expressing the polypeptide A2, receptordependent (XRF) method for the determination in urine of CSA at a concentration of 20-200 μg/ml, including construction of a calibration curve developed with tetramethylbenzidine - TMB, the immobilization of the polypeptide A2 of the solid phase of the tablet, incubation, then the application to him of the analyzed urine samples simultaneously labeled with horseradish peroxidase navs at a dilution of 1:8000, developing TMB, optical density measurements and the values of the calibration curve to establish the quantity of CSA in urine samples, and to determine in the urine of CSA at a concentration over 100 mcg/ml of sample is diluted with a solution of phosphate-saline buffer.

In this receptordependent method of determining NAV in urine solid phase may be a polystyrene surface tablet or nitrocellulose membrane. In this technical solution is also described a method for the qualitative detection of CSA in urine, characterized by the fact that the definition is carried out by application of 30 urine samples onto nitrocellulose membrane with a size of 1 cm2with the first row of dilution standard NAV to construct the calibration curve, sample processing in blocking Rast�'or 2% milk in phosphate-buffered saline labeled with peroxidase by the polypeptide A2, washing the specified buffer solution and staining NAV solution paraphenylenediamine with hydrogen peroxide and the application of the recombinant polypeptide A2 as a reagent for removal of CSA from serum on a column with an affinity sorbent.

This technical solution adopted as a prototype of the claimed group of inventions.

The known method has disadvantages due to the use of a recombinant polypeptide of high molecular weight, which covers a larger area than that of CSA-binding GA modules that might hinder him to contact the molecule of albumin.

The object of the invention is the solution of the actual problem - the creation of a new inexpensive, easy to use, sensitive and specific method for determination of microalbuminuria in patients with diabetes and hypertension, based on the interaction of "protein-protein" using recombinant receptor of CSA-binding polypeptide derived from streptococci, quality cleaning which ensures almost 100% specificity, and test systems that do not require immune serum, highly specific, sensitive enough, not giving false background reactions, economical and affordable to use. Use microcephaly technology in combination with CSA-binding polypeptide to determine MICR�albuminuria can give a rapid assessment of albumin in the urine in a large number of patients and thereby to facilitate screening and monitoring of microalbuminuria.

The claimed group of inventions, United by a single inventive concept, which includes the following objects.

1. Recombinant DNA will get RA3 encoding a recombinant polypeptide A3 having the ability to selectively bind human serum albumin - NAV and having the nucleotide sequence shown in Fig. 3.

2. Recombinant plasmid DNA pQE 30-will get RA3, representing the plasmid pQE 30, bearing a recombinant DNA will get RA3 according to claim 1 and providing a recombinant polypeptide A3.

3. The Escherichia coli strain M 15-A3 producing a recombinant polypeptide A3, derived from the Escherichia coli strain M 15 and containing recombinant plasmid DNA pQE 30-will get RA3.

4. Recombinant A3 polypeptide having the ability to selectively bind NAV and characterized by the amino acid sequence shown in Fig. 4, in which the first 16 amino acid sequence encoded by the plasmid pQE 30, covalently associated with follow-up of 124 amino acids encoded by the sequence of CSA-binding fragment of chromosomal DNA of strain DG 13 Streptococcus group G-G.

5. The test system x-ray fluorescence analysis for the qualitative detection of microalbuminuria, consisting of a matrix of the microchip - nitrocellulose membrane; six standard samples of CSA 100; 50; 25; 12,5; 6,25; 3,125 μg/ml; blocking solution; �combinatio polypeptide according to claim A3 4, labelled with peroxidase; solution paraphenylenediamine with hydrogen peroxide for staining of the membrane.

6. The test system x-ray fluorescence analysis for the quantification of microalbuminuria consisting of polystyrene tablet on a hard surface to which immobilized polypeptide A3 according to claim 4; phosphate-saline buffer solution with tween 20 to wash the tablet; six calibration samples 100; 50; 25; 12,5; 6,25; 3,125 μg/ml to construct a calibration curve of optical density on the concentration of CSA; the Chromogen is 3,3'5,5'-tetramethylbenzidine and 33% hydrogen peroxide for the existence of the number of CSA in the urine.

Technical essence and the achieved when using the claimed group of inventions the technical result consists in the following.

Albumin is the most abundant protein in blood plasma of humans and mammals. In recent decades identified and studied proteins of bacteria possessing receptor activity against NAV.

Streptococci (Streptococcus) groups A, C and G Express surface proteins that bind NAV. (E. Myhre and Kronvall G. Infect. Immun. 27: 6-14 (1980)), Wideback K. et al., Acta Pathol. Microbiol. Immunol. Scand. Sect. B. 91: 373-382 (1983)).

Among these surface proteins of group G streptococci interest is a G protein, a full-sized molecule which has the ability to bind immunoglobulin G (IgG) of human and various MLC�supply, and navs of humans and animals (Bjorck et Mol. Immunol. 24: 1113-1122 (1987)).

