Recombinant plasmid dna pqe30/derf2l coding protein derf2l of mite dermatophagoides farinae and bacterial strain escherichia coli m15/ pqe30/derf2l - producer of such protein

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a recombinant plasmid DNA pQE30/Derf2L and a bacterial strain Escherichia coli M15/pQE30/Derf2L. The characterised recombinant plasmid DNA codes protein Derf2L of a mite Dermatophagoides farinae and consists of BamHI/HindIII - a DNA fragment of a plasmid pQE30 and a DNA sequence coding BamHI/HindIII fragment including gene Derf2L Dermatophagoides farinae. The characterised bacterial strain Escherichia coli M15/pQE30/Derf2L, is produced by transformation of the cells Escherichia coli M15 by the recombinant plasmid DNA pQE30/Derf2L.

EFFECT: presented inventions can be applicable to develop a dust mite diagnosticum, as well as for conducting specific immune therapy.

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The invention relates to biotechnology, in particular genetic engineering, specifically, to obtain the protein Der f 2L of the mite Dermatophagoides farinae, which is one of the most important allergens. Protein Der f 2L can be used to create a test system for Allergy to ticks household dust, as well as for the development of drugs for the specific immunotherapy of Allergy to house dust mites.

Allergies (atopy) is a common disease in the world. From allergies of varying severity affects up to 35% of the population of developed countries. A feature of this disease is a genetic predisposition that leads to early manifestation of disease in childhood eczema with further progression to more severe forms such as seasonal or chronic rhinitis, sinusitis, asthma, atopic dermatitis, or a combination, called "atopic March". Atopy is an allergic immediate-type" and mediated rapid, immediately after the interaction of IgE to the allergen, the degranulation of mast cells on which surface immobilized molecules of IgE. Previously the treatment and relief of Allergy symptoms is absolutely necessary to stop the "atopic March".

The only relatively successful method of pathogenetic treatment of allergies is the specific immunotherapy (SIT), proposed more than 100 years ago. SIT is based on long-term (up to 3 years) subcutaneous injection of very small doses of allergens, which gradually leads to the development of low titers of IgG to these allergens, as well as to the induction of tolerance in T-cells and increase the number of T-regulators that suppress the response to this allergen (Hori, 2010; Ozdemir et al., 2010; Ozdemir et al., 2010). For SIEVES up to the present day use the extract of natural allergens, contains the set of all available causing the Allergy of the body proteins. With the development of genetic engineering technologies in the laboratories of many countries of the world is working on the cloning of genes of different proteins, members of the allergens. This work is complicated by the fact that the number of allergens is great, and the composition of each part of many proteins. In the world practice for the preparation of recombinant proteins are expressing different systems based on different types of organisms used as bioreactors (prokaryotic and eukaryotic). For producing allergens house dust mites using Escherichia coli (Der f I, Takahashi et al., 2000) the yeast Pichia pastoris (Der p 1, Jacques et al., 2002; Der f 1, Yasuhara et al., 2001), transgenic tobacco plants (Der p 1, Buertin et al., 2009), culture of mammalian cells (Der p 1, Massaer et al., 2001), the culture of insect cells (Der p 3, Wayne et al., 2005). It should be noted that so far not described technology in the teaching of recombinant protein Der f 2 mite Dermatophagoidesfarinae.

The invention solves the problem of obtaining recombinant protein Der f 2L mite Dermatophagoidesfarinae.

The problem is solved by constructing recombinant plasmid DNA pQE30/Derf2L that encodes a protein Der f 2L mite Dermatophagoides farinae (3902 BP), and the bacterial strain Escherichia coli M15/ pQE30/Derf2L - producer of the protein Der f 2L mite Dermatophagoides farinae, providing a synthesis of this protein with the level of expression of at least 5% of the total cellular protein.

