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

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

 

The present invention relates to the field of biotechnology, in particular genetic engineering, and can be used in microbiological industry for preparation of semisynthetic cephalosporin antibiotics of the new generation. Available:

recombinant DNA encoding a functionally active hybrid protein (DAOcbd), which consists of oxidase D-amino acids of strain Trigonopsis variabilis BKM Y-2601, merged with Chitinskaya domain of chitinase A1 of 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 strain of E. coli C41(DE3)/pVR1 producing a hybrid protein DAOcbd.

The level of technology

Oxidase D-amino acids (d-Amino acid oxidase, DAO; EC 1.4.3.3.), isolated from yeast .variabilis R.gracilis, industrial enzymes, widely used in the composition of the biocatalysts in the production of semisynthetic beta-lactam antibiotics of the new generation [1]. Due to the need for increased production of this now widely used group of antibiotics improve the known methods of obtaining the necessary for their synthesis enzymes, including using recombinant DNA technologies is of great practical interest.

To date, known recombinant the f plasmid DNA, directing the synthesis of functionally active enzyme DAO Trigonopsis variabilis [2, 3], Rhodotorula gracilis [4, 5] in bacteria [2-5] or yeast [6, 7]. Getting oxidase D-amino acids in heterologous bacterial systems presents a number of difficulties. First, overproduce DAO in the cell has an adverse effect on normal cells host strains of bacteria, since it is associated with release of toxic compounds - hydrogen peroxide, and also leads to suppression of the synthesis of the cell wall of bacteria [3, 4]. Secondly, the recombinant DAO used for biotransformation of cephalosporin antibiotics, should not contain impurities extraneous proteins, in particular beta-lactamase, which complicates the problem of the choice of vector designs for expressii protein and methods of cleaning. Therefore it is important for the development of new systems in the expression of DAO and simplify purification of the enzyme.

As one of the most effective methods of protein purification, today is affine chromatography [8], in recent years, widespread methods expression of recombinant protein products in the form of hybrid proteins containing auxiliary polypeptide specifically binding to a compound, which is used as a ligand in the subsequent protein purification by affinerie. The preparation of recombinant enzymes in the form of a hybrid protein is quite simple and effective method, which has, however, a significant drawback, which is that the modification of the primary structure of the enzyme protein upon binding with the auxiliary component may introduce unwanted changes in its conformation and consequently lead to a decrease or even loss of functional activity. In such situations, it may be possible subsequent cleavage of the hybrid protein (extra stage) on the components under the action of chemical or enzymatic agents. However, the introduction of the procedure of splitting the expressed hybrid protein requires subsequent additional purification. As a result, these potential benefits, simplicity and efficiency are essentially reduced to zero.

Taking into account these features of the receipt of the enzymes in the form of a hybrid protein is more promising to search for combinations of target and auxiliary protein, in which the presence of an additional component in the preparation of the enzyme will not adversely affect the properties of the target product, and the Association intended for purification of the polypeptide of interest protein, in turn, will reduce its specification is eskay binding activity. Except that upon receipt of such a hybrid protein facilitates purification of the target product and ensures the preservation of its activity in the composition of the fused protein, with the right choice of the auxiliary component recombinant enzymes in the form of such hybrid constructions are fully prepared for use as immobilized drug if it is provided by process.

As for oxidase D-amino acids, attempts to carry out her expression in the form of a fused protein is essentially limited to the obtaining of similar bearing 6 additional N-terminal his-tag 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, in the present invention was tasked with designing a hybrid protein comprising oxidase D-amino acids, which stably retaining the full functional activity of both its components, as well as the development of vectors and expression systems for its receipt.

Disclosure of inventions

The task of obtaining any functionally active hybrid protein includes the following required steps:

a) design design the hybrid protein and the gene, assuming optimal from the point of view of preserving the functional activity of the combination of the target and auxiliary protein; b) obtaining and merging using methods of recombinant DNA nucleotide sequences encoding the components of the hybrid protein; C) selecting host cells and the creation of vector constructs for expression of the hybrid protein in a selected heterologous system; d) optimization of conditions for the production and purification of the hybrid protein.