From one of the strains of Peptostreptococcus magnus allocated RAV protein binding of CSA (de Chateau, M., Bjorck L. J. of Biol. Chem. 269: 12147-12151 (1994)). Analysis of its amino acid sequence revealed the plot of 45 amino acid residues responsible for the binding of CSA. This sequence, called the GA module has homology with the CSA-binding region of protein G (Akerstrom, etc. In J of Biol. Chem. 262: 13388-13391 (1987), Sjobring U, etc J Immun. 140: 1595-1599. (1988), Nygren P. et J. Mol. Recognit. 1: 69-74 (1988)).

Unlike RAV protein that is not homologous repeats, a G protein selected from the strain G 148, contains three homologous repetitive sequences, i.e. three GA module.

The emergence of new receptor proteins in bacteria is often associated with gene transfer, their parts or parts of the genome. It is believed that these fragments encode products that are modular elements of proteins. So, it is possible that the CSA-binding GA module of protein RAV occurred during the transfer of the corresponding element from protein G. the structure of the GA module of the G protein and the GA module of protein RAV is the same and consists of three helices (Johansson M et al., J. Mol. Biol. 316: 1083-1099 (2002)).

From a strain of Streptococcus canis 12 DG, separated from cow's milk obtained of CSA-binding protein that contains two GA module. It does not bind IgG, but by the ability to bind NAV is superior to protein G (Sjobring U. Inection and Immun. 60: 3601-3608 (1992)). This property makes it attractive for practical applications.

The study of the structure of CSA-binding proteins is of great importance for the technology of creation of protein reagents that are relevant in immunochemistry, proteomics, biotechnology and clinical diagnostics.

The use of recombinant polypeptide allows you to: eliminate the time-consuming process of preparation of specific antibodies to albumin (for which laboratory animals are used), to avoid non-specific "background" reactions that appear frequently in immunology, used to standardize NAV-binding polypeptide, and thus, to stabilize the whole system analysis.

Almost all strains G, both human and animal produce on its cell surface G protein that binds NAV.

For the first time recombinant NAV receptor, was obtained by Swedish authors by cloning the gene fragment NAV receptor in non-pathogenic host. (R. Nygren, etc., J. Mol. Recognition. 1: 69-74. (1988)). In this work for cloning the AB region (the region responsible for the binding of NAV) was restrictively plasmid pSPG2 enzyme EcoPJ with subsequent processing by the fragment maple, addition of synthetic Sail linker and ligation. When restriction enzymes SalI and PstI was obtained fragment of 640 BP, which is insulated from the Aga�Uzzah and inserted between the same sites in the vector pEML8. The gene fragment was excised from the resulting plasmid using the EcoRI and HindIII sites and inserted into the plasmid pEG, the construction of which is described in the work of Eliasson (Eliasson M et al., J. Biol. Chem. 263: 4323-4327 (1988)).

As a result of this modification was obtained plasmid pB1B2responsible for the CSA-binding activity. From subklona producing navs-binding protein, has been isolated protein. It was purified affinity chromatography. This protein was heterogeneous degradation purified product. The reason for heterogeneity was obviously a quite complicated way its genetic design. Practical application because of the shortcomings of this protein is not found.

In 1996 was prolongirovanne a fragment of the gene encoding the third GA module of protein G strain G148. The obtained protein G148-GA3 was used to study the structure of CSA-binding module. (Kraulis R. et al. FEBS Lett. 378: 190-194 (1996)), i.e. only for research purposes.

In 1992 from CG of animal origin, strain DG 12, was prolongirovanne a fragment of the gene encoding the CSA-binding fragment using plasmid vector RK-2 and determined its nucleotide sequence. (U. Sjobring Infect, and Immun. 60: 3601-3608.(1992)).

Known recombinant NAV-binding receptor polypeptides A1 and A2. Polypeptide A1 was obtained from CG, strain G148 allocated from the person in the Department Molek�polar Microbiology in 1996 (Orlov S. N. biotechnology, etc.. 8: 13-21. (1996)). The strain producing the polypeptide A1, deposited in the collection IEM NWB RAMS, No. 357. A feature of this polypeptide is that it contains of CSA-binding region, covering three GA module. They studied the gene fragment that encodes the polypeptide A1. The study of this receptor polypeptide has proven his ability to communicate exclusively with a major protein of human plasma - NAV. This property polypeptide tried to use in diagnostic assays to determine the concentration of CSA in various biological fluids, particularly in urine. However, when the separation and purification of the recombinant polypeptide A1 of the active reagent in sufficient quantity was difficult, which resulted in having to create a new NAV-binding agent that has greater activity than the polypeptide A1 of protein G.

