RU2310688C1 - RECOMBINANT DNA ENCODING FUNCTIONALLY ACTIVE HYBRID PROTEIN OF D-AMINO ACID OXYDASE WITH CHITIN-COUPLING DOMAIN (DAOcbd), pVR1 RECOMBINANT PLASMID PROVIDING SYNTHESIS THEREOF IN Escherichia coli CELLS AND RECOMBINANT Escherichia coli STRAIN C 41 (DE3)/pVR1 AS PRODUCER OF DAOcbd - Google Patents

Abstract

FIELD: biology, in particular gene engineering, microbiological industry, production of new lactam antibiotics.

SUBSTANCE: constructed is recombinant plasmid of pVR1 expression containing DNA fragment which encodes functionally active hybrid protein (DAOcbd), comprising D-amino acid oxydase of Trigonopsis variabilis BKM Y-2601 strain and chitin-coupling domain of A1 Bacillus circulans domain. As a result of transformation of E.coli strain with disclosed recombinant plasmid and selection of transformed clones new Escherichia coli strain C 41 (DE3)/pVR1 as producer of DAOcbd providing high yield of reconbinant enzyme is obtained. Said active DAO in combination with chitin-coupling domain enables purification of target protein wherein in purification process immobilized enzyme form is obtained.

EFFECT: improved method for purification of target proteins.

3 cl, 3 dwg, 2 tbl, 5 ex

Description

The present invention relates to the field of biotechnology, in particular to genetic engineering, and can be used in the microbiological industry in the production of a new generation of semi-synthetic cephalosporin antibiotics. It is offered:

- recombinant DNA encoding a functionally active fusion protein (DAOcbd), which consists of the D-amino acid oxidase of the Trigonopsis variabilis strain BKM Y-2601 fused to the chitin-binding domain of chitinase A1 Bacillus circulans;

- recombinant plasmid pVR1 containing recombinant DNA encoding DAOcbd, and providing a high level of expression of the hybrid protein in E. coli cells;

- recombinant E. coli strain C41 (DE3) / pVR1 - producer of the DAOcbd fusion protein.

State of the art

D-amino acid oxidases (d-Amino acid oxidase, DAO; EC 1.4.3.3.), Isolated from T. variabilis R. gracilis yeast, are industrial enzymes widely used in biocatalysts in the production of new-generation semi-synthetic beta-lactam antibiotics [1 ]. Due to the need to increase the production volume of this widely used group of antibiotics, the improvement of known methods for producing enzymes necessary for their synthesis, including using recombinant DNA technologies, is of undoubted practical interest.

To date, recombinant plasmid DNAs are known that direct the synthesis of functionally active enzymes DAO Trigonopsis variabilis [2, 3], Rhodotorula gracilis [4, 5] in bacterial cells [2-5] or yeast [6, 7]. Obtaining D-amino acid oxidases in heterologous bacterial systems is fraught with a number of difficulties. First, overproduction of DAO in a cell has an adverse effect on the cells of ordinary host bacterial strains, since it is associated with the release of a toxic compound, hydrogen peroxide, and also leads to a suppression of the synthesis of the bacterial cell wall [3, 4]. Secondly, recombinant DAO preparations used for biotransformation of cephalosporin antibiotics should not contain impurities of extraneous proteins, in particular beta-lactamase, which complicates the problem of choosing vector constructs for protein expression and methods for its purification. In view of this, the task of developing new DAO expression systems and simplifying enzyme purification remains relevant.

Since one of the most effective methods of protein purification today is affinity chromatography [8], in recent years, methods for expressing recombinant protein products in the form of hybrid proteins containing an auxiliary polypeptide that specifically binds to a compound that is used as a ligand for subsequent protein purification have been widely used. affinity chromatography. Obtaining recombinant enzymes in the form of hybrid proteins is a fairly simple and effective method, however, having a significant drawback, which is that modification of the primary structure of the enzyme protein when it is bound to an auxiliary component can introduce undesirable changes in its conformation and, accordingly, lead to a decrease or even loss of functional activity. In such situations, it may be possible to subsequently cleave the fusion protein (additional step) into constituents by chemical or enzymatic agents. However, the introduction of a cleavage procedure for the expressed fusion protein requires subsequent further purification. As a result, such possible advantages of the method as simplicity and efficiency are essentially reduced to zero.

