RU2537006C2 - RECOMBINANT PLASMID pPA-OPRFI DNA CODING HYBRID RECOMBINANT F-I PROTEIN OF Pseudomonas aeruginosa OUTER MEMBRANE, STRAIN Escherichia coli PA-OPRFI PRODUCING HYBRID RECOMBINANT F-I PROTEIN OF Pseudomonas aeruginosa OUTER MEMBRANE AND METHOD FOR PRODUCING HYBRID RECOMBINANT F-I PROTEIN OF Pseudomonas aeruginosa OUTER MEMBRANE - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns recombinant plasmid pPA-OPRFI DNA coding hybrid recombinant F-I protein of Pseudomonas aeruginosa outer membrane, of the bacterial strain E.coli PA-OPRFI producing this hybrid protein, and a method for producing this recombinant protein. The presented plasmid DNA contains a DNA fragment containing a sequence of modified promoter of bacteriophage T5 and two lactose operons; a DNA fragment containing a ribosome entry site, an initiation ATG-codon and a sequence coding six histidines; a DNA fragment containing the full-size sequences of oprF and opri genes of P.aeruginosa; a DNA fragment containing a ribosome entry site, and a DNA fragment containing a lambda t₀ transcription stop region.

EFFECT: presented inventions enables producing the hybrid recombinant F-I protein of Paeruginosa outer membrane for carrying out an immunobiological assay in developing a Pseudomonas aeruginosa vaccine, and also for producing donor immunoglobulins for therapy of active forms of Paeruginosa infection.

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Description

The invention relates to biotechnology, in particular to the production of a hybrid recombinant protein F-I of the outer membrane of Pseudomonas aeruginosa (hereinafter OprF-OprI) for medical purposes, as well as to recombinant strains of Escherichia coli (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 and, in particular, P. aeruginosa. To induce anti-Pseudomonas immunity in our country, drugs were developed based on inactivated bacteria, their components and exoproducts:

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 aeruginosa research institute of vaccines and serums named after Mechnikov, Ufa, a preparation based on P. aeruginosa exotoxin A - Pseudomonas aeruginosa is developed [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 of the proteins of the outer membrane (outer protein - Opr) of 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 proteins F and I of the outer membrane (OprF and OprI). The first protein with a molecular weight of 37.6 kDa forms pores in the plasma membrane, and the second with a molecular weight of 8.8 kDa, which is a lipoprotein, has a structural function. It was shown that these proteins have the highest 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.

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.

An object of the invention is to obtain recombinant plasmid DNA pPA-OPRFI encoding the OprF and OprI proteins of P. aeruginosa and the producer strain of the hybrid recombinant protein P. aeruginosa F-I using E. coli K-12 strain.

This problem is solved by the creation of recombinant plasmid DNA pPA-OPRFI and E. coli strain PA-OPRFI.

Plasmid pPA-OPRFI has 4757 base pairs, characterized by the presence of the following fragments:

Xba I - EcoR I of a 87 bp DNA fragment containing the sequence of the modified T5 bacteriophage promoter and two lactose operons.

EcoR I - BamH I DNA fragment of 57 bp containing the ribosome landing site, the start ATG codon and the sequence encoding six histidines (Arg-Gly-Ser-His-His-His-His-His-His-His-Gly-Ser) .

BamH I - Sad DNA fragment with a size of 1312 base pairs containing fused complete sequences of the oprF and oprI genes of P. aeruginosa.

EcoR I - Sac I DNA fragment of 1369 bp containing the ribosome landing site and the sequence encoding OprF-OprI (consists of a histidine sequence, an OprF protein and an OprI protein).

Hind III - NhE I DNA fragment with a size of 1116 base pairs containing lambda t ₀ region stop transcription.

Plasmid pPA-OPRFI also includes the rrnBT1 transcriptional stop region, the ColEl plasmid replication initiation site, and the gene encoding ampicillin resistance.

Figure 1 shows the scheme of the plasmid pPA-OPRFI.

Figure 2 shows the amino acid sequence OprF-OprI encoded in the plasmid pPA-OPRFI.