The A2 polypeptide was obtained from strain 13 DG G isolated from cow's milk (Supalova T. V. et al., 2012). The strain producing the polypeptide A2, deposited in the collection IEM NWB RAMS, No. 593. It is shown that the polypeptide A2 has a higher NAV-binding activity compared to the polypeptide A1 of the GHS strain of human origin. Created producing strains of E. coli M 15-A2, which allows to obtain the polypeptide A2, which has of CSA-binding activity with high�Kim exit. The polypeptide A2 was used in XRF method for the determination of microalbuminuria. It is characterized by the sequence of 346 amino acids, in which the first 13 amino acid sequence encoded by the plasmid pQE 32, covalently associated with subsequent 333 amino acids encoded by the sequence of a fragment of chromosomal DNA of strain DG 13 G and the identical sequence of the albumin-binding protein from strain DG 12 G (U. Sjobring Infect, and Immun. 60: 3601-3608. (1992)). This sequence consists of 11 amino acids that are compatible with the last part of leader peptide, i.e. it contains a number of hydrophobic residues with a small uncharged amino acid at the end. Then there is a unique sequence of 55 amino acids, denoted by N, followed by two regions, denoted by E1 (28 amino acids) and E2 (29 amino acids). Behind them are two sequences GA modules. In the following sequence, denoted by W, is detected by a sequence of four amino acids (LysProGluVal), repeated 12 times. Nucleotide and amino acid sequence of the albumin-binding protein from strain 13 DG G shown in Fig. 1.

Thus, the molecular weight of the polypeptide A2, the sequence of which consists of 346 amino acids, is 48 kDa, while the sequence containing two of CSA-binding GA fashion�I, only consists of 124 amino acids and encodes a polypeptide with a molecular weight of 14 kDa. The absence of the polypeptide A2 of the optimal conformation may cause steric hindrance in the interaction of albumin with a molecule of large size.

According to the claimed invention, recombinant polypeptide A3 and its producing strains.Recombinant polypeptide A3 contains only a sequence of two of CSA-binding GA module, has a high ability to bind navs and smaller molecular weight. It can be used in the diagnosis when creating specific and cost-effective test systems for the qualitative detection and quantitative determination of microalbuminuria in patients with diabetes and hypertension.

The problem is solved by designing unique primers directed to areas of the gene encoding the polypeptide consisting of the two CSA-binding GA module. For the rapid and simple preparation of recombinant polypeptide A3 for cloning the DNA fragment was used to investigating the expression plasmid pQE. For the creation of producer strain recombinant polypeptide A3 was used a strain of E. coli l5 M. As a result of experimental work this strain has acquired new properties: the ability to Express the recombinant polypeptide A3. The new strain was named E. coli M 15-A3.

In PR�process of work has created a unique recombinant DNA will get RA3, the resulting PCR using chromosomal DNA of strain DG13 CG, unique primers RA1 and RA and subsequent cloning using the expression plasmid pQE-30 (The QIAexpress System, Qiagen, USA).

Was also created recombinant plasmid DNA pQE 30-will get RA3, carrying the recombinant DNA will get RA3, and producing strains of E. coli M 15-A3, which allows under certain conditions to Express the recombinant polypeptide A3.

In the process, the obtained gene fragment encoding a region with two NAV-GA binding modules of strain DG 13 CG, 382 BP for 754 BP (designated as will get RA3, the size of 372 BP) by PCR using primers RA1 and RA and chromosomal DNA of strain DG 13 G.Also, the authors carried out the cloning will get RA3 using gene-expression plasmid pQE-30 and the subsequent transformation of the obtained recombinant plasmid (designated as pQE 30-will get RA3) in heterological system E. coli M 15. The authors have obtained producing strains of recombinant polypeptide A3, designated as E. coli M 15-A3. Also, the authors implemented the expression of the recombinant polypeptide A3 cells of the strain E. coli M 15-A3 with subsequent single-stage affinity purification using Ni-sepharose (GE Healthcare, Sweden).

The preparation of recombinant polypeptide A3.

The source of chromosomal DNA served as the strain DG 13 G (isolated from cow's milk). DNA separation�Naya phenol-chloroform extraction, was used as template in PCR to obtain a fragment will get RA3 gene (372 BP) Oligonucleotide primers presented in table 1.

The underlined links in the nucleotide sequences indicate the restriction sites. Analysis PCR was carried out by separation of DNA fragments in 1% agarose gel using a horizontal electrophoresis (Fig. 2).

The selection of the desired amplificatoare section of DNA was performed using the kit QIAquick Gel Extraction Kit (Qiagen, USA).

The resulting fragment was cloned using gene-expression plasmid pQE 30. In preparation for cloning was performed restriction analysis extracted from the agarose slice will get RA3 (372 BP) and plasmid pQE 30 enzymes BamHI and KpnI with the formation of sticky ends. Products restriction enzymes were separated by horizontal electrophoresis in 1% agarose gel, and then was ligated.

The ligation products was performed calcium transformation of the strain E. coli M 15. The transformation was performed by the method of transformation of Escherichia coli using dense selective medium containing 100 μg/ml ampicillin, 25 µg/ml kanamycin. (Maniatis T, Fritsch E. F., Sambrook J. Molecular cloning: a laboratory manual. - Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 867. (1982)).

Antibiotic-resistant transformants was cleaved into two parallel Petri dishes with Antibes�Omikami. Colonies with one Cup was transferred to a nitrocellulose membrane and revealed with a solution containing 0.2 N NaOH, 0,1% SDS and 0.5% β-mercaptoethanol. The membrane is incubated for 1 hour at room temperature in blocking solution (2 parts 3% milk to 1 part phosphate-buffered saline (PBS) to remove nonspecific binding and then in blocking solution containing NAV, labeled with horseradish peroxidase (Sigma, USA).