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

1) constructed a recombinant DNA encoding a hybrid protein (DAOcbd), in which the sequence oxidase D-amino acids of strain Trigonopsis variabilis (TvDAO) merged with Chitinskaya domain of chitinase A1 of Bacillus circulans;

2) constructed recombinant plasmid pVR1 containing recombinant DNA encoding a hybrid protein, and provides a synthesis DAOcbd in cells of Escherichia coli with high yield;

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

The choice Chitinskaya on the exchange of chitinase A1 of Bacillus circulans (CBD) as an auxiliary polypeptide was due to several reasons: (a) used in this case for the purification of chitin is a natural compound, and is cheaper compared to many other media for affinity chromatography; b) the system is an enzyme-CBD-chitin, as a rule, increases the stability of the protein preparation (enzyme immobilized on chitin medium through chitinase domain can be used many times);) in many known cases of the use of Chitinskaya domains in combination with the active proteins they do not adversely impact on the enzymatic activity. Since the latter fact was not obvious in relation to specific enzyme (DAO), the level of enzyme activity DAO after merging with Chitinskaya domain was previously determined by experiment, which confirmed the correctness of the choice made.

The nucleotide sequence encoding chitinase domain of chitinase A1 of Bacillus circulans (SEQ ID No. 12), obtained from commercially available plasmids pTYB-4.

Complete coding sequence of a gene TvDAO (SEQ ID No. 6) was obtained from strain Trigonopsis variabilis BKM Y-2601 by PCR amplification of the gene region DAO using primers tv1 (SEQ ID No. 1) and tv2 (SEQ ID2), it is specific to the N - and C-terminal region, and then removing from the received sequence of the intron by PCR with primers tv3 (SEQ ID No. 4) and tv5 (SEQ ID No. 5).

For Paul the continuity, encoding a hybrid protein, was first constructed intermediate vector pETcbd (figure 1), which is a commercial vector rate (Novagen), containing in polylinker coding sequence Chitinskaya domain (cbd), which is then the restriction sites NcoI and XhoI was embedded sequence (SEQ ID No. 6), encoding TvDAO, with the formation of plasmid pETDAOcbd (figure 2), including the full sequence of the hybrid gene (SEQ ID No. 13) for protein TvDAOcbd

Recombinant plasmid pVR1 designed for expression TvDAOcbd, was obtained by sublimirovanny full sequence of the hybrid gene (SEQ ID No. 13) from the vector pETDAOcbd in our custom-designed intermediate plasmid pET-TetI, representing the vector RET (Novagen), in which the gene of resistance to ampicillin resistance gene replaced to tetracycline.

By transforming cells of Escherichia coli strain C41(DE3) [10] constructed a plasmid pVR1, selection and cultivation of clones transformed with high-level synthesis of functionally active hybrid protein derived recombinant strain C41(DE3)/pVR1 producing a hybrid protein consisting of TvDAO, merged with Chitinskaya domain of chitinase A1 of Bacillus circulans. Synthesis TvDAOcbd in the resulting recombinant strain is under cultivation in conventional selective media with the addition of the inducer isopropyl-D (IPTG) or lactose. The level of synthesis TvDAOcbd in strain C41(DE3)/pVR1 is about 100 mg/l under the title culture 1×109cells/ml and the specific activity of the enzyme in the composition of the obtained hybrid - 85 u/mg protein.

Thus, the present invention includes three objects.

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

A second object of the recombinant plasmid pVR1 for synthesis of a hybrid protein TvDAOcbd in Escherichia coli cells and consisting of a DNA fragment with the sequence SEQ ID No. 13, the coding called hybrid protein, and the NdeI/XhoI fragment of plasmid pET-TetI, obtained by replacing the gene of beta-lactamase in the vector RET gene for resistance to tetracycline (TetR) in the reverse orientation.

The third object is a recombinant Escherichia coli strain C41(DE3)/pVR1 producing functionally active hybrid protein TvDAOcbd.

A brief description of the figures.

Figure 1 - Physical and genetic map of the vector pETcbd. The indicated position of indicator restriction enzymes cut sites, sites of promoter and terminator RNA polymerase of phage T7 (T7-promoter and T7), the region of the beginning of replication (f1 origin), the gene of resistance to ampicillin (Bla Amp)gene CBD, patch, encoding exegetically tag (HIS tag).

Figure 2 - the Physical and the mental and genetic map of the vector pETDAOcbd. The position of the gene DAOcbd, other symbols as in figure 1.

Figure 3 - Physical and genetic map of the vector pVR1. The position of the gene of resistance to tetracycline (TetR), other symbols as in figure 1.