Strain E. coli PA-OPRFI obtained by transformation of cells of the parent strain of E. coli K-12 containing the plasmid pREP4, using traditional genetic engineering technology.

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

Cultural and morphological characters.

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 signs.

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

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

Amino acids, glycerin, carbohydrates are used as a carbon source.

Antibiotic resistance. Cells are resistant to ampicillin (up to 200 μg / ml) and to kanamycin (up to 100 μg / ml).

Strain E. coli PA-OPRFI - producer of OprF-OprI.

The method, conditions and composition of the medium for storage of the strain:

L-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-OPRFI 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 you to obtain highly purified protein OprF-OprI, consisting of full-sized highly immunogenic amino acid sequences of membrane proteins OprF and OprI P. aeruginosa.

The method includes the following steps:

Culture in a medium of E. coli strain PA-OPRFI;

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 OprF-OprI using dialysis.

Assessment of the protective properties of purified OprF-OprI.

The optimal conditions for the individual stages of the production of recombinant OprF-OprI are as follows:

Cell destruction 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 a temperature of 4 ° C by stepwise dialysis against 50 mm Tris-HCl buffer solution (pH 9.0).

The yield of recombinant OprF-OprI as a result of applying the method in the optimal mode is at least 40 mg of purified OprF with 1 l of culture medium, the expression level of OprF-OprI is at least 100 mg with 1 l of culture medium.

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

Example 1. Obtaining recombinant plasmids pPA-OPRFI.

The technology for plasmid pPA-OPRFI includes the following steps:

Genomic DNA Isolation,

Amplification of OprF and oprIP genes. aeruginosa using PCR,

Insertion of amplified sequences into a plasmid vector.

Any strain of Pseudomonas aeruginosa can be used to isolate genomic DNA.

The P. aeruginosa cell culture was stored in a lyophilized state. To obtain a live culture, the ampoule was opened and 1 ml of Mueller-Hinton broth was added (casein acid hydrolyzate - 17.5 g / l, beef extract - 300.0 g / l, soluble starch - 1.5 g / l). The suspension was transferred into a 10 ml tube and incubated for 4 hours at 37 ° C. Then, using a microbiological loop, the tubes were inoculated with a dashed line with a beveled agarized (1.5% bactoagar) medium (Mueller-Hinton broth). Culture tubes were incubated for 16 hours at 37 ° C.

The resulting biomass was carefully removed with a microbiological loop from the surface of the agar medium and transferred to a microcentrifuge tube. Bacterial biomass was dissolved in 10 mM Tris-HCl buffer solution (pH 8.0). Then, a solution (10%) of sarcosyl was added to a final concentration of 0.5% and a solution (20 mg per ml) of proteinase K to a final concentration of 20 μg per ml. The mixture was incubated for 1 hour at a temperature of + 50 ° C with stirring.

To precipitate DNA from the solution, 2.5 volumes of ethanol and 1/10 volume of a 3 M sodium acetate solution were added. The tube was incubated at a temperature of -70 ° C for 15 minutes, and then centrifuged for 10 minutes at 16,000 g and a temperature of + 4 ° C. The supernatant was removed and the precipitate was thoroughly washed with 70% alcohol. After washing, the precipitate was dried in air.

After drying in air, the precipitate was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

When PCR of each gene used a pair of primers, one (direct) of which corresponded to the beginning, and the second (reverse) was complementary to the end of the gene.

The forward primer for oprF had the following structure (5'-3 '): AAG GAT CCA TGA AAC TGA AGA ACA CCT TAG, and the reverse: AAA GAT CTT TTG GCT TCA GCT TCT AC. The forward primer contained the BATH I restriction site at the 5'end, and the reverse restriction site Bgl II.

The forward primer for opr1 had the following structure (5'-3 '): AA GGA TCC AAC AAC GTT CTG AAA TTC TC, and reverse TTG AGC TTC TAC TTG CGG CTG GCT TTT TCC. The forward primer contained the BamH I restriction site at the 5'end, and the reverse restriction site Sac I.

