RU2529359C2 - RECOMBINANT PLASMID DNA pPA-OPRF-ETA CODING SYNTHESIS OF RECOMBINANT PROTEIN OPRF-ETA Pseudomonas aeruginosa, STRAIN Escherichia coli PA-OPRF-ETA PRODUCING RECOMBINANT PROTEIN OPRF-ETA Pseudomonas aeruginosa AND METHOD FOR PREPARING RECOMBINANT PROTEIN OPRF-ETA Pseudomonas aeruginosa - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology. The recombinant plasmid DNA pPA-OPRF-ETA having a size of 8007 base pairs codes the synthesis of recombinant protein OprF-ETA Pseudomonas aeruginosa. The plasmid contains the following fragments: Xba I-Hind III DNA fragment having a size 177 base pairs; Hind III-Hind III DNA fragment having a size of 1059 base pairs; Hind III-Xho I DNA fragment having a size of 1598 base pairs; Sph I-Hind III DNA fragment having a size of 425 base pairs; EcoR I - BamH I DNA fragment having a size of 57 base pairs. There are also presented the bacterial strain Escherichia coli PA-OPRF-ETA producing protein OprF-ETA and prepared by transforming the parent bacterial strain Escherichia coli BL21(DE3) of plasmid DNA pPA-OPRF-ETA and a method for preparing the above protein OprF-ETA.

EFFECT: group of inventions enables preparing the target product weighing approximately 100 kDa.

3 cl, 3 dwg, 1 tbl, 4 ex

Description

The invention relates to biotechnology, in particular to the production of a hybrid recombinant protein comprising the amino acid sequences of the outer membrane protein F and Pseudomonas aeruginosa exotoxin A (hereinafter OprF-ETA recombinant protein) for medical purposes, as well as to recombinant Escherichia coli strains (hereinafter E. coli) and plasmids for its production.

Pseudomonas aeruginosa (hereinafter P.aeruginosa) is one of the main causative agents of purulent-inflammatory diseases in humans, in particular, causes hospital infections. Pseudomonas infection is widespread due to its unpretentiousness in relation to nutrients and environmental conditions, as well as high resistance to chemotherapeutic agents and antibiotics.

Active immunization may be one of the effective means of protection against opportunistic microorganisms, in particular P. aeruginosa. To induce anti-Pseudomonas immunity, preparations based on inactivated bacteria, their components and exoproducts were developed in our country: polyvalent pseudomonas corpuscular vaccine [1-3], pioimmunogen [4-5], pseudomonas vaccine [6-9]. For various reasons, no drugs are currently registered for use in clinical practice, including surface Pseudomonas aeruginosa antigens.

For immunoprophylaxis and immunotherapy of Pseudomonas infections in the Research Institute of Vaccines and Serums named after Mechnikov, Ufa developed a drug based on exotoxin A P. aeruginosa - Pseudomonas aeruginosa toxoid [10-11]. But the release of this drug has been suspended due to commercial difficulties.

The difficulties in creating effective immunopreparations against Pseudomonas aeruginosa are that the pathogenesis of infections developing with its participation is due to the influence of a number of pathogenicity factors, among which it is difficult to single out the most significant for the development of vaccine preparations. During infection, when the pathogen penetrates the tissues of the macroorganism, the primary immune reactions are formed mainly on the surface antigens of the bacterial cell (components of the membrane and cell wall). When studying surface proteins, it was found that some proteins of the outer membrane (Opr) P.aeruginosa possess the greatest immunogenicity, which are responsible for intercellular transport and preservation of the structure of the bacterial membrane. The most immunologically studied major pore-forming protein F of the outer membrane (hereinafter OprF) with a molecular weight of 37.6 kDa, which has a high immunogenicity. These data led studies on the development of vaccines based on purified membrane proteins [12]. However, the preparation of such preparations is problematic, since the protein content of the outer membrane is negligible relative to the total proteins of the bacterial cell.

