RU2430161C2 - RECOMBINANT PLASMID DNA pQe30_PS-CFP2/TurboYFP_MBP7, CODING PROTEIN PS-CFP2/TurboYFP_MBP7, STRAIN OF Escherichia coli BL21(DE3)/pQe30_PS-CFP2/TurboYFP_MBP7 - SAID PROTEIN PRODUCENT AND METHOD OF OBTAINING PROTEIN PS-CFP2/TurboYFP_MBP7 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: in order to obtain protein PS-CFP2/TurboYFP_MBP7, constructed is recombinant plasmid DNA PS-CFP2/TurboYFP_MBP7 with size 4916 bp, coding hybrid protein, containing sequence of proteins PS-CFP2 and Turbo YFP, bound with fragment of myelin basic protein 80-104. Composition of plasmid DNA also includes promoter of T5 PHK-polymerase transcription, site of ribosome binding; fragment of DNA plasmid gen of β-lactamase, determining stability of Escherichia coli cells to ampicillin, as genetic marker. Obtained plasmid DNA is used to transform cells of Escherichia coli strain BL21(DE3) to obtain strain-producent of hybrid protein PS-CFP2/TurboYFP_MBP7. In order to obtain protein PS-CFP2/TurboYFP_MBP7 cultivation of strain-producent of Escherichia coli BL21 (DE3)/pQe30_PS-CFP2/TurboYFP_MBP7 is performed, cells are destroyed and target protein is purified by method of affine and gel-filtration chromatography.

EFFECT: invention makes it possible to increase biosensor sensitivity and stability and extend its specificity in respect to pool of catalytic antibodies.

3 cl, 7 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of biotechnology, and may find application in medicine to create an innovative biomarker of multiple sclerosis, as well as a diagnostic and diagnostic criterion.

In the last decade, the role of the B-cell response, accompanied by the formation of antibodies to its own antigens, has become increasingly important in the etiology and pathogenesis of autoimmune diseases. Among the pathologies of an autoimmune nature, a special place is occupied by multiple sclerosis (PC), a chronic neurodegenerative disease that leads to the destruction of the myelin sheath of nerve fibers. At the moment, there is no doubt that autoantibodies produced on the major component of the myelin sheath, the main myelin protein (MBM), are one of the important pathogenic factors that affect the degradation of nerve fibers in the central nervous system in PC. Diagnosis of PC and prognostic criteria for the development of the disease need improvement and additional verification. The current complex of functional tests, based on the clinical picture of the course of the disease and forming the basis for creating a unified patient disability scale (EDSS), also needs additional quantitative refinement. One of the main components of the diagnostic complex is the data of magnetic resonance imaging (MRI) of the brain of patients. However, the foci of demyelination detected using this method are not always an absolutely clear evidence of the development of PC, because can be caused by other neurodegenerative and post-traumatic processes.

A variety of methods are known for producing a biosensor for various highly toxic pathogens based on a two-protein fluorescence system (US Pat. No. 7495069, publ. 2009, No. 7183066, publ. 2007; application WO / 2000/073437, publ. 2000). Also known is a biosensor with the principle of resonant energy transfer to various highly toxic pathogens based on a two-protein fluorescent system (given in PCT / CA 2000/000620, published in 2000). A common serious drawback of these sensors is the inability to measure the activity of a particular type of proteinase — catalytic antibodies specific for the myelin basic protein present in the blood serum of patients with multiple sclerosis.

Known closest to the claimed biosensor (Ponomarenko N. A. "Catalytic antibodies - proteinases", M., 2008, abstract of the dissertation for the degree of Doctor of Biological Sciences, p. 49). Serious disadvantages of this biosensor are insufficient accuracy and low stability.

The objective of the invention is the creation of a biosensor based on genetic constructs for measuring the activity of catalytic antibodies specific for the myelin basic protein present in the blood serum of patients with multiple sclerosis.