Conjugation of horseradish peroxidase with NAV held periodate method (Primal G. Immunological methods: M. Medicine: 438-439. (1987)). Then the membrane was sequentially washed with blocking solution and PBS. Peroxidase activity showed a solution paraphenylenediamine at a concentration of 1 mg/ml with 0.15% hydrogen peroxide. From the transformants with the most pronounced expression of CSA-binding activity were isolated recombinant plasmids using Mini-prep plasmid DNA purification kit (Qiagen, USA). The presence of recombinant DNA was confirmed by PCR with the original primers RA1 and RA2 Plasmid DNA pQE 30-will get RA3 was used as the matrix in PCR with primers pQE1 and pQE2. Amplificat was isolated after electrophoresis in 1% agarose gel and sequenced.

Totals coincided with the known nucleotide sequence of the coding region with two of CSA-binding GA module. (U. Sjobring Infect, and Immun. 60: 3601-3608. (1992)).

Fig. 3 presents the nucleotide pic�egovernance, consisting of 372 BP recombinant DNA will get RA3 and 48 BP plasmid pQE 30. Bold sequence of CSA-binding GA module.

Fig. 4 presents the amino acid sequence consisting of 140 amino acid residues, in which 124 the amino acid A3 polypeptide encoded by the recombinant DNA will get RA3 and the first 16 amino acids of the plasmid pQE 30. Bold sequence of CSA-binding GA module.

The culture of the strain E. coli M 15 containing a recombinant plasmid pQE 30-will get RA3, designated as E. coli M 15-A3 were cultured in liquid BHI medium (Brain Heart Infusion Broth, Gibco, USA) with the addition of antibiotics (ampicillin (100 μg/ml) and kanamycin (25 µg/ml)) to late logarithmic growth phase (OD600=0.7 to 0.9). Then the expression of recombinant protein was induced by adding isopropyl-beta-D-thiogalactopyranoside (IPTG), and cells were cultured for another 4 hours. After that, the cells were besieged by centrifugation, washed with lytic buffer A (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 20 mm imidazole, pH 8.0) and suspended in the same buffer by adding protease inhibitor, phenylmethylsulfonyl (PMSF), to a concentration of 1 mm. Suspension cells were opened by sonication and centrifuged. The supernatant was applied onto a column Packed with Ni-separate. After the protein was associated with Ni-separate, column �raavali buffer A to remove unbound proteins. Recombinant protein was suirable buffer B (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 250 mm imidazole, pH to 8.0). After affinity chromatography protein were dialyzed 18 hours against distilled water.

Selected recombinant polypeptide was analyzed by the method of SDS polyacrylamide gel electrophoresis, which allowed to conclude about a satisfactory purification of the polypeptide and also on its molecular weight, comparing the mileage A3 polypeptide with mileage of proteins of known molecular mass (Precision Plus Protein standards (161-0373), Bio-Rad, USA). The molecular weight of the polypeptide A3 was equal to (14,0±0,5) kDa (Fig. 5).

Thus, recombinant A3 polypeptide contains the amino acid sequence of CSA-binding polypeptide CTG strain DG 13 containing 124 amino acids, covalently linked with 16 amino acid residues encoded by pQE 30.

Study of peculiarities of interaction of the recombinant polypeptide A3 with NAV as compared to a similar reaction with the polypeptide A2

The resulting characterization of the interaction of recombinant molecules A3 with NAV. The results allow to extend the scope of application of the polypeptide A3, expanding its use in the creation of diagnostics, for affine selection of CSA and for the liberation of serum from NAV.

Structural differences recombinant�s polypeptides A2 and A3 make recombinant polypeptide A3 is more preferable when used in a test system for the determination of microalbuminuria.

A comparison of the receptor activity of recombinant polypeptides A2 and A3 in relation to NAV, represented by the following samples: 1) NAV - polyclonal human serum albumin, labeled with horseradish peroxidase. 2) unlabeled Polyclonal NAV.

It is established that the polypeptide A3 and the A2 polypeptide, has the ability to selective binding of CSA used in any of the variants of statement of ELISA (direct ELISA, indirect ELISA, inhibition ELISA, competitive inhibition). Structural differences between the two polypeptides that are implemented by methods of molecular genetics, contribute to a significant differentiation of recombinant polypeptides A2 and A3 of CSA-binding properties.

In the case of direct ELISA polypeptides A2 and A3 were immobilized on the solid phase. With estimated the number associated with them labeled NAV. Fig. 6 shows the graph reflecting a comparison of CSA-binding activity of polypeptides A2, and A3. The analyzed polypeptides A2 and A3 have the NAV-binding activity against labeled NAV, and the number of CSA associated with A3 polypeptide, about the same as in the case of polypeptide A2. Polypeptides A2 and A3 encoded by different fragments of the gene and having a different molecular weight, are obviously, the structural differences that determine NAV-recepto�ing the activity of each of them. It can be assumed that implementation of this ability is influenced by the conditions of statement of ELISA, namely the presence of the polypeptide on the solid phase or in solution. The impact of this factor is explained by the presence of peptides spatial configuration, which is different for molecules that circulate freely in solution or fixed on a solid phase.