Exotoxin A is one of the main pathogenetic factors in the development of generalized forms of infections caused by P. aeruginosa [13, 14]. Exotoxin A has a molecular weight of about 70 kDa and is a secreted protein, consisting of a polypeptide chain of 613 amino acids in length, forming the following domains: N-terminal domain I (amino acid residues 1-252) necessary for interaction with the surface of target cells; domain II (amino acid residues 253-404) providing effective transfer of the toxin into the cytosol of cells; C-terminal domain III (amino acid residues 405-613), which is responsible for cytotoxic function [15]. The mechanism of this process is as follows: exotoxin A recognizes and interacts with the a2-macroglobulin receptor, also called a protein related to the low density lipoprotein receptor (LRP1) or CD91 present on epithelial and antigen-presenting cells. After binding to the receptor, the toxin enters the cell via receptor-mediated endocytosis, where domain II is cleaved in the REDLK region using a furin protease. Domain III, which has toxic activity, catalyzes the inactivation of eukaryotic elongation factor 2 (eEF-2) using ADP-ribosylation, which leads to inhibition of protein synthesis and cell death [16]. To protect the body from damage by exotoxin A, it is possible to use preparations of toxoid, that is, a purified native toxin chemically treated to eliminate toxicity and preserve immunogenicity. Domestic preparations based on exotoxin A created for immunotherapy and immunoprophylaxis are atoxic forms of protein obtained by purification of P.aeruginosa culture toxin-containing medium, followed by detoxification [17–20]. It was shown that after their use in the human and animal body, the formation of protective antibodies occurred. However, the complexity of the technological stages of obtaining, cleaning, and neutralizing, as well as the non-standard methods for assessing the specific activity of drugs, determine the feasibility of creating vaccines based on recombinant toxoid, as well as developing adequate standard methods for assessing the specific activity of the drug.

Recently, when creating vaccine preparations, much attention has been paid to the use of genetic engineering methods, the essence of which is to integrate the genes responsible for the synthesis of individual protective antigens in pathogens into the genome of a non-pathogenic microorganism (for example, E. coli or Saccharomyces cerevisiae). During the cultivation of the obtained producers, the target products (recombinant proteins) are synthesized in large quantities, which are subjected to simple purification and can be used to prepare the vaccine. This strategy allows you to isolate and use the most immunogenic components of the bacterium. At the same time, the vaccine production process excludes contact with cultures of pathogenic microorganisms, which is a significant technological advantage that ensures personnel safety.

To date, there is evidence of successful use in vaccine preparations created using recombinant proteins, including the amino acid sequences of P. aeruginosa outer membrane proteins. In Germany, a hybrid protein was obtained consisting of 190-342 amino acid residues of OprF and 21-83 amino acid residues of Oprl. To obtain the producer of this protein, the parent strain E. coli K-12 and the vector plasmid pGEX were used. The recombinant protein had an additional histidine sequence at the N-terminus and was purified by affinity chromatography on a nickel-activated sorbent. The resulting preparation was immunogenic and contributed to the protection of burn patients from sepsis caused by Pseudomonas aeruginosa [21].

Earlier, we obtained two recombinant forms of the P. aeruginosa OprF protein (full-size and C-terminal regions, including 192-342 amino acid residues). It was shown that these proteins had immunogenic properties, their serum had a bacteriostatic effect, which was shown in vitro. Recombinant forms of the OprF protein, as well as their immune sera, contributed to the protection of mice from experimental infection caused by P. aeruginosa [22]. In order to develop a drug designed to stimulate antitoxic immunity, that is, to neutralize such a significant pathogenicity factor as exotoxin A, we obtained recombinant exotoxin A [23] and its atoxic forms [24].