Recombinant plasmid DNA pQE30/Derf2L encoding a protein Der f 2L mite Dermatophagoides farinae, characterized by the following features (Figure 1):

consists of 3902 P.O.;

encodes the amino acid sequence of Der f 2L D. farinae with 6 histidine residues HaN-end (170 amino acid residues, SEQ No. 5-6);

contains: the promoter of bacteriophage T5, the transcription terminator tobacteriophage lambda, the gene for β-lactamase, which determines the stability of the transformed plasmid pQE30/Derf2L cells to ampicillin, the site of initiation of ColE1 replication; unique recognition sites of restriction endonucleases, with the following coordinates: XhoI - 2, EcoRI - 89, BamIII - 146, HindIII - 629, NheI - 749, NdeI - 1841.

Expression of Der f 2L D. farinae in cells of E. coli is accompanied by accumulation of recombinant allergen in the bacterial cytoplasm as insoluble Taurus inclusion. For his selection, the use of denaturing agents. The presence of AMI is kislotno a sequence of six histidine residues facilitate purification of the recombinant protein on Ni-NTA agarose.

To obtain strain-producer of recombinant allergen Der f 2L D. farinae competent cells of Escherichia coli M15 transformed with recombinant plasmid pQE30/Derf2L.

The obtained Escherichia coli strain M15/pQE30/Derf2L characterized by the following features.

Morphological features. Cells are small rod-shaped, gram-negative, risperadone, 1×3 μm.

Cultural characteristics. During growth on solid medium LA colonies are round, smooth, translucent, shiny, grey, smooth edge, the diameter of the colonies 1-3 mm; the pasty consistency. Growth in LB liquid medium is characterized by a smooth blurred with the formation of light draught.

Physical and biochemical characteristics. Cells grow at temperatures 4-42°C (optimum 37°C) With optimum pH of 6.8 to 7.2. As the source of nitrogen used as a mineral salt in ammonium form, and organic compounds in the form of peptone, tryptone, yeast extract, amino acids. As the carbon source used amino acids, glycerol, carbohydrates.

Resistance to antibiotics. Cells are resistant to ampicillin (200 μg/ml), due to the presence of the plasmid pQE30/Derf2L gene beta-lactamase, and kanamycin (100 μg/ml), due to the presence of the strain in 5 Ml of the plasmid pREP4, carrying the gene for resistance to kanamycin and encodes a repressor of transcription.

The E. coli strain M15/pQE30/Der£2L provides the t inducible synthesis of recombinant allergen Der f 2L D. farinae in an amount not less than 5% of total cellular protein.

The obtained recombinant protein Der f 2L D. farinae can be used to develop test systems for the presence of IgE to the clamp household dust, as well as preparations for the SITH.

The invention is illustrated graphics.

Figure 1 shows the physical map of recombinant plasmid pQE30/Derf2L;

figure 2 - purification of recombinant Der f2L D. farinae using metal-affinity chromatography on Ni-NTA agarose under denaturing conditions (8M urea), the rate of elution of 0.5 ml/min Check the optical density of the eluate at 280 nm, the arrow indicates the peak corresponding to the protein yield Der f2L D. farinae;

figure 3 - electrophoregram lysates of cells of the producer strain E. coli M15/pQE30/Der£2L before and after induction (lanes 1-2), fractions of elution (lane 3-5), track M - protein molecular weight markers; the arrow indicates the protein Der f2L D. farinae (15% polyacrylamide gel).

1. Hori S. Developmental plasticity ofFoxp3+regulatory T cells. Curr Opin Immunol. 2010 Oct;22(5):575-82.

2. Gurung P, Kucaba TA, Schoenberger SP, Ferguson TA, Griffith TS. TRAIL-expressing CD8+ T cells mediate tolerance following soluble peptide-induced peripheral T cell deletion. J Leukoc Biol. 2010 Dec; 88(6):1217-25.