Given these features of obtaining enzymes in the form of hybrid proteins, it seems more promising to search for such a combination of the target and auxiliary protein in which the presence of an additional component in the enzyme preparation does not adversely affect the properties of the target product, and the combination of the polypeptide for purification with the protein of interest in its in turn, will not lead to a decrease in its specific binding activity. In addition, upon receipt of such a hybrid protein, the purification of the target product is simplified and its activity in the composition of the fusion protein is fully preserved, with a successful choice of an auxiliary component, recombinant enzymes in the form of such a hybrid design are fully prepared for use as an immobilized preparation, if this is provided for by the technological process.

As for the D-amino acid oxidase, attempts to express it in the form of a fusion protein are essentially limited to obtaining an analogue carrying 6 additional N-terminal histidine residues, which are traditionally used for subsequent chromatographic purification of many recombinant proteins. However, it is known that the obtained protein was not stable and was prone to aggregation and inactivation [9]. Therefore, the present invention was tasked with constructing a hybrid protein comprising a D-amino acid oxidase in which the full functional activity of both its components would be stably preserved, as well as the development of expression vectors and expression systems for its production.

Disclosure of invention

The solution to the problem of obtaining any functionally active hybrid protein includes the following necessary steps:

a) the design design of the gene of the hybrid protein, suggesting an optimal combination of the target and auxiliary protein from the point of view of maintaining functional activity; b) obtaining and combining, using recombinant DNA methods, nucleotide sequences encoding components of a fusion protein; c) selection of host cells and creation of vector constructs for expression of the hybrid protein in a selected heterologous system; d) optimization of the conditions of production and purification of the hybrid protein.

The goal to obtain a functionally active hybrid protein based on DAO, suitable for use as an immobilized preparation, was achieved due to the fact that

1) a recombinant DNA encoding a fusion protein (DAOcbd) was constructed in which the D-amino acid sequence of the Trigonopsis variabilis strain (TvDAO) is fused to the chitin-binding domain of A1 of Bacillus circulans chitinase;

2) a recombinant plasmid pVR1 was constructed, containing recombinant DNA encoding a fusion protein and providing DAOcbd synthesis in E. coli cells in high yield;

3) as a result of transformation of cells of E. coli strain C41 (DE3) by expressing plasmid pVR1, a recombinant Escherichia coli strain C41 (DE3) / pVR1 was obtained, characterized by a high level of inducible synthesis and stable production of the DAOcbd fusion protein.

The choice of the chitin-binding domain of chitinase A1 Bacillus circulans (CBD) as an auxiliary polypeptide was determined by a number of reasons: a) the chitin used in this case for purification is a natural compound and is also cheaper than many other affinity chromatography carriers; b) the enzyme-CBD-chitin system, as a rule, increases the stability of the protein preparation (an enzyme immobilized on a chitin carrier through a chitin-binding domain can be used repeatedly); c) in many cases of the use of chitin-binding domains known to us in combination with active proteins, they did not adversely affect the enzymatic activity. Since the latter circumstance was not obvious in relation to a specific enzyme (DAO), the level of enzymatic activity of DAO after combining with a chitin-binding domain was previously determined experimentally, as a result of which the correctness of the choice was confirmed.

The nucleotide sequence encoding the chitin-binding domain of Bacillus circulans A1 chitinase (SEQ ID NO: 12) was obtained from the commercial plasmid pTYB-4.

The complete coding sequence of the TvDAO gene (SEQ ID No. 6) was obtained by us from the Trigonopsis variabilis BKM Y-2601 strain by PCR amplification of the DAO gene region using primers tv1 (SEQ ID No. 1) and tv2 (SEQ ID2) specific for its N- and C-terminal regions, and subsequent removal of the intron from the obtained sequence using PCR with primers tv3 (SEQ ID No. 4) and tv5 (SEQ ID No. 5).