PCR was performed under the following reaction conditions: 50 mM KCl, 10 tM 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 oprF gene amplification process consisted of the following stages: heating at 94 ° С for 3 min and 30 PCR cycles (94 ° С - 1 min, 30 sec 65 ° С - 1 min, 72 ° С - 1 min) and incubation at 72 ° C for 2 minutes The oprI gene amplification process consisted of the following stages: heating at 94 ° С for 3 min and 30 PCR cycles (94 ° С - 1 min, 30 sec 65 ° С - 1 min, 72 ° С - 20 sec) and incubation at 72 ° С for 40 sec.

In the first case, the obtained DNA fragment had a size of about 1 kb, and in the second about 0.25 kb. The first amplification was treated with restriction enzymes BamH I and Bgl II, and the second BamH I and Sac I. In parallel with the restriction enzymes BamH I and Sac I, the vector pQE-30 (QIAGEN) was processed.

Restrictions were purified on an agarose gel and combined into a single construct during the ligation reaction. When selecting recombinant clones, which was carried out using restriction analysis and sequencing, samples with constructs representing the oprF gene fused to the oprI gene located in direct orientation in the vector plasmid were selected. As a result, a recombinant plasmid pPA-OPRFI of 4757 base pairs in size was obtained, in which the DNA of the fusion gene is flanked by the restriction sites BamH I and Sac I (Fig. 1).

Example 2. Obtaining a strain of E. coli PA-OPRFI - producer of OprF-OprI.

The E. coli PA-OPRFI producing strain was obtained by transforming the cells of the parent E. coli K12 strain containing the pREP4 plasmid with the recombinant pPA-OPRFI plasmid followed by selection of the recombinant clones in an ampicillin and kanamycin medium at 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 ampicillin and kanamycin at concentrations of 100 and 50 μg per ml, respectively, was inoculated with a bacterial colony. Grown in a thermostated 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). 5 μl of a 1 M aqueous solution of a chemical inducer of isopropyl-β-d-thiogalactopyranoside (IPTG) was added. 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 (PAGE) using the Laemmli method. (fig. 3).

An analysis of the biomass obtained after induction of the expression of the recombinant gene revealed the presence of a specific product that was absent in the cells of the producer grown without the addition of IPTG. The molecular weight of the synthesized recombinant product was between 45 and 55 kDa. The calculated mass for recombinant OprF-OprI corresponded to 47.9 kDa. According to the results of electrophoresis, it was found that the amount of recombinant protein was at least one third of all cellular proteins.

Recombinant proteins showed high specificity when interacting with polyclonal hyperimmune rabbit serum to P. aeruginosa and with polyclonal hyperimmune rabbit serum to purified recombinant proteins OprF and OprI (Fig. 3).

Example 3. Obtaining OprF-OprI from E. coli strain PA-OPRFI.

Obtaining OprF-OprI was carried out in 4 stages:

Stage 1. Cultivation of E. coli PA-OPRFI strain with induction of recombinant gene expression.

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

3 stage. Chromatographic purification of OprF-OprI.

4th stage. Dissolution and renaturation of OprF-OprI.

Cultivation of E. coli PA-OPRFI strain with induction of recombinant gene expression.

Inoculated E. coli strain PA-OPRFI in 500 ml of LB medium containing ampicillin and kanamycin at concentrations of 100 and 50 μg per ml, respectively. Cultivation was carried out for 12 hours at a temperature of 37 ° C in a thermostated 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 ampicillin and kanamycin at concentrations of 100 and 50 μg per ml, respectively. 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-OPRFI 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 OprF-OprI.

To the clarified lysate, 20 ml of a suspension of Ni-agarose (QIAGEN) was added thereto 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 OprF-OprI.

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 0.59 calculated using the OMIGA program was used.

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

Example 4. Protective properties of OprF-OprI.

The protective properties of purified OprF-OprI were evaluated in a mouse model.

For immunization, an aluminum hydroxide gel (State Unitary Enterprise Immunopreparation) was added to the purified recombinant protein preparation at the rate of: 1 mg protein - 3 mg aluminum hydroxide. The protein concentration in the preparation was adjusted to the required value with phosphate-buffered saline. Sorption was carried out during the day at 4-7 ° C.