3. Ozdemir C, Akdis M, Akdis CA. T-cell response to allergens. Chem Immunol Allergy. 2010; 95:22-44.

4. Takahashi K, Takai T, Yasuhara T, Yuuki T, Ohtake Y, Yokota T, Okumura Y. Production ofenzymatically and immunologically active Der f 1 in Escherichia coli. Int Of ACS Allergy Immunol. 2000; 122(2):108-14.

5. Jacquet, A., M. Magi, H. Petry, A. Bollen. High-level expression of recombinant house dust mite allergen Dr p 1 in Pichia pastoris. Clin.& Exp.Allergy. 2002. 32: 1048-1053.

6. Yasuhara T., Takai T., Yuuki So, Okudair H., Okumuki Y. Cloning and Expression ofcDNA Encoding the Complete Prepro-Form of an Isoform of Der f 1, the Major Group 1 Allergen from House Dust Mite Dermatophagoides farinae Bioscience, Biotechnology, and Biochemistry. 2001; 65(3): 563-569.

7. Buertin D., H.Chabre, B.Olagnier, A.Didierlaurent, M.-N.Couret, D.Comeau, E.Wambre, H.Laparra, L.Van Overtvelt, F.Montandoni, T.Batard, V.Jonval, A.Lorphelin, C.Merle, C.Berrouet, L.Parry, V.Gomord, R.Van Ree, P.Moingeon. Production of native and modified recombinant Der p 1 molecules in tobacco plants. Clinical & Experimental Allergy. 2009; 39(5): 760-770.

8. Massaer, M. et al. High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage. Int. Arch. Allergy Immunol. 2001; 125: 32-43.

The invention is illustrated by the following examples.

Example 1. Obtaining the sequence of the gene encoding the protein Der f2L D. farinae.

Gene protein Der f2L amplified from cDNA obtained by reverse transcription of total RNA mites Dermatophagoides farinae, isolated using Trizol reagent (Invitrogen). In the first stage were used for amplification primers 1 (SEQ # 1) and 2 (SEQ No. 2). All PCR reaction mixture in a volume of 25 µl containing 10 pmol of primers, and reagents according to the Protocol of PCR with KARA HIFI polymerase (Kara Biosystems). Cycle amplification involves the denaturation of DNA at 95°C (1 min), annealing at 62°C (1 min) and elongation at 72°C (1 min). Just spend 30 cycles of the reaction. The reaction mixture is applied onto an agarose gel for separation by electrophoresis and the desired fragments cut out from the gel, and then the DNA extracted from the gel with p the power set QIAEX II Gel Extraction Kit (Qiagen). After amplification and purification the fragment of the clone in the vector pTZ57R/T using a set of reagents InsTAclone PCR cloning kit (Fermentas). After checking clones and determination of their nucleotide sequences from the clone carrying the correct gene protein Der f2L, conduct the second stage of amplification with primers 3 (SEQ No. 3) and 4 (SEQ No. 4), bearing the recognition sites of the restriction endonucleases BamHI/HindIII. After amplification, the received fragment of the clone in the vector pTZ57R/T using a set of reagents InsTAclone PCR Cloning Kit. This is followed by the selection of clones carrying the sequence of the gene encoding the gene of the protein Der f2L, flanked by restriction sites BamHI/HindIII.

Example 2. Construction of recombinant plasmid DNA pQE30/Derf2L (figure 1).

500 ng of plasmid DNA pTZ57RT/Derf2L containing gene Der f2L (SEQ No. 5), treated with restrictase BamHI and HindII (SibEnzyme) and from the resulting hydrolysate is isolated in a 2%agarose gel fragment length 483 BP, containing the gene Der f2L.

500 ng of plasmid DNA pQE30 treated with restrictase BamHI and HindII (SibEnzyme) and from the resulting hydrolysate is isolated in a 1%agarose gel vector DNA length 3419 BP

The obtained fragment and vector DNA are combined using a ligase reaction 10 ál of buffer for ligation (SibEnzyme)containing 1 unit of T4 DNA ligase. 1 μl of the reaction mixture used to transform 100 μl of competent cells XL-1 Blue. /10 cells used for transformation, plated on LB-agar containing 100 μg/ml ampicillin. From grown clones secrete the target plasmid DNA pQE30/Derf2L and analyze it by processing a set of restriction endonucleases HindIII and BamHI, followed by electrophoretic analysis of the lengths of restriction fragments in a 1.5% agarose gel.