The conformation change may hypothetically lead to an increase or decrease in the ability of molecules to bind NAV. To check this assumption, used indirect and inhibitory ELISA.

When the indirect ELISA results showed that the polypeptides in the solution bind NAV with about the same activity (Fig. 7). Inhibition ELISA showed that the polypeptides A2 and A3, while in solution, prevented the interaction of labeled NAV with adsorbed molecules A2 and A3 in the range of 70-94% (Fig. 8).

Observed peculiarities in the behavior of proteins in the ELISA conditions. The polypeptides of A2 and A3 is able to interact with navs as in solution (inhibitory and indirect ELISA) and in the adsorbed state (direct ELISA). This property allows their use as immunochemical reagents in ELISA, and, as in immobilized form, and revealing the role of molecules in the presence of the enzyme label.

Thus, the degree of binding observed for the polypeptides A2 and 3 in terms of direct and indirect ELISA, about the same.

The method for determining the excretion of CSA with urine, i.e. the creation of test systems for the determination of microalbuminuria

Conducted research on the interaction of the polypeptide A2 and A3 with NAV showed that recombinant molecules possess NAV-binding capacity. This property served as the basis for creating test systems to determine the concentration of CSA in the urine.

For screening and monitoring patients with diabetes and hypertension requires simultaneous analysis of several tens of samples. To solve this problem, the possibility of developing a method that combines the use of stable highly specific recombinant NAV-binding polypeptide and technology of microchips. When creating a test system for the qualitative detection of microalbuminuria is usually used antibodies to albumin were replaced by a recombinant receptor polypeptide, and the principle of the enzyme immunoassay was transformed into a powder XRD.

As a matrix for the deposition of urine samples of patients with nephropathy was chosen nitrocellulose membrane. On the nitrocellulose membrane, the size of 1 cm2were inflicted 30 urine samples, using automated calibrator. The first row on the membrane corresponded to the standard drug dilutions NAV to construct the calibration curve(100; 50; 25; 12,5; 6,25; 3,125 µg/ml). Membrane�was on preincubation in blocking solution (2% milk, diluted in PBS) with peroxidase-labeled NAV-binding polypeptide A3. Then the membrane was washed and stained with a solution of paraphenylenediamine with hydrogen peroxide. On the membrane clearly manifested spots, varying degrees of staining corresponded with various concentrations of albumin in the standard samples and the urine samples (Fig. 9). Having a reading device and a corresponding computer program, it is possible to obtain a quantitative estimation of albumin in the urine, and simultaneously in a large number of samples.

To create a test system for the quantitative determination of microalbuminuria preferred competitive ELISA method, as this method contains a minimal number of operations requiring lower consumption of reagents and can be easily automated. Replacement of antibodies to albumin on recombinant polypeptide has created certain advantages as it allowed to build up from the laborious process of preparation of specific antisera, used to standardize the recombinant polypeptide and, thereby, to unify the entire system of analysis. In addition, the use of recombinant receptor polypeptide helped to avoid the background reactions, potential for immunological reactions.

Previously this approach for analysis of albumin was not used.

The principle of XRF method for quantitative�governmental definition of microalbuminuria is as follows: on a hard surface tablet immobilities recombinant NAV-binding polypeptide (A2 or A3) which are applied to analyze urine samples simultaneously labeled with NAV. In such a formulation albumin sample and labeled NAV competing for the CSA-binding polypeptide immobilized on a solid phase. The concentration of albumin in the sample is inversely proportional to the enzymatic activity of the solid phase.

When developing test systems were chosen conditions of the analysis, which provides the necessary sensitivity and specificity. As a result of the experiments was chosen working dilution of labeled NAV 1:8000 at a concentration of immobilized polypeptide 0.5 μg/ml. According to serial dilutions of unlabeled NAV constructed calibration curve.

After the selection of the optimal conditions were building a calibration curve of optical density on the concentration of CSA in the standard samples(5.0; 10; 20; 25; 50, 100 and 200 μg/ml). The concentration of CSA found in the urine by the values of optical density on the calibration curve.

Multiple repetitions of this experiment revealed that when using the A3 polypeptide encoded by the gene fragment of 420 BP was responsible only for binding with the NAV area with two GA modules, the points lie exactly on a straight and very accurately determined the concentration of albumin in the urine. With regard to the use of the polypeptide A2, which has a much greater molecular weight and covering a large region�St', than of CSA-binding GA module, points corresponding to the calibration samples, which do not always lie on a straight line, which hampered the precise determination of the concentration of albumin in the urine.

Thus, were able to show that recombinant polypeptide A3 can be used as a highly specific reagent for the quantitative determination of albumin in urine. The minimum concentration of CSA that can be identified in urine, was 5 μg/ml and a maximum of 200 mcg/ml Urine with higher concentrations of albumin should be diluted with PBS. As a result, the sensitivity of this detection method allows to detect the amount of albumin in the urine within, including the excretion of albumin in the urine in norm, i.e., up to 20 mcg/ml and in the range of microalbuminuria from 20 to 200 μg/ml. the Method is extremely accurate.