To obtain a sequence encoding a fusion protein, the intermediate vector pETcbd (Fig. 1) was first constructed, which is a commercial vector pET23a (Novagen) containing, in the region of the polylinker, the coding sequence of the chitin binding domain (cbd), into which it then follows the restriction sites NcoI and XhoI a sequence was inserted (SEQ ID No. 6) encoding TvDAO to form the plasmid pETDAOcbd (FIG. 2) including the complete hybrid gene sequence (SEQ ID No. 13) for the TvDAOcbd protein

The recombinant plasmid pVR1, designed for the expression of TvDAOcbd, was obtained by subcloning the complete sequence of the hybrid gene (SEQ ID No. 13) from the vector pETDAOcbd into our constructed intermediate plasmid pET-TetI, which is the pET21 vector (Novagen), in which the ampicillin resistance gene is replaced tetracycline resistance genome.

By transforming cells of the Escherichia coli strain C41 (DE3) [10] with the constructed plasmid pVR1, selecting and culturing transformant clones with a high level of synthesis of a functionally active hybrid protein, the recombinant strain C41 (DE3) / pVR1, a producer of a hybrid protein consisting of TvDAO, was fused with the chitin-binding domain of chitinase A1 Bacillus circulans. The synthesis of TvDAOcbd in the resulting recombinant strain is carried out by cultivation on conventional selective media with the addition of an isopropyl-D-thiogalactoside inducer (IPTG) or lactose. The level of TvDAOcbd synthesis in strain C41 (DE3) / pVR1 is about 100 mg / l with a culture titer of 1 × 10 ⁹ cells / ml, and the specific activity of the enzyme in the resulting hybrid is 85 units / mg protein.

Thus, the present invention includes three objects.

The first object is recombinant DNA, which encodes a hybrid protein (DAOcbd), consisting of Trigonopsis variabilis D-amino acid oxidase and Bacillus circulans chitin-binding domain A1, and is characterized by the nucleotide sequence of SEQ ID No. 13.

The second object is the recombinant plasmid pVR1, which provides the synthesis of the TvDAOcbd fusion protein in Escherichia coli cells and consists of a DNA fragment with the sequence SEQ ID No. 13 encoding the fusion protein and an NdeI / XhoI fragment of the plasmid pET-TetI obtained by replacing the beta-lactamase gene in the vector pET23, the tetracycline resistance gene (TetR) in the reverse orientation.

The third object, a recombinant strain of Escherichia coli C41 (DE3) / pVR1, is a producer of the functionally active TvDAOcbd fusion protein.

A brief description of the figures.

Figure 1 - Physical and genetic maps of the vector pETcbd. The positions of the indicator restriction sites, the regions of the promoter and terminator of RNA polymerase of phage T7 (T7-promoter and T7), the region of origin of replication (f1 origin), the resistance gene for ampicillin (Bla Amp), the CBD gene, the site encoding the hexohistidine tag (HIS) are indicated tag).

Figure 2 - Physical and genetic maps of the vector pETDAOcbd. The position of the DAOcbd gene is indicated, the remaining designations are as in Fig. 1.

Figure 3 - Physical and genetic maps of the vector pVR1. The position of the tetracycline resistance gene (TetR) is indicated, the remaining designations are as in FIG. 1.

The implementation of the invention

In the implementation of the invention, in addition to the methods described in detail in the following examples, the methods well-known to those skilled in the art described in the manuals of molecular biology and genetic engineering were used [11, 12].

Example 1. PCR amplification of the DAO Trigonopsis variabilis gene sequence.

The primary structure of the DAO gene of Trigonopsis variabilis and its mRNA is known (access number in GenBank Z80895). This circumstance allows us to design the “forward” and “reverse” primers for PCR amplification of the desired gene region. In this case, the following oligonucleotides were used as primers:

5'-TGATAGGCAAATCCGTGCTC_-3 '"direct" tv1 primer (SEQ ID No. 1) and

5'-GTAAACACGTCGCAGTCGTC-3 '"reverse" tv2 primer (SEQ ID No. 2).

The genomic DNA of Trigonopsis variabilis BKM Y-2601, isolated by traditional methods, is denatured by heating at 100 ° C for 5 minutes, placed on ice and subjected to 30 PCR cycles.

Mix for PCR (50 μl):

5 μl of 10-fold Taq-SE PCR buffer (Sibenzyme);

5 μl of genomic DNA (200 ng / μl);

5 μl of 3 μM tv1 primer;

5 μl of 3 μM tv2 primer;

5 μl of 2.5 mm dNTP of each type;

25 μl of deionized water;

1 μl Taq-SE polymerase (5 u / μl, Sibenzyme).