Finally the structure of the recombinant DNA pQE30/Derf2L confirmed by determining the nucleotide sequence in the region of the embedded fragment containing the gene Der f2L D. farinae.

Example 3. Selection of recombinant protein Der f 2L D. farinae and determining the productivity of the producer strain Der f2L D. farinae.

Recombinant plasmid DNA pQE30/Derf2L transform competent cells of Escherichia coli 5 Ml (Qiagen). For this to 100 ál of competent cells, add 10 PG plasmid DNA pQE30/Derf2L, mixed well and incubated in ice for 30 minutes and Then the cells subjected to heat shock for 60 sec at 42°C, then placed on ice and incubated for 5 minutes Then to the cell suspension, add 900 ál environment SOB cells and incubated for 90 min at 37°C With good aeration. The cells are then seeded in 100 μl on Petri dishes with solid nutrient medium containing ampicillin (100 μg/ml) and kanamycin (50 μg/ml). The result producing strains Der f2L D. farinae.

For the recombinant protein Der f 2 in E.coli providerproperty the preparation of recombinant proteins. For preparative separation of proteins 1000 ml of LB medium containing 100 mg/ml ampicillin and 50 mg/ml kanamycin, inoculant 50 ml overnight culture of cells of the strain E. coli M15/pQE30/Derf2L. Induction spend 3 hours at a concentration of 1.5 mm IPTG. Cells are harvested by centrifugation for 30 min at 5000 rpm and the precipitate resuspended in 30 ml of buffer B (8M urea, 0.1m NaH2PO4, 0,01M Tris(hydroxymethyl)aminomethane, pH 8). In this buffer, the cells are lysed 30 min, conduct ultrasonic treatment (10 min) and the lysate centrifuged 10 min at 12000 rpm Conducting metal-affinity chromatography on a column with Ni-NTA agarose: column volume of 10 ml is applied to the lysate at a flow rate of 0.5 ml/min Column is washed with buffer to reduce the optical density to background values, then the buffer (composition same as that of the buffer, but pH 6.3) to remove the speaker is not specific associated protein. Recombinant protein elute with buffer E (the same composition as the buffer, but pH 4.5) (figure 2). The obtained fractions analyzed by electrophoresis in 15% SDS page. Gel paint Kumasi G-250 (figure 3). The chromatogram calculate the yield of recombinant protein. Protein Der f 2L is 5% of the total cellular protein (figure 2). The result was obtained 110 mg of protein Derf2L.

Example 4. Analysis of the binding of recombinant protein Der f 2L D. farinae by immunoglobulin E man.

For analysis svyazyvanie the recombinant proteins with IgE used vysokotochnoye serum of patients with allergies. Analysis was performed by the method of immunoassay according to the following procedure. In wells contributed recombinant allergens (5 μg/ml, 100 μl) and the extract from the ticks were incubated overnight at +4°C. were blocking with 2% solution of bovine serum albumin (BSA) in phosphate buffer (FB) for 1 hour at room temperature. To the wells were added dilutions (1/50, 1/250, 1/1250 and 1/6250) patients ' serum, and incubated 1 hour at room temperature. After washing CFT with 0,05% Tween-20 (FB-T), in holes made solution of antibodies against human IgE conjugated to horseradish peroxidase (Sigma), incubated 1 h at room temperature, washed and added to the TMB substrate (Sigma). After the staining reaction was stopped by adding 10% hydrochloric acid and perform the accounting results by measuring the optical density at a wavelength of 450 nm.

The results of the analysis of the binding of recombinant proteins, obtained in E. coli, and IgE are presented in table 1.