An object of the invention is to obtain a recombinant plasmid DNA pPA-OPRF-ETA encoding a hybrid recombinant protein comprising the amino acid sequences of the F membrane protein and exotoxin A Pseudomonas aeruginosa (OprF-ETA), and the producer strain of the recombinant protein OprF-ETA using E. coli strain BL21 (DE3).

This problem was solved by the creation of recombinant plasmid DNA pRA-OPRF-ETA and E. coli strain PA-OPRF-ETA.

Plasmid pPA-OPRF-ETA has 8007 base pairs and is characterized by the presence of the following fragments:

Hind III DNA fragment 1059 base pairs in size containing the P. aeruginosa oprF gene sequence.

Hind III - Xho I - DNA fragment with a size of 1598 base pairs, encoding the first two and part of the third domain of P. aeruginosa exotoxin A.

Xba I - Hind III - a 177 bp DNA fragment containing the sequence of the ribosome landing site and the sequences encoding the start codon and six histidines.

Sph I - Hind III - DNA fragment 425 base pairs in size containing the modified T7 bacteriophage promoter, lactose operon, ribosome landing site sequence and sequences encoding a start codon and six histidines.

EcoR I - BamH I DNA fragment with a size of 57 base pairs containing the ribosome landing site, the starting ATG codon and the sequence encoding six histidines.

Figure 1 shows the scheme of the plasmid pPA-OPRF-ETA.

Figure 2 shows the amino acid sequence of the recombinant protein OprF-ETA encoded in the plasmid pPA-OPRF-ETA.

Figure 3 presents the analysis of protein products obtained by expression in E. coli cells of sequences of recombinant genes located in the plasmid pPA-OPRF-ETA.

Table 1 presents data on the analysis of the protective properties of the recombinant protein OprF-ETA from a live virulent culture of P.aeruginosa strain RA 103.

The E. coli strain PA-OPRF-ETA was obtained by transforming cells of the parent strain of E. coli BL21 (DE3) using traditional genetic engineering technology.

Strain E. coli PA-OPRF-ETA is characterized by the following features:

Cultural and morphological features

The cells are small, straight, thickened, rod-shaped, gram-negative, non-spore.

Cells grow well on simple nutrient media. When growing on Difco agar, the colonies are round, smooth, convex, cloudy, shiny, gray, the edges are even. When growing in liquid media (on a minimal medium with glucose or LB broth) they form an intense smooth turbidity.

Physico-biological characteristics

Aerobe. The temperature range of growth is 4-42 ° C at optimum pH in the range of 6.5-7.5.

As a nitrogen source, the microbe uses both mineral salts in ammonium and nitrate forms, and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc.

The microbe uses amino acids, glycerin, carbohydrates as a carbon source. Cells are resistant to kanamycin (up to 100 μg / ml). Strain E. coli PA-OPRF-ETA - producer of the recombinant protein OprF-ETA. The method, conditions and composition of the medium for storage of the strain:

LB broth with 10% glycerol and antibiotics at -70 ° C in ampoules and in a lyophilized state in ampoules at 4 ° C.

The E. coli strain PA-OPRF-ETA was identified by the Bergi Identifier (1974) as a strain of the species E. coli.

A feature of the proposed method is the development of technology that allows to obtain complex highly purified P.aeruginosa antigen in a relatively simpler way and in a shorter time, compared with traditional methods for producing bacterial proteins. At the same time, the surface antigen necessary for stimulating immunity against a microbial cell and the toxoid necessary for stimulating immunity to neutralize exotoxin A are combined in one protein preparation. The combination of two antigens in one protein preparation contributes to a more unified method for their preparation.

The method includes the following steps.

Nutrient cultivation of E. coli strain PA-OPRF-ETA; Precipitation of biomass by centrifugation.

The destruction of the cells of the microorganism upon dissolution in denaturing buffer containing 8 M urea.

Purification by affinity chromatography on nickel-activated sorbents. Renaturation of purified recombinant toxoid by dialysis.

The optimal conditions for the individual stages of the production of recombinant toxoid are as follows.