The implementation of the invention

When carrying out the invention, in addition to the methods disclosed in detail in the following examples used are well known in the art the techniques described in the manuals of molecular biology and genetic engineering [11, 12].

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

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

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

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

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

Mixture for PCR (50 ál):

5 ál of 10x Taq-SE PCR buffer (SibEnzyme);

5 ál of genomic DNA (200 ng/ál);

5 μl of 3 μm primer tv1;

5 μl of 3 μm primer tv2;

5 μl of 2.5 mm dNTP each type;

5 μl of deionized water;

1 μl of Taq-SE polymerase (5 u/ál, SibEnzyme).

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

After amplification, 5 µl of the PCR mixture is analyzed by electrophoresis in 1% agarose gel and identify homogeneous fragment size of about 1 TPN. Fragment isolated from the gel using the set Wizard PCR Preps Kit and subjected to automated sequencing using primers tv1, tv2. Its sequence is presented here as SEQ ID No. 3.

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

Used primers limit fragment of the DAO gene, including the beginning of the 2nd exon, located at position +125 sequence SEQ ID No. 3, and the termination codon TAG, located at position +1170 the same sequence, and add to this sequence initiation codon ATG broadcast and plot 8 encoding amino acids 1 exon. Primers are also sites for restricted NcoI and XhoI, missing in the coding sequence of the gene DAO. 1 ng obtained at the first stage PCR fragment is subjected to 25 cycles of PCR amplification with primers tv5/tv3. From agarose gel isolated fragment size of about 1 TPN and is sequenced. The nucleotide sequence of this fragment is represented in SEQ ID is 6.

Example 2. Obtaining a DNA sequence that encodes a hybrid protein, and containing the expression vector pVR1.

Gene hybrid protein DAOcbd was obtained by combining fragments encoding individual domains of the protein, as part of one plasmid construction.

2.1. Construction of intermediate plasmid pETcbd.

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

Received ligase mixture was used to transform competent cells of Escherichia coli strain XL1-Blue. From the obtained ampicillin-resistant transformants were isolated plasmid DNA, were selected positive clones by PCR screening with primers CBD_R and T7prom (SEQ ID No. 10) and were detected education PCR product size 268 BP

Several positive clones were checked by sequencing using primers T7prom and T7term (SEQ ID No. 11) and selected the clone with the insertion CBD fragment (SEQ ID No. 12), does not contain non-specific mutations. Plasmid contained in the cells of the selected clone was designated as pETcbd (Fig).

2.2. Obtaining plasmids pETDAOcbd.

In vector pETcbd the restriction sites NcoI/XhoI built a DNA fragment from the genome of TvDAO(SEQ ID No. 6). Selection of the desired clones was performed using restrictive analysis of preparations of plasmid DNA by the formation of fragment sizes 1266 and 3586 gel hydrolysis NdeI+XhoI. One of the selected vectors with a molecular mass 4852 P.N. identified as pETDAOcbd and used in further work. Inserting a gene of the hybrid protein in the received vector, sequenced with primers T7 prom, T7term, CBD_F and CBD_R to confirm the identity of the obtained gene sequence of the hybrid protein of the expected (SEQ ID No. 13).

2.3. The construction of the intermediate vector pET-TetR.

Plasmid rat was replaced with the gene of resistance to ampicillin resistance gene for tetracycline. "Insert", carrying the gene of resistance to tetracycline, was obtained from the plasmid pKRP12[13]. To do this, 2 μg of plasmid DNA restriction enzyme hydrolyzed in the > PST and was isolated from agarose gel, the fragment size of 1200 BP resulting fragment ligated with the hydrolyzed site > PST a plasmid RET in the reaction mixture containing 1 μl of the solution restricciones 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 5-fold leagues the EIT buffer (Gibco-BRL), 5 μl water and 1 μl T4 DNA ligase (1 u/ál, SibEnzyme). The mixture is incubated for 14 hours at 12°C, then warmed up for 15 min at 65°C, cooled in ice and 5 μl of the mixture was used to transform competent cells of Escherichia coli strain JM109 [14]. Selected tetracycline-resistant clones sensitive to ampicillin. Additional selection of the desired clones was performed using restrictive analysis of preparations of plasmid DNA by the formation of fragment sizes 1873, 2132, 2721 BP during the hydrolysis EcoRI+ > PST). In the selected clones was determined by the orientation of the insertion marker Tetrby simultaneous treatment with restrictase ClaI+XhoI. The formation of fragments with a size of 1.7 TPN and 5 TPN indicates a "straight" orientation of the insertion marker Tetrand fragments of size of 2.7 TPN and TPN 4 - about "reverse". A plasmid with a "straight" orientation marker was designated as pET-TetF, and a plasmid with the "reverse" orientation - as a pET-TetR. In future work, we used the vector pET-TetR.