Table 1
Analysis of the binding of recombinant protein Der f2L with serum IgE Allergy sufferers.
Syvorotkina antibodies (RIDA AllergyScreen, R-Biopharm, Germany) The results of ELISA
D. pteronyssinusD. farinaeextract of house dust mitesrDerf2L (E. coli)
The experimental group of people with a positive reaction to extract mites
182342,99/a 4.749,66/a 4.9++
2203,69/3,04,10/3,0--
3237>100/6,0>100/6,0++++++
428350,99/5,051,40/5,0--
572727,68/4,326,29/4,2--
668653,99/5,052,35/5,0++ ++
7605>100/6,0>100/6,0++++

The results show that the recombinant protein Der f 2L D. farinae binds to IgE in sera of patients. Thus, the claimed technical solution allows to obtain allergen capable of interacting with immunoglobuline E man, with his level of bacterial synthesis is not less than 5% of the total cellular protein. This allows you to get in milligramme quantities of recombinant Der f 2L D. Farinae to create a test system for determining allergies to house dust mites, as well as the development of medications for the SITH allergies to house dust mites.

1. Recombinant plasmid DNA pQE30/Derf2L encoding a protein Der f 2L mite Dermatophagoides farinae (3902 BP), consisting of BamHI/HindIII - fragment DNA plasmids pQE30 length 3419 P.O. containing T5-promoter of bacteriophage T5, the transcription terminator tobacteriophage lambda, the gene for β-lactamase, which determines the stability of the transformed plasmid pQE30/Derf2L cells to ampicillin, the site of initiation of ColE1 replication; and an artificial DNA sequence encoding a BamHI/HindIII fragment length 483 BP containing the gene Der f2L Dermatophagoides farinae as SEQ No. 5, after the broadcast, giving the polypeptide chain is as SEQ No. 6; containing a unique recognition sites of restriction endonucleases, with the following coordinates: XhoI - 2, EcoRI - 89, BamHI - 146, HindIII - 629, NheI - 749, NdeI - 1841.

2. The bacterial strain Escherichia coli M15/pQE30/Derf2L producing protein Der f 2L mite Dermatophagoides farinae, obtained by transformation of competent cells of Escherichia coli M15 recombinant pQE30 plasmid DNA/Derf2L.



 

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EFFECT: invention enables to obtain a novel enzyme which is suitable for efficient production of lipids and fatty acids.

13 cl, 5 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns preparing a genetic construct providing a synthesis of p35d recombinant protein in Escherichia coli cells. There are presented: recombinant plasmid DNA pQE-p35d providing the synthesis of p35d recombinant protein of cowpox virus and containing in accordance with physical and genetic map presented on Fig. 2: pQE30 plasmid vector, a fragment coding MRGSHHHHHHG oligopeptice and a fragment of 17 base pairs, coding a fragment of p35 protein of cowpox virus within 1 to 239 amino acid residues (Fig.1a); Escherichia coli XL1Blue/pQE-p35d B-1252 bacterial strain that is a producer of p35d recombinant protein of cowpox virus, containing recombinant plasmid DNA pQE-p35d deposited in the Collection of Bacteria, Bacteriophages and Fungi of FBUN GNTs VB Vector, registration No. B-1252, and p35d recombinant protein of cowpox virus.

EFFECT: solutions may be used to engineer the test systems and to prepare orthopoxvirus split vaccines.

3 cl, 7 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: recombinant strains Rhodococcus erythropolis 37 p16-Ami RNCIM Ac-1937 and Rhodococcus erythropolis HX7 p16-Ami RNCIM Ac-1938 are constructed, constitutively producing the enzyme acylamidase with acylating activity. Also, the method of synthesis of N-substituted acrylamides is created, in particular N-isopropylacrylamide, and N-dimethylaminopropylacrylamide, using these strains as a biocatalyst.

EFFECT: invention enables to improve the efficiency of preparing N-substituted acrylamides.

3 cl, 1 tbl, 1 dwg, 9 ex

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