Conditions for PCR: 94 °, 5 '(denaturation), 94 °, 30 "; 50 °, 30"; 72 °, 1 '(amplification).

After amplification, 5 μl of PCR mixture was analyzed by electrophoresis in 1% agarose gel and a homogeneous fragment of about 1 kb was detected. The fragment was isolated from the gel using the Wizard PCR Preps Kit and subjected to automatic sequencing using tv1, tv2 primers. Its sequence is presented here as SEQ ID No. 3.

Next, the intron is removed from the obtained DNA fragment, for which it is subjected to repeated PCR amplification with primers tv5 (SEQ ID No. 4) and tv3 (SEQ ID No. 5).

The primers used limit the DAO gene fragment, which includes the beginning of the 2nd exon located at position +125 of SEQ ID NO: 3 and the TAG termination codon located at position +1170 of the same sequence, and the ATG translation initiation codon and region are added to this sequence encoding 8 amino acids of the 1st exon. The primers also carry restriction enzyme sites NcoI and XhoI, which are absent in the coding sequence of the DAO gene. 1 ng of the PCR fragment obtained in the first step is subjected to 25 cycles of PCR amplification with tv5 / tv3 primers. A fragment of about 1 kb is isolated from the agarose gel and sequenced. The nucleotide sequence of this fragment is presented in SEQ ID No. 6.

Example 2. Obtaining a DNA sequence encoding a fusion protein, and containing its expression vector pVR1.

The DAOcbd fusion protein gene was obtained by combining fragments encoding individual protein domains in a single plasmid construct.

2.1. Construction of the intermediate plasmid pETcbd.

First, a plasmid was obtained bearing the CBD domain of B.circulans chitinase A1 under the control of the T7 phage promoter of pET23a vector. Plasmid pTYB4 (New England Biolabs) was used as the source of the CBD domain sequence. Using primers CBD_F (SEQ ID No. 8) and CBD_R (SEQ ID No. 9) on a pTYB4 plasmid matrix using Pfu polymerase, a unique 0.2 kb PCR fragment was obtained that was isolated from an agarose gel. 100 ng of the obtained fragment was hydrolyzed with 5 units of restriction enzymes NdeI and BamHI (Fermentas) and ligated with hydrolyzed NdeI / BamHI vector pET23a using T4 DNA ligase.

The competent ligase mixture was transformed with competent cells of the Escherichia coli strain XL1-Blue. Plasmid DNA was isolated from the obtained ampicillin-resistant transformants, “positive” clones were selected by PCR screening with primers CBD_R and T7prom (SEQ ID No. 10), and the formation of a 268 bp PCR product was detected.

Several “positive” clones were checked by sequencing using T7prom and T7term primers (SEQ ID No. 11) and a clone with the insertion of a CBD coding fragment (SEQ ID No. 12) containing no non-specific mutations was selected. The plasmid contained in the cells of the selected clone was designated as pETcbd (Figure 1).

2.2. Obtaining the plasmid pETDAOcbd.

A DNA fragment with the TvDAO gene (SEQ ID No. 6) was inserted into the pETcbd vector at the NcoI / XhoI restriction sites. The selection of the desired clones was carried out using restriction analysis of plasmid DNA preparations to form fragments of 1266 and 3586 bp in size. upon hydrolysis of NdeI + XhoI. One of the selected vectors with a molecular weight of 4852 bp designated as pETDAOcbd and used in further work. The insertion of the hybrid protein gene in the resulting vector was sequenced from T7 prom, T7term, CBD_F and CBD_R primers to confirm the identity of the obtained hybrid protein gene sequence expected (SEQ ID No. 13).