The destruction of cells is carried out as a result of dissolution of biomass in an 8 M urea buffer solution.

Removal of insoluble components of the cell is carried out by centrifugation.

Renaturation is carried out at 4 ° C by stepwise dialysis against 50 mM Tris-HCl buffer solution (pH 9.0).

The yield of recombinant OprF-ETA protein as a result of applying the method in the optimal mode is at least 20 mg of purified recombinant OprF-ETA protein with 1 l of culture medium, the expression level of recombinant OprF-ETA protein is at least 30 mg with 1 l of culture medium.

The essence and advantages of the claimed group of inventions is illustrated by the following examples.

Example 1. Obtaining a recombinant plasmid DNA pPA-OPRF-ETA

The basis for the preparation of the plasmid pPA-OPRF-ETA DNA was the plasmid construct designed for expression of the recombinant atoxic form of exotoxin A P.aeruginosa [24]. This construct was plasmid pET28 (b) with the P. aeruginosa exotoxin A gene inserted at the Hind III and Xho I restriction sites with a deletion of the region encoding 106 C-terminal amino acid residues.

A plasmid construct designed for expression of recombinant OprF was used as a matrix for obtaining the oprF gene [22]. When amplifying the oprF gene with the aim of incorporating it into the construct with the toxoid gene, the following primer pair was used: 5'-GAT AAG CTT ATG AAA CTG AAG AAC ACCTTA G and 5'-CAT AAG CTT CTT GGC TTC AGC TTC TAC. The first primer corresponded to the beginning of the oprF gene, and the second was complementary to the end of the oprF gene. Both primers contained an additional Hind III restriction site (underlined) at the 5'-end.

The amplification process of the oprF gene consisted of the following stages: heating at 94 ° C for 3 min; 30 cycles of PCR (94 ° C - 1 min, 30 sec 65 ° C - 1 min, 72 ° C - 1 min) and incubation at 72 ° C for 2 min. PCR was performed under the following reaction conditions: 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, 1.5 mM MgCl, 0.120 mM dNTP, (pH 9.0). For 50 μl of the reaction mixture used 3 units. TAQ polymerase (Fermentas) and 0.1 μg of genomic DNA. The reaction was carried out in a Tertsik thermocycler. The amplification process consisted of the following stages: heating at 94 ° C for 3 min; 30 cycles of PCR (94 ° C - 1 min, 30 sec 65 ° C - 1 min, 72 ° C - 1 min) and incubation at 72 ° C for 2 min.

Amplified DNA was digested with restriction enzyme Hind ° III. At the same time, the plasmid construct designed for expression of the recombinant atoxic form of P. aeruginosa exotoxin A was processed with the same restriction enzyme.

Both restrictions were purified on an agarose gel and combined into a single construct in a ligation reaction. As a result, the recombinant plasmid pRA-OPRP-ETA was obtained (Fig. 1). The amino acid sequence of the recombinant protein obtained by translation of the inserted fusion genes in the plasmid pPA-OPRF-ETA (which was carried out in the OMIGA computer program) is shown in the second figure (Fig. 2).

Example 2. Obtaining a strain of E. coli PA-OPRF-ETA - producer of the recombinant protein OprF-ETA

The E. coli PA-OPRF-ETA producing strain was obtained by transforming the cells of the parent E. coli strain BL21 (DE3) with the recombinant plasmid pPA-OPRF-ETA, followed by selection of the recombinant clones in a medium containing kanamycin (50 μg / ml) at a temperature of 37 ° C.

The primary selection of recombinant plasmids was performed using restriction analysis and sequencing.