Example 1. Obtaining the DNA fragment will get RA3 PCR.

To 0.25 µg of genomic DNA extracted phenol-chromosomal extraction from strain 13 DG G added 10 micromoles each of specific primers flanking the sequence under consideration, the buffer with magnesium for polymerase, 0.2 mm of the four deoxyribonucleotides, the volume was adjusted with water to 25 µl and added 0.5 ál thermostable DNA polymerase. On the surface of the liquid was added 40 μl of mineral oil. The tubes were placed in-line amplifier and Inc�was boravali at 94°C 2 min. The PCR program consisted of: denaturation at 94°C for 30 sec, primer annealing at 60°C for 1 min and synthesis at 72°C - 1 min. This cycle was repeated 30 times, after which the mixture was treated at 72°C 10 min, we used the oligonucleotide primers listed in Table 1. PCR products were separated in 1% agarose gel in a horizontal electrophoresis (Fig. 2). The allocation amplificatoare section of DNA was performed using the kit QIAquick Gel Extraction Kit (Qiagen, USA). Analysis of the size of the resulting DNA fragment was carried out, based on the comparison of its electrophoretic mobility the electrophoretic mobility of the marker of molecular weights (100 BP DNA marker, the Helicon).

Fig. 2 shows the èlektroforegramme amplificatoare of the DNA fragment will get RA3), where 1 is the PCR product, 2-100 BP DNA marker (from top to bottom: 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 nucleotide pairs).

As a result of PCR with primers RA1 and RA was obtained fragment of 372 BP, as shown in Fig. 2.

Example 2. Cloning of the DNA fragment will get RA3 using gene-expression plasmid pQE 30.

Plasmid pQE 30 and the fragment obtained by PCR was treated with two restriction enzymes BamHI and KpnI, which led to the formation of sticky ends. Products restriction enzymes were separated by horizontal electrophoresis in 1% agarose gel and then isolated from the agarose using a set� "QIAquick Gel Extraction Kit (Qiagen, USA). During cloning to 5 μl of the DNA fragment will get RA3 was added 1 μl of the DNA fragment pQE 30 obtained after electrophoresis and cut out from the agarose, 2 µl of ten-fold ligase buffer and 1 µl ligase of phage T4. The volume was adjusted with distilled water to 20 ál. The mixture was incubated at 10° C for 12 hours. As a result of the cloning was obtained recombinant plasmid, designated as pQE 30-will get RA3 carrying recombinant DNA (designated as will get RA3), consisting of 372 BP and 48 BP fragment of the plasmid pQE-30.

Example 3. The transformation of a culture of E. coli M 15 plasmid DNA pQE 30-will get RA3.

The recombinant plasmid was transformed into the heterologous system of E. coli M 15. As a positive control in parallel, we performed transformation of E. coli M 15 original plasmid DNA pQE 30.

Genetic marker plasmid DNA pQE 30 is a marker amp, encoding beta-lactamase that provides resistance to ampicillin cells carrying this plasmid. Cell culture E. coli M 15 were cultured in BHI broth (Brain Heart Infusion Broth, Gibco, USA) with kanamycin (25 μg/ml) for 12 hours at 37° C and vigorous stirring. Then the inoculum-passaged to a new volume of the same medium (10 ml of medium 0.1 ml of inoculum) and incubated at 37° for 2-3 hours with stirring to OD600=0,3. Grown cells in a volume of 1.5 ml was centrifuged for 1 min at 12000 about�/min., and the resulting precipitate was suspended in 200 µl of a solution of 0.1 M CaCl2. The mixture was kept in ice for 1 hour. After centrifugation the precipitate was resuspended in 187, 5 µl 0.1 M CaCl2and 64.5 µl of distilled water. Then in a test tube made of 0.2 μg of plasmid DNA and the mixture incubated on ice for 1 hour. The mixture was stirred for 2 min at 42°C and again the mixture was transferred to ice for 10 min. then to the mixture was added 1 ml of BHI broth and incubated 1 hour at 37°C. After precipitation by centrifugation (8000 rpm for 1 min), the cells were sown on cups with 1% L-agar (Difco, USA) containing ampicillin (100 μg/ml) and kanamycin (25 µg/ml). After 18 hours of cell growth at 37°C was carried out the selection of clones of transformants.

Antibiotic-resistant transformants were transferred into two parallel Petri dishes with antibiotics. Colonies with one Cup was transferred to a nitrocellulose membrane and revealed with a solution containing 0.2 N NaOH, 0,1% SDS and 0.5% β-mercaptoethanol. The membrane was incubated for 1 hour in blocking solution containing NAV, labeled with horseradish peroxidase (Sigma, USA). Then sequentially washed with blocking solution and PBS. Peroxidase activity showed a solution paraphenylenediamine at a concentration of 1 μg/ml with 0.15 M hydrogen peroxide.