The problem is solved by:

for the construction of a 4916 bp pQe30_PS-CFP2 / TurboYFP_MBP7 recombinant plasmid DNA encoding the PS-CFP2 / TurboYFP_MBP7 fusion protein containing the sequence of PS-CFP2 and Turbo YFP proteins connected by a fragment of the main myelin protein 80-104 and having the amino acid sequence SEQ ID NO: 1, where the recombinant plasmid DNA has a physical map shown in figure 1, and consists of:

a DNA fragment containing a T5 RNA polymerase transcription promoter, a ribosome binding site, and a PS-CFP2 / TurboYFP_MBP7 fusion protein sequence;

a DNA fragment of the plasmid pQe30 containing the transcriptional terminator of the T5 RNA polymerase of the bacteriophage T5 and the site of replication initiation;

- a DNA fragment of the β-lactamase gene that determines the resistance of ampicillin to Escicichin coli transformed with the plasmid pQe30_PS-CFP2 / TurboYFP_MBP7, as a genetic marker;

also due to the Escherichia coli strain BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 - producer of the PS-CFP2 / TurboYFP_MBP7 hybrid protein obtained by transformation of Escherichia coli BL21 (DE3) cells of the recombinant plasmid DNA pQe2BPo-Turbo plasmid pQP_BYo-PS;

and due to the method of producing the PS-CFP2 / TurboYFP_MBP7 protein, including culturing the producer strain Escherichia coli BL21 (DE3) / pQe30 PS-CFP2 / TurboYFP_MBP7, cell disruption and purification of the PS-CFP2 / TurboYFP_MBP7 protein by affinity and gel chromatography.

The biosensor's advantages are a significantly expanded gamma of detectable pathogenic antibodies due to the introduced flanking amino acid residues and, at the same time, increased stability in solution due to a significant decrease in the physical interaction of fluorophore proteins. Compared to the known biosensor, both the sensitivity of the bisensor and its specificity with respect to the pool of catalytic antibodies have expanded. The innovative properties of the biosensor allow it to be used for the entire circle of patients with multiple sclerosis (both for progressive and remitting stages), which could not be achieved earlier. The introduced amino acid insertions also significantly increased the physicochemical properties of the biosensor. Reduced molecular interference of fluorophore proteins increases the biosensor stability and the quantum yield of resonant energy transfer. The technical result achieved consists in improved accuracy, high stability of the biosensor during storage and analysis procedure, which, in turn, significantly increased the efficiency and ease of diagnosis. The high sensitivity and selectivity of the biosensor reduces the number of false-positive results and allows you to confidently distinguish between multiple sclerosis and other neurodegenerative diseases during diagnosis.

The advantage of the invention is, firstly, that as a result of the method, a protein is obtained containing a fragment of the myelin basic protein between two fluorescent proteins, which is an FRET pair. It was clearly shown that the first introduced flanking amino acid sequences are extremely important for the binding and subsequent hydrolysis of the biosensor by catalytic pathogenic antibodies. The introduction of "anchor" end residues significantly improved the physicochemical characteristics of the product. The low toxicity of the target protein for the producer strain allows to achieve high yields of the hybrid protein (about 20% of the total cellular protein). The advantage of this method is the simplicity of EPeFRET cleaning technology. The high level of expression and its extremely high solubility and stability in aqueous solutions allow purification in two stages to obtain product homogeneity of more than 98%. For the biosynthesis of a recombinant protein, optimal regulatory elements that control its expression are used: a promoter and terminator for the highly processive T5 bacteriophage RNA polymerase, a consensus bacterial ribosome binding site, and start and stop codons.