2.4. Obtaining expression vector pVR1.

To obtain a vector expression of the hybrid protein DAOcbd of plasmids pETDAOcbd were isolated fragment NdeI/XhoI size 1200 mo and embed it in hydrolyzed by restitutae NdeI/XhoI vector pET-TetR. The obtained plasmid was designated as pVR1 (figure 3).

Example 3. Obtaining recombinant strain - producer of the hybrid protein TvDAO cbd.

Received recombi is based the plasmid pVR1 transformed strain E. coli C41(DE3) [10]. The selection of this strain as the recipient was made on the results of preliminary analysis that compared the productivity of the strains E. coli BL21(DE3) [F-, ompT, hsdSB(rIn-, mIn-), 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 with T7 promoter and improves the coupling between transcription and translation of target genes under the control of T7 promoter [10, 16].

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

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

On the basis of the obtained data was selected clone (No. 6), characterized best by the accumulation of biomass and the highest output functionally active enzyme (table 1), which was marked C41(DE3)/pVR1 and used to produce recombinant strain.

Table 1
Growth parameters of culture and activity oxidase from individual transformants of strain C41(DE3) with plasmid pVR1
Individual clonesThe growth of culture, A600The DAO activity, units/mg proteinOutput DAO, IU/ml
No. 1of 5.41511
No. 24,8119
No. 35,01210
No. 45,81715
No. 55,11618
No. 66,72021

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

Morphological features: the Cells have an elongated rod-like shape, when the division is not packouts.

Cultural characteristics:

Cells grow well on commonly used nutrient media. The generation time of about 60 min in liquid LB-medium. 2-2,5% nutrient agar "Difco" formed round, smooth, yellowish colonies with smooth edges. When grown in liquid LB and YT environments formed smooth intense turbidity.

Physiological and biochemical characteristics:

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

The level of synthesis DAOcbd (according to the definition of activity in the samples of biomass producer strain) is about 100 mg/l under the title culture 1×109cells/ml

Example 5. The hybrid protein purification and analysis activity immobilized on chitin medium TvDAOcbd.

The resulting biomass recombinant strain (5 g) is suspended in 20 ml of buffer a containing 100 mm sodium phosphate, pH 7.5, 5 mm 2-mercaptoethanol, 2 mm EDTA, 0.3% setitimer ammonium bromide (BECOMING), and was destroyed by ultravioletgaia. The supernatant was collected debris washed with buffer a and were discarded.

Ballast proteins from the supernatant precipitated with ammonium sulfate (30% saturation), recombinant DAOcbd of the supernatant 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 of chitin granules (2×5 cm). The column was washed with buffer B until the disappearance of the absorption at A280was taking an aliquot of the sorbent and used to determine the activity of the oxidase (α-Metacity method) and total protein. The stages of purification and immobilization are shown in Table 2.

Table 2
Purification and immobilization DAOcbd
Cleanup stepTotal protein (mg)The oxidase activity (µmol/min)Output %The degree of purification (fold)
General

(E)
Unit (U/mg)
Cell-free extract90516290181001
Sulfate-ammonium residue (60%)325consumed totals 13,03040802,2
Immobilization of chitin sorbent125105908565the 4.7
Note: experience has taken 10 g of wet biomass E.coli

Some us specific activity of the hybrid protein DAOcbd in an immobilized state is 85 u/mg protein, which essentially corresponds to the known from the literature indicators specific activity (95 u/mg) is not immobilized recombinant DAO, expressed in E. coli in the form of a simple (non-hybrid) protein [3, 7]. This allows us to conclude that created the design of the chimeric protein is functionally active and fully retains all the properties of both the components. Getting active DAO in the proposed combination with Chitinskaya domain facilitates purification of the target protein, and the result of the cleaning process is formed immobilized form of the enzyme, suitable for direct use in the process, and the subsequent "prishivki" to other media, in particular to further improve the stability and durability of the enzyme preparation.