Thus, the obtained data allow to draw a conclusion that as a result of CL�to increase recombinant clones, bearing the plasmid pQE 30-will get RA3 with recombinant DNA will get RA3.

Example 4. The purification of the recombinant polypeptide A3.

The culture of the strain E. coli M15-A3 were grown in BHI broth with addition of ampicillin at a concentration of 100 μg/ml and kanamycin at a concentration of 25 µg/ml overnight with vigorous stirring. Then the cell-passaged with 800 ml of the same medium and incubated at 37°C for 2-3 hours under vigorous stirring until the OD600=0.7 to 0.9. The expression of recombinant protein was induced by adding a solution of isopropyl-beta-D-thiogalactopyranoside to final concentration of 2 mm, after which the cells were incubated under the same conditions for another 4 hours. The resulting cell suspension was centrifuged at 4000 rpm for 20 min. the Supernatant was decanted, and the cells suspended in the buffer ((20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 20 mm imidazole, pH to 8.0), adding a protease inhibitor to phenylmethylsulfonyl (PMSF) to a final concentration of 1 mm. For the lysis of cells was used three ultrasonic treatment at 4°C for 20 sec. with a break of 40 seconds. in an ultrasonic disintegrator (UZDN-U.2, England). The cell lysate was centrifuged at 20000 rpm and 4°C for 20 min. the Supernatant was passed through 0.45 µm filter (Millipore, USA) and then applied to a column of Ni-separate, pre-balanced b�from A. Next, the column was washed with the same buffer until then, until the values of the OD280coming out of the solution were not more than 0.01. Protein was suirable solution of buffer B (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 250 mm imidazole, pH to 8.0). The eluate was collected in fractions of 500 μl and measured in them the values of the OD280. Fractions with the highest OD values280were pooled and were dialyzed against distilled water for 12 hours. Fig. 5 presents the èlektroforegramme recombinant polypeptide A3.

Fig. 5. shows the èlektroforegramme recombinant polypeptide A3, where 1 - a preparation of purified protein A3, 2 - molecular weight marker, (from top to bottom: 250, 150, 100, 75, 50, 37, 25, 20, 16, 10 kDa). The molecular weight of the polypeptide A3 was determined by comparing the mileage of protein A3 with mileage of proteins of known molecular mass (Precision Plus Protein standards (161-0373), Bio-Rad, USA). The molecular weight of the protein A3 is equal to (14±0,5) kDa.

Example 5. Binding of serum labeled NAV adsorbed polypeptides A2 and A3.

Analysis of CSA-binding activity carried out by direct ELISA. For the IFA used the tablets NUNC MaxiSorb (Denmark).

Cooking divorced drugs labeled NAV was carried out using a solution of the FSB. The removal of unbound reagents were performed three times washing tablets with PBS with 0.05% tween 20 (FSBT)

Polypeptides A2 and A3, diluted in PBS, pH 8.0 to 1 μg/ml, were absorbed on the tablet for 18 hours at 4°C. After three laundering in tablets made of diluted labeled NAV. Incubation was performed at 37°C for 1 hour. Further tablets 3 times washed from unbound reagents. The activity of the enzyme label horseradish peroxidase was determined using the Chromogen - tetramethylbenzidine (TMB) dissolved in 0.1 M citrate-phosphate buffer, pH 5.0 with addition of hydrogen peroxide. The reaction was terminated by addition of 2N sulfuric acid. The optical density was determined at a wavelength of 450 nm on multiscale.

It is established that the polypeptide A3, like the A2 polypeptide, has roughly the same high of CSA-binding activity, as demonstrated in Fig. 6.

Fig. 6 shows a comparison of the efficiency of binding of the polypeptide A2 and A3 with NAV, where the black bar - A2 polypeptide, white columns, polypeptide A3, the abscissa axis is the dilution of labeled NAV, the ordinate is the optical density (OD450).

Example 6. Binding of serum labeled NAV polypeptides A2 and A3 in the solution.

In the case of indirect ELISA on solid phase were absorbed unlabeled navs, to which a solution of joined molecules A2 or A3, in turn linking from a solution of peroxidase-labeled NAV.

For the IFA used �lansey NUNC MaxiSorb (Denmark).

Cooking divorced drugs labeled NAV was carried out using a solution of the FSB. In this case, were sensibilized NAV diluted in PBS, for 18 h at 4°C. Unbound navs were washed three times by adding 150 μl PBST to each well was added in two-fold dilutions of the recombinant polypeptide, starting at 20 µg/ml, were incubated for 1 hour at 37°C and again three times washed PBST. Polypeptide bound to a NAV determined by using the conjugate NAV with peroxidase. The results were recorded as in the direct method.

In the experiments used in parallel at least four rows of titration. The results showed that the polypeptides A2 and A3 in the solution bind NAV is about the same (Fig. 7).

Fig. 7 presents a comparison of the effectiveness of binding in solution polypeptides A2 and A3 with NAV, where the solid curve is the polypeptide A2, dashed line - polypeptide A3, the abscissa axis is the concentration of polypeptides ng/ml, ordinate - optical density (OD450).