The construction of a new plasmid DNA pQe30_PS-CFP2 / TwboYFP_MBP7 containing the fluorescent proteins PS-CFP2 and TurboYFP connected by the OBM80-104 fragment (EPeFRET) is carried out by replacing the original linker with single-stage cloning using two oligonucleotides. The selection of positive clones is carried out using PCR using specific primers and subsequent restriction analysis of the isolated plasmid DNA. The structure of a gene encoding a fusion protein containing an OBM fragment 80-104 is determined by sequencing according to the Sanger method. EPeFRET expression is carried out in E. coli strain BL21 (DE3). When the undergrowth of the culture is 37 ° C after reaching an optical density of OD _{600 of} 0.6-0.8 units, the cell suspension is transferred to room temperature and incubated for 24-36 hours to mature the chromophore. The soluble protein fraction is prepared as follows. The cell lysate is applied onto Talon Superflow metal chelate sorbent previously balanced with buffer at a rate of 1 ml / min. Then the column is washed with 100-120 volumes of a solution containing 50 mm sodium phosphate, 300 mm NaCl, pH 7.0. Recombinant protein is eluted with a 50 mM EDTA solution at a rate of 0.5 ml / min. Next, gel filtration is performed on a Superdex 75 column in a PBS / TBS solution at a rate of 0.5 ml / min. The resulting protein solution is kept for a week at a temperature of 10 ° C with 0.05% sodium azide for the final maturation of the chromophore. Fluorescence measurements were carried out on a Saru Eclipse fluorometer (Varian) and a Tecan Genios (Tecan) plate reader at an excitation and emission wavelength of 405 and 538 nm, respectively.

Recombinant plasmid DNA pQe30_PS-CFP2 / TurboYFP_MBP7 encoding an EPeFRET fusion protein containing the OBM 80-104 peptide is characterized by the following features:

- has a molecular size of 4916 bp .;

- encodes a hybrid protein EPeFRET containing a modified sequence of proteins PS-CFP2 and Turbo, connected by a fragment of the main myelin protein 80-104;

- consists of a DNA fragment containing a T5 RNA polymerase transcription promoter, a ribosome binding site and a sequence of a PS-CFP2 / TurboYFP MBP7 hybrid polypeptide containing a sequence of PS-CFP2 and Turbo proteins connected by a fragment of the myelin 80-104 basic protein and a plasmid DNA fragment pQe30 containing a transcriptional terminator of T5 RNA polymerase, a replication initiation site and a β-lactamase gene;

- has a unique combination of features: promoter and terminator of transcription of RNA polymerase of bacteriophage T5; ribosome binding site; an artificial gene encoding a PS-CFP2 / TurboYFP MBP7 fusion protein comprising a sequence of PS-CFP2 and Turbo beige linked by a fragment of myelin basic protein 80-104; a β-lactamase gene that determines the resistance of ampicillin to the cells transformed with plasmid pQe30_PS-CFP2 / TurboYFP_MBP7.

To obtain the producer strain of the hybrid protein EPeFRET, plasmid DNA preparation pQe30_PS-CFP2 / TurboYFP_MBP7 transforms the competent cells of Escherichia coli BL21 (DE3) and selects clones capable of expressing the recombinant protein. The presence and level of expression of recombinant proteins is monitored by denaturing PAAG electrophoresis.

Cells grow at temperatures from 4 ° C to 40 ° C with optimum pH from 6.8 to 7.5. As a source of nitrogen, both mineral salts in the ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. Amino acids, glycerin, carbohydrates are used as a carbon source. Cells are resistant to ampicillin (up to 500 μg / ml) due to the presence of the β-lactamase (bla) gene in plasmid pQe30_PS-CFP2 / TurboYFP_MBP7.

The advantages of the E. coli BL21 (DE3) strain as the basis for creating the producer strain are the use of bacteria with the Lon OmpT phenotype, which excludes the possibility of proteolytic cleavage of the synthesized recombinant protein and contamination of the drug with the most active E. coli proteases. The T5-RNA polymerase gene is integrated into BL21 (DE3) chromosomal DNA, which, together with the use of the T5 promoter and T5 terminator in the pQe30 PS-CFP2 / TurboYFP_MBP7 plasmid, provides fast and efficient protein production by E. coli cells upon isopropylthio-β-D induction -galactoside or lactose.

E. coli BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 cells are a good biosensor producer due to the unique plasmid pQe30_PS-CFP2 / TurboYFP_MBP7 introduced into them. Upon induction with isopropylthio-β-D-galactoside, an effective biosynthesis of the EPeFRET hybrid protein occurs, which accumulates in the cells in an amount of 20-25% of the total bacterial protein.