2.3. Construction of the intermediate vector pET-TetR.

In plasmid pET23, the ampicillin resistance gene was replaced by the tetracycline resistance gene. An “insert” carrying the tetracycline resistance gene was obtained from plasmid pKRP12 [13]. For this, 2 μg of plasmid DNA was hydrolyzed with restriction enzyme PstI and a 1200 bp fragment was isolated from agarose gel. The resulting fragment was ligated with the PETI site hydrolyzed plasmid pET23 in a reaction mixture containing 1 μl of a solution of a restricted vector with a concentration of 20 ng / μl, 1 μl of a solution of a fragment with a concentration of 50 ng / μl, 2 μl of a 5-fold ligase buffer (Gibco-BRL) 5 μl of water and 1 μl of T4 DNA ligase (1 unit / μl, Sibenzyme). The mixture was incubated for 14 hours at 12 ° C, then heated for 15 min at 65 ° C, cooled in ice, and 5 μl of the mixture was used to transform competent cells of the Escherichia coli strain JM109 [14]. Tetracycline resistant ampicillin sensitive clones were selected. Additional selection of the desired clones was carried out using restriction analysis of plasmid DNA preparations (by the formation of fragments of 1873, 2132, 2721 bp in size during EcoRI + PstI hydrolysis). In the selected clones, the “orientation” of the Tet ^r marker insert was determined by simultaneous treatment with restriction enzymes ClaI + XhoI. The formation of 1.7 TPN and 5 TPN fragments indicates a “direct” orientation of the Tet ^r marker insert, and 2.7 TPN and 4 TPN fragments indicate a “reverse” orientation. A plasmid with a “direct” marker orientation was designated as pET-TetF, and a plasmid with a “reverse” orientation was designated as pET-TetR. In further work, the vector pET-TetR was used.

2.4. Obtaining the expression vector pVR1.

To obtain the expression vector for the DAOcbd fusion protein from the plasmid pETDAOcbd, a 1200 bp NdeI / XhoI fragment was isolated and inserted into the NETI / XhoI restriction enzyme-digested pET-TetR vector. The resulting plasmid was designated as pVR1 (FIG. 3).

Example 3. Obtaining a recombinant strain producing a hybrid protein TvDAO cbd.

(DE3)], BL21(DE3)/plysS [15] и C41(DE3). Штамм C41(DE3) является производным от BL21(DE3) и несет дополнительную мутацию, снижающую уровень транскрипции с Т7 промотора и улучшающую сопряжение между транскрипцией и трансляцией целевых генов, находящихся под контролем Т7 промотора [10, 16].The obtained recombinant plasmid pVR1 transformed the E. coli strain C41 (DE3) [10]. The choice of this strain as a recipient was made according to the results of a preliminary analysis, which compared the productivity of strains of E. coli BL21 (DE3) [F-, ompT, hsdS _B (r _B- , m _B- ), dcm, gal,

(DE3)], BL21 (DE3) / plysS [15] and C41 (DE3). Strain C41 (DE3) is a derivative of BL21 (DE3) and carries an additional mutation that reduces the level of transcription from the T7 promoter and improves the conjugation between transcription and translation of target genes controlled by the T7 promoter [10, 16].

Separate clones of transformants were grown in 50 ml of medium at a temperature of 30 ° С and low aeration, optimal for DAO production [17]. After the culture reached a density of 1.6 OE (A ₆₀₀ ), the IPTG inducer was introduced to a final concentration of 1 mM and incubation continued for another 18 hours. At the end of cultivation, the growth parameters of the culture and enzyme activity in bacterial cells were determined, as described previously [17]. To obtain a coarse extract, the cell pellet was destroyed by ultrasound with periodic cooling in ice (4 times for 30 sec with an interval of 1 min) and the cell debris was separated by centrifugation (13000 rpm, 10 min).

50 μl of the resulting clarified lysate were incubated with 250 μl of 10 mM D-alanine in 50 mM phosphate buffer, pH 8.0, for 15 or 30 minutes, the reaction was stopped by adding 100 μl of a saturated solution of 2,4-dinitrophenylhydrazine in 2N HCl and after 5 min to the solution was added 300 μl of 3M NaOH and 2.5 ml of distilled water. After incubation for 10 min at room temperature, the absorbance of the solution was measured at 550 nm, which was compared with a calibration curve constructed using standard concentrations of pyruvic acid. The specific activity was expressed in units of the enzyme (μmol of the substrate for 1 min at 25 ° C) per mg of total protein of the clarified extract. The total protein content was determined by the Bradford method [11].

Based on the data obtained, a clone (No. 6) was selected, characterized by the best biomass accumulation and the highest yield of functionally active enzyme (Table 1), which was designated C41 (DE3) / pVR1 and used to obtain the recombinant strain.