For the production of inhibitory ELISA polypeptides A2 and A3 at a concentration of 1 μg/ml were immobilized on the tablet. Thereto was added a pre-preincubating a mixture of the corresponding polypeptide and divorced labeled NAV (at a ratio of 1:1, incubation at room temperature for two hours) Incubation of immobilized polypeptides with a mixture of polyp�of Prigov and labeled NAV was performed for 1 hour at 37°C. The values of optical density was calculated the percentage of inhibition of the interaction of labeled NAV with adsorbed molecules A2 and A3 each of the polypeptides in the solution (Fig. 8). Polypeptides A2 and A3, while in solution, prevented the interaction of labeled NAV with adsorbed molecules A2 and A3 in the range of 70-94%.

Fig. 8 presents a comparison of the effectiveness of binding of CSA adsorbed polypeptides A2 and A3 and polypeptides A2 and A3 in solution, where the diamonds - A2 polypeptide, triangles - A3 polypeptide, the abscissa axis is the dilution of labeled NAV, the ordinate is the percentage inhibition, %.

Example 7. The test system for the qualitative detection of microalbuminuria.

For the qualitative detection of microalbuminuria was created the microchip.As the matrix of the microchip for the application of urine samples of patients were selected nitrocellulose membrane. On the nitrocellulose membrane, the size of 1 cm2were applied 30 urine samples, using automated calibrator. The first row on the membrane corresponded to the standard drug dilutions NAV, 6 two-fold dilutions of CSA, starting with 100 µg/ml: 100; 50; 25; 12,5; 6,25; 3,125 μg/ml to construct the calibration curve. The membrane was preincubation in blocking solution (2% milk diluted in PBS) with peroxidase-labeled NAV-binding polypeptide A3. Then the membrane was washed with�camping and stained with a solution of paraphenylenediamine with hydrogen peroxide. On the membrane clearly manifested spots, varying degrees of staining corresponded with various concentrations of albumin in the standard samples and the urine samples (Fig. 9).

Fig. 9 shows a nitrocellulose membrane is shown by the spots, which correspond to the concentration of CSA in urine samples of patients where the first row of dilution of a standard preparation of CSA, other numbers of urine samples of patients.

Example 8. The test system for the quantitative determination of microalbuminuria.

Quantitative determination of microalbuminuria by XRF method was conducted as follows: on a hard surface polystyrene tablet immobilizovana polypeptide A2 or A3, which struck analyzed urine samples simultaneously labeled with NAV. In such a formulation albumin sample and labeled NAV competing for the CSA-binding polypeptide immobilized on a solid phase.

The concentration of albumin in the sample is inversely proportional to the enzymatic activity of the solid phase. Conditions of the analysis, which provides the necessary sensitivity and specificity, the following: as a result of the experiments was chosen as the working dilution of the labeled NAV 1:8000 at a concentration of immobilized polypeptide A2 or A3, 5 µg/ml. After the selection of the optimal conditions were building a calibration curve of optical�tion density on the concentration of CSA in the standard samples (5.0; 10; 20; 25; 50, 100 and 200 μg/ml). The concentration of albumin in the urine may be guided by the values of optical density on the calibration curve.

The technical solution can be implemented using known technology and hardware.

1. Recombinant DNA will get RA3 encoding a recombinant polypeptide A3 having the ability to selectively bind human serum albumin - navs, and having the nucleotide sequence shown in Fig. 3.

2. Recombinant plasmid DNA pQE 30-will get RA3, representing the plasmid pQE 30, bearing a recombinant DNA will get RA3 according to claim 1, and providing a recombinant polypeptide A3.

3. The Escherichia coli strain M 15-A3 producing a recombinant polypeptide A3, derived from the Escherichia coli strain M 15 and containing recombinant plasmid DNA pQE 30-will get RA3.

4. Recombinant A3 polypeptide having the ability to selectively bind NAV and characterized by the amino acid sequence shown in Fig. 4, in which the first 16 amino acid sequence encoded by the plasmid pQE 30, covalently associated with follow-up of 124 amino acids encoded by the sequence of CSA-binding fragment of chromosomal DNA of strain DG 13 Streptococcus group G-CG.

5. The test system x-ray fluorescence analysis for the qualitative detection of microalbuminuria, SOS�Mr sage from the matrix of the microchip - the nitrocellulose membrane; six standard samples of CSA 100; 50; 25; 12,5; 6,25; 3,125 μg/ml; blocking solution; recombinant A3 polypeptide according to claim 4, labelled with peroxidase; solution paraphenylenediamine with hydrogen peroxide for staining of the membrane.

6. The test system x-ray fluorescence analysis for the quantification of microalbuminuria consisting of polystyrene tablet on a hard surface to which immobilized polypeptide A3 according to claim 4; phosphate-saline buffer solution with tween 20 to wash the tablet; six calibration samples 100; 50; 25; 12,5; 6,25; 3,125 μg/ml to construct a calibration curve of optical density on the concentration of CSA; the Chromogen is 3,3'5,5'-tetramethylbenzidine and 33% hydrogen peroxide for the existence of the number of CSA in the urine.



 

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9 cl, 11 dwg, 1 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

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