The biosynthesis of the product is carried out as follows: cells of the E. coli strain BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 are grown in a rich medium (for example, 2 × YT) with the addition of 100-200 μg / ml ampicillin. In an undergrowth culture at 37 ° C, after reaching an optical density of OD _{600 of} 0.6-0.8 units, protein synthesis is induced with isopropyl-β-D-galactoside at a concentration of 0.1-1.0 mM, then the cell suspension is transferred to room temperature and incubated 24-36 hours for the maturation of the chromophore. An increase in the yield of recombinant protein can be achieved by forced aeration of the culture fluid and the use of enriched culture media (for example, with the addition of glucose).

The method for producing EPeFRET protein from producer cells includes the following stages: separation of bacteria from the culture medium by centrifugation, their destruction by one of the commonly used methods (ultrasonic disintegration, chemical lysis using detergents and chaotropic agents), separation from inclusion bodies and water-insoluble cell components, and purification of the target protein by affinity and gel permeation chromatography.

The identity of the EPeFRET protein to the claimed one is established by the methods of PAGE-electrophoresis in the Tris-Tricin system, MALDI time-of-flight mass spectrometry, sequencing of the N-terminal amino acid sequence according to the Edman method, as well as testing for cleavage by natural proteases and highly purified catalytic antibodies. The degree of purification of EPeFRET is determined by the methods of PAGE-electrophoresis in Tris-tricin system and MALDI time-of-flight mass spectrometry. The yield of EPeFRET obtained by the claimed method with a purity of not less than 95% is not less than 4 mg / l of culture.

The invention is illustrated by drawings.

Figure 1. Physical map of the recombinant plasmid pQe PS-CFP2 / TurboYFP MBP7. BamHI, BglII, HindIII - restriction sites; T5 promoter - transcription promoter; PS-CFP2 is a sequence encoding a fluorescent cyan protein; YSP sequence encoding a fluorescent yellow protein, MBP7 is a sequence encoding the sequence of the main myelin protein 80-104.

Figure 2. Schematic diagram of the FRET approach to the study of the kinetics of abzymatic reaction. Below shows the change in the fluorescence spectrum when the biosensor is triggered.

Figure 3. Inhibition curves of abzymatic and enzymatic hydrolysis of EPeFRET with the Trx fusion protein with the OBM80-104 fragment (A), its mutant R97A (B), Sorakhone (C) and the carrier protein (D).

Figure 4. Schematic representation of the proportion of patients with various forms of PC (RR - remitting, PP / SP - progressive), patients with other neurodegenerative diseases (OND), as well as healthy donors whose antibodies showed catalytic activity measured by the EPeFRET biosensor.

The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA pQe30_PS-CFP2 / TurboYFP_MBP7. 2 genes of the PS-CFP2 and turboYFP proteins are integrated into the original pQe30 plasmid (Qiagen) using the Quick change method as described in (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK, and Pease, LR (1989 ) Gene 77 (1), 51-59).

First, standard PCR was performed from the original plasmids pPS-CFP2-C and pTurboYFP-C containing the PS-CFP2 and turboYFP genes, respectively, from the following primers:

Both PCR is carried out under the following conditions:

T = 94 ° C - 5 minutes

1 cycle

T = 94 ° C - 30 seconds

T = 60 ° C - 30 seconds

T = 72 ° C - 1 minute

25 cycles

A Taq / Pfu polymerase mixture (Fermentas) was used in a 10/1 ratio.

Next, PCR is carried out using previously obtained PCR products without using primers under the following conditions:

The composition of the reaction mixture:

PCR - product of the PS-CFP2 gene - 2 μl (0.75 μg DNA)

PCR - product of the turboYFP gene - 2 μl (0.75 μg DNA)

reaction buffer for herculase polymerase - 0.6 μl

high-performance polymerase - Herculase (Amersham) - 0.1 μl

deoxyribonucleotides - 0.6 μl (0.1 mm each)

water mQ - 0.7 μl

PCR mode:

T = 94 ° C - 5 minutes

1 cycle

T = 94 ° C - 30 seconds

T = 55 ° C - 30 seconds

T = 72 ° C - 15 minutes

5 cycles

Next, standard PCR is carried out using the PCR obtained in the previous one.

product as a matrix in a dilution of 1/50

T = 94 ° C - 5 minutes

1 cycle

T = 94 ° C - 30 seconds

T = 60 ° C - 30 seconds

T = 72 ° C - 1.5 minutes

25 cycles

Use a mixture of Taq / Pfu polymerases (Fermentas) in the ratio

10/1

The obtained PCR product and plasmid pQE30 are subjected to restriction using BamHI and HindIII enzymes with further ligation, the resulting construct is called pQe_PS-CFP2 / turboYFP_Bgl.

Next, the plasmid pQe_PS-CFP2 / twboYFP_Bgl is subjected to restriction using the BglII enzyme and further ligation with the oligonucleotide corresponding to the OBM80-104 fragment treated with preliminarily restriction endonucleases BglII and BclI. A construct containing an encephalitogenic fragment of MBP 80-104 was called pQe_PS-CFP2 / turboYFP_MBP7.

Example 2. The expression of fusion proteins is carried out in the E. coli strain BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 (producer of the hybrid protein PS-CFP2 / TurboYFP_MBP7 obtained by transformation of Escherichia coli strain BL21 (DE3) cells with the unique recombinant plasmid PSP-pQPPQPPQPPQQPPQPPQQP2PQF2 / TurboYFP_MBP7). In an undergrowth culture at 37 ° C after reaching an optical density of OD ₆₀₀ 0.7 units, the cell suspension is transferred to room temperature and incubated for 36 hours to mature the chromophore. The soluble protein fraction is prepared as follows. The supernatant is applied to Talon Superflow metal chelate sorbent previously equilibrated with buffer at a rate of 1 ml / min. Then the column is washed with 120 volumes of a solution containing 50 mm sodium phosphate, 300 mm NaCl, pH 7.0. Recombinant protein is eluted with a 50 mM EDTA solution at a rate of 0.5 ml / min. Next, gel filtration is performed on a Superdex 75 column in a PBS / TBS solution at a rate of 0.5 ml / min. The resulting protein solution is kept for a week at a temperature of 10 ° C with 0.05% sodium azide for the final maturation of the chromophore. Fluorescence measurements were carried out on a Saru Eclipse fluorometer (Varian) and a Tecan Genios (Tecan) plate reader at an excitation and emission wavelength of 405 and 538 nm, respectively.

All chromatographic isolation steps are carried out on devices for high-speed and high-performance liquid chromatography (FPLC, HPLC) firms Gilson and Waters (Millipore).

Isolation of polyclonal IgG antibodies from human and mouse sera is carried out according to the following scheme. Initially, the antibody fraction was precipitated with 50% ammonium sulfate for 24 hours. The reprecipitation procedure is repeated three times. The proteins thus obtained were diluted 1:10 with respect to the volume of the initial serum with PBS and applied at a rate of 0.5 ml / min on a HiTrap Protein-G Sepahrose column (Amresham, United States). The column is then washed with 100 volumes of application buffer at a rate of 2 ml / min to separate non-specifically adsorbed whey proteins. The class G immunoglobulin fraction was eluted from the column with a 100 mM glycine-HCl pH 2.6 solution and immediately neutralized with a 2 M Tris base solution to pH 7.5.

The isolation of MBP-binding antibodies is carried out by the method of affinity chromatography using the myelin basic protein immobilized on sepharose as a sorbent. For this, the total IgG fraction is applied to the immobilized MBP at a rate of 0.05 ml / min in PBS. The column is then washed with 90 volumes of application buffer at a rate of 2 ml / min. The elution of MBP-binding antibodies from the sorbent is carried out with a 100 mm solution of glycine-Hcl pH 2.6 and neutralized with a solution of Tris base to pH 7.5.

Identification of antibody preparations and assessment of their purity is carried out using the method of denaturing polyacrylamide gel electrophoresis followed by silver staining and enzyme-linked immunosorbent assay.