Table 1 Growth parameters of culture and oxidase activity in individual transformants of strain C41 (DE3) with plasmid pVR1 Individual Clones Culture Growth, A600 DAO activity, units / mg protein Yield DAO, units / ml No. 1 5,4 fifteen eleven Number 2 4.8 eleven 9 Number 3 5,0 12 10 Number 4 5.8 17 fifteen Number 5 5.1 16 eighteen Number 6 6.7 twenty 21

Example 4. Characterization of the recombinant E. coli strain C41 (DE3) / pVR1

Morphological signs: Cells have an elongated rod-shaped shape, do not bud when dividing.

Cultural traits:

Cells grow well on commonly used culture media. The generation time is about 60 minutes in a liquid LB medium. On 2-2.5% nutrient agar "Difco" round, smooth, yellowish colonies with smooth edges are formed. When grown on liquid LB and YT media, intense even turbidity forms.

Physiological and biochemical signs:

The optimum cultivation temperature is from 25 to 30 ° C, the optimum pH is 7.6. The source of nitrogen is organic compounds (in the form of tryptone, yeast extract).

The DAOcbd synthesis level (according to the activity determination in the biomass samples of the producer strain) is about 100 mg / L with a culture titer of 1 × 10 ⁹ cells / ml.

Example 5. Purification of the hybrid protein and analysis of the activity of TvDAOcbd immobilized on a chitin carrier.

The resulting recombinant strain biomass (5 g) was suspended in 20 ml of buffer A containing 100 mM sodium phosphate, pH 7.5, 5 mM 2-mercaptoethanol, 2 mM EDTA, 0.3% cetyltrimethyl ammonium bromide (CTAB), and was destroyed by ultrasound disintegration. The supernatant was collected, debris was washed with buffer A and discarded.

Ballast proteins from the supernatant were precipitated with ammonium sulfate (30% saturation), recombinant DAOcbd from the supernatant was precipitated with ammonium sulfate (60% saturation). The precipitate was dissolved in 10 ml of buffer B (10 mm sodium pyrophosphate, pH 8, 5 mm 2-mercaptoethanol, 2 mm EDTA, 10% glycerol, 300 mm NaCl) and applied to a column with chitin granules (2 × 5 cm). The column was washed with buffer B until absorption disappeared on A ₂₈₀ , an aliquot of the sorbent was taken and used to determine oxidase activity (α-keto acid method) and total protein. The stages of purification and immobilization are shown in Table 2.

table 2 Purification and immobilization of DAOcbd Cleaning stage Total protein (mg) The activity of oxidase (μmol / min) Exit, % The degree of purification (times) Total
(E) Specific (E / mg) Cell-free extract 905 16290 eighteen one hundred one Ammonium sulphate precipitate (60%) 325 13030 40 80 2.2 Chitin Sorbent Immobilization 125 10590 85 65 4.7 Note: 10 g of wet E.coli biomass were taken in the experiment.

The specific activity of the DAOcbd fusion protein determined by us in the immobilized state is 85 u / mg protein, which essentially corresponds to the literature on the specific activity (95 u / mg) of the non-immobilized recombinant DAO expressed in E. coli as a simple (not hybrid) protein [3, 7]. This allows us to conclude that the created design of the chimeric protein is functionally active and fully preserves the properties of both its constituent components. The preparation of an active DAO in the proposed combination with a chitin-binding domain significantly facilitates the purification of the target protein, and as a result of the purification process, an immobilized form of the enzyme is formed, suitable both for direct use in the technological process and for subsequent “sewing” to other carriers, in particular for additional increase the stability and durability of the enzyme preparation.

Claims (3)

1. Recombinant DNA that encodes a functionally active fusion protein (DAOcbd), consisting of D-amino acid oxidase Trigonopsis variabilis and the chitin-binding domain of chitinase A1 Bacillus circulans, and is characterized by the nucleotide sequence of SEQ ID No. 13. 2. Recombinant plasmid pVR1, providing synthesis of the TvDAOcbd fusion protein in Escherichia coli cells and consisting of the DNA fragment representing the recombinant DNA according to claim 1 (SEQ ID No. 13) encoding the fusion protein and the NdeI / XhoI fragment of the pET-TetR plasmid obtained by replacing the beta-lactamase gene in the pET23 vector with the tetracycline resistance gene (Tet ^r ) in the reverse orientation. 3. Recombinant strain of Escherichia coli C41 (DE3) / pVR1-producer of a functionally active hybrid protein TvDAOcbd.

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