1. Recombinant DNA which encodes a functionally active hybrid protein (DAOcbd), consisting of oxidase D-amino acids Trigonopsis variabilis and Chitinskaya domain of chitinase A1 of Bacillus circulans, and characterized by nucleotide sequence SEQ ID No. 13.

2. Recombinant plasmid pVR1 for synthesis of a hybrid protein TvDAOcbd in Escherichia coli cells and consisting of a DNA fragment representing the recombinant DNA according to claim 1 (SEQ ID No. 13)encoding a named hybrid protein, and the NdeI/XhoI fragment of plasmid pET-TetR obtained by replacing the gene of beta-lactamase in the vector RET gene for resistance to tetracycline (Tetr) in the reverse orientation.

3. Recombinant Escherichia coli strain C41(DE3)/pVR1 producing functionally active hybrid protein TvDAOcbd.



 

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3 cl, 6 dwg, 7 ex

FIELD: molecular biology, biochemistry, medicine, oncology.

SUBSTANCE: invention relates to DNA sequences found in analysis of mDNA from squamous carcinoma cellular lines of different origin wherein these DNA sequences represent transcripts from rearranged genes SCCA1 and SCCA2. Result of rearrangement is formation of fused gene consisting of exon 2-7 of gene SCCA1 and exon 8 of gene SCCA2, or exons 2-7 of gene SCCA2 and exon 8 of gene SCCA1. Prepared expressing vectors comprising above said combinations of exons of two genes provide synthesis of corresponding fused protein in host-cell. Proposed sequences of nucleic acids and genetic constructions based on thereof represent novel agents for diagnosis squamous carcinomas.

EFFECT: valuable biological and medicinal properties of transcripts.

8 cl, 9 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to isolated DNA encoding human peptide related to urocortin and peptide named as urocortin II. These peptides are relative with corticotropin-releasing factor and involves in mechanisms for initiation of hypophysis-suprarenal responses for the stress. Pharmaceutical composition comprising such peptide in combination with acceptable vehicle can be used in treatment of pathophysiological states, such as enhanced body temperature, appetite disorder, congestive cardiac insufficiency, stress, anxiety state and undesirable low levels of ACTH.

EFFECT: valuable medicinal properties of urocortin proteins.

33 cl, 27 dwg, 2 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: method involves determining complex formed between HCV anticore antigen and NS3/4a-antibody capable of recognizing both HCV- antigens and antibodies available in sample when using common solid base and solid substrates usable in immunoassay.

EFFECT: high accuracy and sensitivity of hepatitis C diagnosis.

47 cl, 8 dwg, 10 tbl

FIELD: biotechnology, molecular biology, medicine.

SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.

EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.

23 cl, 71 dwg, 12 tbl, 17 ex

FIELD: biotechnology, peptides, genetic engineering.

SUBSTANCE: invention relates to constructing nucleic acid molecule comprising functional in starch-containing plant tissues promoter, fragment encoding transit peptide for translocation of useful peptide into amyloplast, fragment encoding useful peptide, region encapsulating into starch and terminator. In insertion into plant genome DNA molecule expresses hybrid polypeptide comprising the desirable protein encapsulated into starch matrix. Prepared vegetable material can be used, for example, in manufacturing fodders for mammals, fishes and poultries. Also, invention can be used in food industry.

EFFECT: valuable properties of nucleic acid and polypeptides.

15 cl, 19 dwg, 9 tbl, 7 ex

FIELD: genetic engineering, pharmaceutical and medical-biological industry.

SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.

EFFECT: improved preparing method, valuable biological properties of polypeptide.

23 cl, 67 dwg, 1 tbl, 35 ex

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

SUBSTANCE: constructed is recombinant plasmid DNA pETTvDAO2 providing high level of DNA fragment which encodes D-amino acid oxydase of Trigonopsis variabilis yeast in Escherichia coli cells. As a result of transformation of E.coli strain with disclosed recombinant plasmid and selection of transformed clones new recombinant Escherichia coli strain C41(DE3)/pETTvDAO2 as producer D-amino acid oxydase is obtained.

EFFECT: improved method for producing of cephalosporin antibiotics.

2 cl, 4 dwg, 2 tbl, 7 ex

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