Kinetic measurements are carried out using various concentrations of EPeFRET (0.25 to 30 μM) in a solution of PBS for antibodies or TBS-CaCl ₂ in the case of enzymes. The acid buffer for cathepsin D is not applicable due to a sharp decrease in the yield of substrate fluorescence under such conditions. Final concentrations of antibodies, trypsin, and matrix-3 metalloprotease are 80, 40, and 30 nM, respectively. Cathepsin D is inactive at similar concentrations. Inhibition is carried out at a constant concentration of EPeFRET of 1 μM and a varying concentration of inhibitors from 0.001 to 100 μM in about an order of magnitude. The concentration of antibodies take 80 nm, trypsin and MMP-3 - 10 nm. The reaction is carried out in a volume of 120 μl in black 96-hole dies, the measurement is carried out at 405/535 nm, respectively.

The kinetic constants of the hydrolysis of EPeFRET by autoantibodies, trypsin and matrix metalloprotease-3, as well as the inhibition of this reaction by fusion proteins and Sorahone, a well-known drug for PC, which is a copolymer of lysine, glutamic acid, alanine and tyrosine, are presented in table 1 and figure 2 and 3.

Table 1 Type of biocatalyst Kinetic constants of hydropysis EPeFRET Inhibition of EPEFRET hydrolysis reaction (IC ₅₀ , μM) K _m (µM) k _cat sec ^-1 k _cat / K _m
(sec ^-1 M ^-1 ) OBM80-104 OBM80-104 R97A Trx Chorahon Trypsin (3.5 ± 0.4) · 10 ^-1 8.8 ± 1.5 (2.5 ± 0.7) · 10 ⁵ 3.1 ± 0.7 1.8 ± 0.6 2.9 ± 0.3 (2.0 ± 0.1) · 10 ¹ MMP-3 6.4 ± 0.8 (2.4 ± 1.0) 10 ^-1 (3.8 ± 1.8) · 10 ⁴ 8.2 ± 0.8 (1.2 ± 0.1) · 10 ¹ (1.5 ± 0.1) · 10 ¹ (2.0 ± 0.1) · 10 ¹ Abzyme 7.6 ± 0.9 (1.4 ± 0.2) · 10 ^-2 (1.8 ± 0.5) · 10 ³ (7.0 ± 2.0) · 10 ^-3 (9.0 ± 1.0) 10 ^-2 Not valid (1.0 ± 0.2) · 10 ^-1

The catalytic efficiency of the studied biocatalysts decreased by about an order of magnitude in the series of trypsin - MMP-3 - abzyme. The nature of the inhibition of EPeFRET cleavage by fusion proteins of thioredoxin with the OBM80-104 fragment, its mutant, carrier protein, and Sorachone is radically different in the case of catalysis by abzymes and model proteases. If, for catalytic antibodies, the ability to inhibit in the series Trx-OBM80-104, Trx-OBM80-104 (R97A), Sorakhone, Trx decreases from IC ₅₀ 7.0 · 10 ^-3 to> 100 μM, then for enzymes the calculated IC ₅₀ values are in the range from 3 to 20 μM, regardless of the type of inhibitor. The effective action of Sorakhone on trypsin is apparently due to the high (up to 29%) content of lysine residues in this copolymer.

Example 3. To characterize the substrate specificity of antibodies, FRET sensors are incubated with immunoglobulins (total IgG fraction) isolated from the blood serum of 26 patients with multiple sclerosis, 22 patients with other neurodegenerative diseases (OND), 11 healthy donors (healthy donors, HD) and SJL autoimmune mice with EAE induced MBP immunization. Frozen serum samples were provided by the Moscow Regional Research Institute (MONIKI) and the Clinical Hospital of the Russian Academy of Sciences. The catalytic and binding activity of autoantibodies was characterized for 26 patients with multiple sclerosis, 22 patients with other neurodegenerative diseases, and 11 healthy donors. The age of patients with PC ranged from 16 to 72 years (mean 39.5 years, interquartile value from 28 to 48 years). The EDSS parameter varied from 0 to 6.5 units (average 3.2 ± 1.8). The clinical diagnosis was verified in accordance with international criteria for Poser's classification (Poser, C.M. and V.V. Brinar, Diagnostic criteria for multiple sclerosis. Clin Neurol Neurosurg, 2001. 103 (1); p.1-11) and confirmed by laboratory studies. The cohorts of patients with other neurodegenerative diseases and healthy donors had an average age of 65 years (interquartiles from 39 to 72 years) and 33.5 years (interquartiles from 26 to 47 years), respectively. The necessary permits were obtained from each patient in accordance with the regulatory documents of the Ministry of Health of the Russian Federation. Statistical analysis was performed using the SPSS 13 program using the non-parametric Mann-Whitney U test and Spearman rank correlation.