Further, the selection of producers was carried out according to the ability of the controlled synthesis of recombinant protein in cells. For this, 3 ml of LB medium containing kanamycin at a concentration of 50 μg per ml was inoculated with a bacterial colony. Grown in a thermostatically controlled shaker with an orbital rotation speed of 250 rpm for 12-16 hours and a temperature of 37 ° C. Then, 100 μl of the grown culture was added to 5 ml of fresh medium and incubated under previous conditions for 2–3 hours until the culture reached the active growth phase (0.6 optical units at a wavelength of 600 nm). Added 5 μl of a 1 M aqueous solution of a chemical inducer of isopropyl-P-d-thiogalactopyranoside (hereinafter IPTG). Incubation was continued under the previous conditions for 4 hours, after which the resulting biomass was besieged.

As a control, a producer bacterial culture incubated under the same conditions, but without the addition of IPTG, was used. Protein products were analyzed by polyacrylamide gel electrophoresis according to the Laemmli method.

As a result of expression of the pPA-OPRF-ETA construct in E. coli cells of strain BL21 (DE3), a synthesis of a recombinant product with a mass of about 100 kDa was observed (Fig. 3A). The estimated mass of the recombinant OprF-ETA protein was 105 kDa. The recombinant protein had an additional amino acid sequence at the N-terminus, including six histidine residues, which allowed them to be purified in Ni-Sepharose columns. The purified product turned out to be specific for immunoblotting by reaction with rabbit serum against the recombinant OprF protein and against the Pseudomonas toxoid, to the same extent as with the individual recombinant proteins against which the serum data were obtained (Fig. 3B and 3B).

As a control, a producer bacterial culture incubated under the same conditions, but without the addition of IPTG, was used. Protein products were analyzed by polyacrylamide gel electrophoresis according to the Laemmli method followed by Coomassie R-250 staining (Fig. 3A).

As a result of expression of the pPA-OPRF-ETA construct in E. coli cells of strain BL21 (DE3), a synthesis of a recombinant product with a mass of about 100 kDa (Track A3) was observed. The estimated mass of the recombinant OprF-ETA protein was 105 kDa. Protein products in the experimental tracks were compared with the proteins of the weight protein marker applied to track A1. At the same time, producer proteins obtained by growing biomass without induction of expression (i.e., without adding IPTG) did not contain a specific product (track A2). The recombinant protein had an additional amino acid sequence at the N-terminus, including six histidine residues, which allowed them to be purified in Ni-Sepharose columns (Track A4).

The immunological specificity of the recombinant proteins was evaluated in immunoblotting. Figure 3B shows the nitrocellulose membrane after immunoblotting with serum to recombinant OprF, and Figure 3B shows the nitrocellulose membrane after immunoblotting with serum to recombinant toxoid.

The purified product turned out to be specific for immunoblotting by reaction with rabbit serum against the recombinant OprF protein (Fig. 3B, track 6) and against the Pseudomonas toxoid (Fig. 3B, track 8), to the same extent as with individual recombinant proteins (positive controls in immunoblotting) against which serum data were obtained. The purified recombinant OprF protein was loaded in the fifth track (Fig. 3B, track 5), and the purified recombinant toxoid in the seventh track (Fig. 3B, track 7).

Example 3. Obtaining recombinant protein OprF-ETA from E. coli strain PA-OPRF-ETA

Obtaining recombinant protein OprF-ETA was carried out in 4 stages:

Stage 1. Cultivation of E. coli strain PA-OPRF-ETA with induction of expression of the recombinant gene.

2 stage. Obtaining cell lysate of E. coli strain PA-OPRF-ETA after expression of the recombinant gene.

3 stage. Chromatographic purification of recombinant OprF-ETA protein.

4th stage. Dissolution and renaturation of the recombinant OprF-ETA protein.

Cultivation of E. coli strain PA-OPRF-ETA with induction of expression of the recombinant gene.

Inoculated E. coli strain PA-OPRF-ETA in 500 ml of LB medium containing kanamycin at a concentration of 50 μg per ml. Cultivation was carried out for 12 h at a temperature of 37 ° C in a thermostatically controlled shaker with an orbital rotation speed of 250 rpm.