White model mice weighing 18-22 g were used as model animals. The immunization dose was 50 μg of protein per animal. The volume of drug administered was 0.5 ml. Drugs were administered to animals intraperitoneally. The scheme of double immunization with a two-week interval was used. Non-immunized mice of the same batch were used as a culture control. In addition, the recombinant OprF protein P. aeruginosa was used as a reference preparation.

Two weeks after the last immunization, in order to induce an experimental infection, the mice were intraperitoneally infected in a volume of 0.5 ml with various doses (in two steps) of a live virulent culture of P. aeruginosa (strain RA-170015) grown on Hottinger agar medium. Immunized mice were infected with the following doses: 50, 100, 200, 400 and 800 million microbial cells (mk) / 0.5 ml. Infection of control mice was carried out at lower doses: 25, 50, 100, 200 million MK / 0.5 ml. The calculation of dead and surviving animals was carried out for five days with the subsequent determination of LD ₅₀ , which was calculated according to the Kerber formula in the modification of Ashmarin-Vorobiev: LD ₅₀ = logA-log2 (B ₁ / C ₁ + ... + B _n / C _n ) -0 , 5, where A is the maximum infectious dose in the experiment, B is the number of animals that died in the group, C is the initial number of animals in the group, n is the number of groups (Table 1).

Recombinant proteins, as a result of immunization, protected mice from experimental intraperitoneal infection. Moreover, the hybrid recombinant protein (OprF-OprI) showed better results. The efficiency index (IE) of the protective properties of the recombinant OprF-OprI protein was 4.9, which was one and a half times higher than the same result for the OprF protein (IE 3.0).

Table 1 Study of the protective properties of recombinant proteins A drug Dose of infection million mk./0.5 ml. The number of mice that died / survived LD ₅₀ IE OprF-OprI 800 7/3 324.9 4.9 400 6/4 200 3/7 one hundred 2/8 fifty 0/10 Orprf 800 9/1 200 3.0 400 8/2 200 6/4 one hundred 3/7 fifty 0/10 Control (intact mice) 200 8/2 66 - one hundred 5/5 fifty 4/6 25 4/6 12.5 0/10

Information sources

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4. Stanislavsky ES, Joo I. Vaccines against Pseudomonas aeruginosa infections: results and research prospects // Bacterial antigens. Proceedings - Moscow. - 1982. - S.3-14.

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Claims (3)

1. Recombinant plasmid DNA pPA-OPRFI, encoding a hybrid recombinant FI protein of the outer membrane of P. aeruginosa with a size of 4757 base pairs, containing: Xba I - EcoR I DNA fragment with a size of 87 base pairs containing the sequence of the modified T5 bacteriophage promoter and two lactose operons; EcoR I - BamH I DNA fragment 57 bp containing the ribosome landing site, ATG start codon and six histidine coding sequence (Arg-Gly-Ser-His-His-His-His-His-His-His-Gly-Ser) ; BamH I - Sac I DNA fragment with a size of 1312 base pairs, containing full-length sequences of the oprF and opri genes P.aeruginosa obtained by polymerase chain reaction (PCR); EcoR I - Sac I DNA fragment of 1369 bp containing the ribosome landing site and the sequence encoding the OprF and OprI proteins (consists of the histidine sequence and the OprF and OprI proteins themselves); Hind III-NhE I DNA fragment 1116 base pairs in size, containing lambda t ₀ transcriptional stop region. 2. The bacterial strain E. coli PA-OPRFI is a producer of a hybrid recombinant protein F-I of the outer membrane of P. aeruginosa, obtained by transformation of the parent strain of bacteria Escherichia coli K12 recombinant plasmid DNA pPA-OPRFI according to claim 1. 3. The method of producing a hybrid recombinant protein FI of the outer membrane of P. aeruginosa using the E. coli PA-OPRFI strain of bacteria transformed by genetic engineering methods according to claim 2, including culturing it in a nutrient medium, destroying 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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