SJL autoimmune mice were obtained from Harlan nursery (UK) and kept in a vivarium of the Institute of Bioorganic Chemistry RAS branch in Pushchino under sterile conditions that minimize contact of the immune system with external antigens (Specific Pathogen-Free status). The age of the mice used in the experiments varies from 5 weeks to 12 months. EAE induction in model mice is carried out according to the standard method {Current protocols on CD. [cited; computer laser optical discs], Coligan, J.E. and National Institutes of Health (U.S.), Current protocols in immunology. 1992, New York: Greene Pub. Associates and Wiley-Interscience. 5 v.). Mice aged 6 to 8 weeks are immunized with an emulsion of 50 μg antigen (MBP bull) in complete Freund's adjuvant containing 4 mg / ml heat inactivated M. tuberculosis. Immunization is carried out twice with a weekly interval. The day before and two after the first immunization, 400 ng pertussis toxin is intraperitoneally administered to mice. Mice are monitored for the development of EAE daily, blood collection is done by decapitation in 2-3 weeks.

The catalytic properties were exhibited by antibodies isolated from 77% of patients with progressive and 85% with remitting type of PC. Among patients with neurodegenerative diseases and healthy donors, the proportion of positive catalytic response is 9% and 0%, respectively (figure 4). The exception is two cases of osteochondrosis, in which the presence of catalytic degradation of the OBM peptide under the influence of autoantibodies was noted. Moreover, these patients are characterized by a significant duration of the pathological process and serious neuronal lesions. The specificity of hydrolysis is controlled by the degradation of recombinant proteins by bovine trypsin, cathepsin D and matrix metalloprotease 3 (MMP-3).

Claims (3)

1. 4916 bp recombinant plasmid DNA pQe30_PS-CFP2 / TurboYFP_MBP7 encoding the PS-CFP2 / TurboYFP_MBP7 fusion protein containing a sequence of PS-CFP2 and Turbo YFP proteins linked by a myelin 80-104 basic protein fragment and having the SEQ amino acid sequence ID NO: 1, where the recombinant plasmid DNA has a physical map shown in figure 1, and consists of:
a DNA fragment containing a T5 RNA polymerase transcription promoter, a ribosome binding site, and a PS-CFP2 / TurboYFP_MBP7 fusion protein sequence;
a pQe30 plasmid DNA fragment containing the transcriptional terminator of the T5 bacteriophage T5 RNA polymerase and a replication initiation site;
of the β-lactamase gene DNA fragment determining the resistance of ampicillin to Escicichin coli plasmid pQe30_PS-CFP2 / TurboYFP_MBP7 transformed by the plasmid as a genetic marker. 2. The strain Escherichia coli BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 is a producer of the hybrid protein PS-CFP2 / TurboYFP_MBP7 obtained by transforming cells of the Escherichia coli strain BL21 (DE3) recombinant plasmid DNA pQe30_PSBFFP1P1F1PPP1P1PFP1P1. 3. The method of producing protein PS-CFP2 / TurboYFP_MBP7, comprising culturing the producer strain Escherichia coli BL21 (DE3) / pQe30_PS-CFP2 / TurboYFP_MBP7 according to claim 2, cell disruption and protein purification PS-CFP2 / TurboYFP_MBP7 by affinity chromatography and gel-chromatography .

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