The resulting culture was aseptically introduced into a laboratory fermenter with 10 L of sterile LB medium containing kanamycin at a concentration of 50 μg per ml. Cultivation in the fermenter was carried out at a temperature of 37 ° C, a dissolved oxygen concentration of 30%, a stirrer revolutions of 300 rpm.

When the culture density reached 0.6 optical units at a wavelength of 600 nm, 10 ml of a 1 M IPTG aqueous solution was introduced into the fermenter. Incubation was continued under previous conditions for 4 hours.

Obtaining cell lysate of E. coli strain PA-OPRF-ETA after expression of the recombinant gene.

Biomass was besieged in a Beckman J2-21 centrifuge (JA-14 rotor) at a rotation speed of 3000 rpm for 10 minutes. The precipitate was collected and dissolved in one liter of the following solution: 8 M urea; 0.1 M NaH ₂ PO ₄ ; 0.01 M Tris-HCl; pH is 8.0. The biomass was dissolved in an orbital shaker at a rotation of 250 rpm for 12 hours at room temperature.

Then the lysate was freed from undissolved cell fragments in a Beckman J2-21 centrifuge (JA-14 rotor) with a rotation speed of 15,000 rpm at room temperature for one hour. The supernatant was transferred to a clean, sterile flask.

Chromatographic purification of recombinant OprF-ETA protein.

To the clarified lysate, 20 ml of a suspension of Ni-agarose (QIAGEN) was added and incubated for 1 hour at room temperature in an orbital shaker with gentle shaking (40 rpm).

The suspension was passed through five stages through a chromatography column (Bio-Rad) with a volume of 250 ml and a diameter of 5 cm in five steps. As a result, the lysate liquid phase was removed from the column and the sorbent with the bound recombinant protein remained.

Then two liters of washing solution of the following composition were passed through the column: 8 M urea; 0.1 M NaH ₂ PO ₄ ; 0.01 M Tris-HCl; pH 6.3.

To elute the recombinant protein, 15 ml of the first elution solution and ten times 15 ml of the second elution solution were passed through the column ten times, collecting each fraction in a separate tube. The elution solutions had the same composition as the washing solution, only the first elution solution had a pH of 5.9 and the second of 4.5.

Aliquots of the collected fractions were performed by polyacrylamide gel electrophoresis. After analysis of the results of electrophoresis, fractions containing purified protein were selected.

Dissolution and renaturation of the recombinant OprF-ETA protein

The purified recombinant protein was dissolved in 50 mM Tris-HCl (pH 9.0) using stepwise dialysis at 4 ° C and stirring the buffer on a magnetic stirrer. For this, fractions containing purified protein were pooled and transferred into a dialysis bag.

A tied dialysis bag was placed in a container containing 1 liter of the following solution: 6 M urea; 50 mM Tris-HCl; pH is 9.0. After two hours, the contents were alternately changed to buffer solutions containing 4, 2 and 1 M urea.

Then, a clean buffer solution (50 mM Tris-HCl, pH 9.0) was poured into the dialysis container. It was changed three times every two hours.

The protein content in the preparation was determined spectrophotometrically at a wavelength of 280 nm. When calculating the concentration of recombinant proteins, an extinction coefficient of 0.85 calculated using the OMIGA program was used.

Preparations of purified recombinant OprF-ETA protein were stored in 5 ml aliquots in sterile tubes at -70 ° C.

Example 4. Evaluation of the protective properties of the recombinant protein OprF-ETA

To assess the protective properties of the OprF-ETA recombinant protein, a live virulent culture of P. aeruginosa strain PA 103 was used, which is characterized by a high level of exotoxin synthesis and is similar to most strains found in the clinic.

The study of the recombinant protein OprF-aTox3 was carried out in comparison with individual proteins OprF and aTox3. Four groups of animals were formed for this purpose. The first group consisted of mice injected with 50 μg of the OprF-aTox3 fusion protein. The second and third groups included animals that received preparations of recombinant proteins separately (OprF and a Tox3). The fourth group, consisting of intact mice, was the control. Protein preparations were administered twice at a two-week interval. Two weeks after the course of immunization, mice were injected with different doses (from 12.5 to 200 million microbial cells) of a live virulent P.aeruginosa culture of the highly toxicogenic strain RA-103. The control group was injected from 6.25 to 100 million microbial cells (million MK). Counting of dead and surviving animals was carried out over the next seven days (table. 1). For the group of intact mice, LD ₅₀ corresponded to 30.8 million mk; in the group of animals immunized with toxoid - 61.6 million MK; OprF - 66 million sq.m .; fusion protein 93.3 million mk

The immunizing dose was 50 μg. In each group, 20 individuals were present. After a course of immunization, mice were injected with 5 to 160 million microbial cells of a live virulent culture of P. aeruginosa strain RA 103. Fallen animals were counted for five days (Table 2). LD ₅₀ in the group of intact mice (control) amounted to 12.3 million microbial cells. In the group of immunized mice, the LD ₅₀ value reached 32.5 million microbial cells. Thus, the performance index was 2.6.

The efficacy indices (IE) of the protective properties (ratio of LD ₅₀ for immunized mice to LD ₅₀ in the control group) of toxoid and OprF were 2.0 and 2.1. Immunization with the recombinant protein OprF-aTox3 showed an increase in protective properties. IE in this case was 3.0 (Table 1).

Information sources

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Table 1 Study of the protective properties of the recombinant protein OprF-ETA. Group of animals Dose of infection, million The number of mice that died / survived LD ₅₀ , million m / k IE Immunized OprF-ETA 200 8/2 93.3 3.0 one hundred 4/6 fifty 2/8 25 1/9 12.5 1/9 Immunized with toxoid (aTox3) 200 9/1 61.6 2 one hundred 7/3 fifty 3/7 25 2/8 12.5 1/9 Immunized OprF 200 8/2 66 2.1 one hundred 6/4 fifty 4/6 25 2/8 12.5 1/9 Control (intact mice) one hundred 9/1 30.8 - fifty 7/3 25 4/6 12.5 2/8 6.25 0/10

Claims (3)

1. Recombinant plasmid DNA pPA-OPRF-ETA, encoding the synthesis of recombinant protein OprF-ETA Pseudomonas aeruginosa, size 8007 base pairs, containing: Xba I-Hind III DNA fragment size 177 base pairs, containing the sequence of the ribosome landing site and the sequence encoding the start codon and six histidines; Hind III-Hind III DNA fragment with a size of 1059 base pairs containing the sequence of the oprF gene P.aeruginosa; Hind III-Xho I DNA fragment with a size of 1598 base pairs, encoding the first two and part of the third domain of P. aeruginosa exotoxin A; Sph I-Hind III DNA fragment 425 bp in size containing the modified T7 bacteriophage promoter, lactose operon, ribosome landing site sequence and sequences encoding a start codon and six histidines; EcoR I - BamH I DNA fragment with a size of 57 base pairs containing the ribosome landing site, the ATG codon start codon and the sequence encoding six histidines, and the expression of this construct results in the synthesis of a recombinant product with a mass of about 100 kDa. 2. The bacterial strain Escherichia coli PA-OPRF-ETA is a producer of the recombinant protein OprF-ETA P. aeruginosa obtained by transformation of the parent strain of bacteria Escherichia coli BL21 (DE3) recombinant plasmid DNA pRA-OPRF-ETA. 3. A method of producing a recombinant OprF-ETA P.aeruginosa protein using the E. coli strain PA-OPRF-ETA according to claim 2, including culturing it in a nutrient medium, disrupting microorganism cells, purification of the obtained product using affinity chromatography on nickel-activated sorbents and dissolution with renaturation of the purified preparation under native conditions using stepwise dialysis.

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