RU2793753C1 - RECOMBINANT PLASMID DNA pPA-FlicC ENCODING SYNTHESIS OF RECOMBINANT FLAGELLIN-C PSEUDOMONAS AERUGINOSA, STRAIN ESCHERICHIA COLI PA-FlicC - PRODUCER OF THE HYBRID RECOMBINANT PROTEIN AND A METHOD FOR PRODUCING SAID PROTEIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates in particular to production of a recombinant FlicC protein, consisting of the amino acid sequence of P. aeruginosa flagellin-C, strain RA 103, intended for use as a component of a recombinant vaccine, as well as to the recombinant plasmid pPA-FlicC and recombinant strain E.coli PA-FlicC for the synthesis of the recombinant FlicC protein. The method for obtaining FlicC according to this invention, including the cultivation of E. coli PA-FlicC in a nutrient medium, the destruction of microorganism cells, the isolation of inclusion bodies, the removal of impurities soluble in 8 M urea, the dissolution of the recombinant FlicC protein in a buffer solution with 6 M guanidine chloride and the transfer of the recombinant FlicC protein to native conditions using dialysis excludes the use of chromatography, which uses expensive nickel-activated sorbents, and the proposed procedure itself is faster.

EFFECT: faster procedure of obtaining recombinant FlicC protein.

3 cl, 4 dwg, 5 ex

Description

The invention relates to biotechnology, in particular to the production of a recombinant protein consisting of the amino acid sequence of flagellin-C of Pseudomonas aeruginosa strain RA 103 (hereinafter referred to as the recombinant FlicC protein) intended for use as a component of a recombinant vaccine, as well as to the construction of a recombinant plasmid and the creation of a recombinant strain Escherichia coli (hereinafter E. coli) intended for the synthesis of the recombinant FlicC protein.

Pseudomonas aeruginosa (hereinafter P. aeruginosa) up to 15-20% of all nosocomial infections [1, 2]. One third of all lesions of the genitourinary system in urological patients, 20-25% of purulent surgical infections and primary gram-negative bacteremia are due to P. aeruginosa [3]. Pseudomonas aeruginosa is the cause of infectious diseases in immunocompromised people, including pneumonia and cystic fibrosis. Infections caused by P. aeruginosa respond poorly to antibiotic therapy due to the frequent isolation of multiresistant strains [4]. P. aeruginosa has many pathogenicity factors, some of which are widely used in the development of antipseudomonal vaccines. Currently, in vaccine design technology, more and more attention is paid to the use of genetic engineering methods. In the laboratory of protective antigens of the Federal State Budgetary Scientific Institution NIIVS them. I.I. Mechnikov, recombinant proteins (OprF, OprI, OprL) of the outer membrane of P. aeruginosa, a deletion variant of exotoxin A, and fused recombinant proteins including the amino acid sequences of the above proteins (OprF-Oprl, OprF-aTox, OprF-aTox-OprI) were obtained and studied. their immunobiological properties [5].

Recently, the interest of researchers in the P. aeruginosa flagellin protein family has increased. Flagellin is a bacterial protein, the main component of bacterial flagella. As an important factor in the virulence of P. aeruginosa, flagellin is involved in the formation of host responses to pathogen invasion by activating innate and adaptive immune response factors [6]. It is now known that flagellin is a ligand for the innate immune system receptor TLR5 (toll-like receptor 5) and/or NAIPS (Nod-like receptor apoptosis-inhibitory protein 5) receptors, which are members of the family of cytosolic receptors NLRs (nucleotide binding and oligomerization domain-like receptor). The formation of the flagellin-TLR5 complex stimulates the development of the body's defense responses. It is known that administration of flagellin to mice had a protective effect against the pathogenic effects of toxicants, bacteria, viruses, and radiation [7, 8]. Of particular interest is the study of adjuvant (immunostimulatory) properties of flagellin proteins in combination with recombinant protective antigens, which is associated with the stimulation of innate immune response factors by them. It was shown that the inclusion of flagellin in the composition of recombinant proteins contributed to an increase in the production of cytokines and activation of antigen-presenting cells. An in vivo study confirmed this phenomenon: immunization of mice with this flagellin-containing protein induced a specific T-cell response. At the same time, the introduction of flagellin potentiated the development of adaptive immunity not only in young, but also in old animals [9, 10, 11].

Closest to the claimed technical solution (prototype) is the method described in Goudarzi et al. dated 2009 [10]. Synthesis of the recombinant protein was carried out using the plasmid pET-22b (Novagen). In this method, the flicC gene obtained from the genome of P. aeruginosa strain 8821M was cloned. The disadvantage of this method is that the purification of the recombinant protein by Goudarzi et al. was carried out using affinity chromatography on a nickel-activated sorbent, which increases the cost of obtaining this protein.

The objective of the invention is to construct a recombinant pPA-FlicC plasmid DNA encoding a recombinant P. aeruginosa FlicC protein; creation of a producer strain of recombinant P. aeruginosa FlicC protein using E. coli strain K-12 and development of a method for purification of recombinant P. aeruginosa FlicC protein.

To solve this problem, a recombinant plasmid DNA pPA-FlicC is proposed, encoding the synthesis of the recombinant Pseudomonas aeruginosa FlicC protein, 4584 base pairs in size, which is cleaved as a result of hydrolysis by restriction nucleases BamHI and HindIII into DNA fragments with sizes of 1161 and 3423 base pairs; containing the sequences of the modified bacteriophage T5 promoter and two lactose operons; ribosome landing site; start ATG codon; a sequence encoding six histidines; lambda t0 transcription stop region; transcription stop region; site of initiation of replication of the plasmid ColE1; a gene encoding resistance to ampicillin; in this case, as a result of the expression of this construct, the synthesis of a recombinant product with a mass of about 40.7 kDa occurs.

In another aspect of the invention, a bacterial strain of Escherichia coli PA-FlicC is provided - a producer of recombinant P. aeruginosa FlicC protein with a molecular weight of 40.7 kDa, obtained by transforming the parent E. coli bacterial strain K-12 with recombinant plasmid DNA pPA-FlicC according to claim 1 .

In another aspect of the invention, a method is provided for obtaining a recombinant P. aeruginosa FlicC protein using the E. coli PA-FlicC strain according to claim 2, including its cultivation in a nutrient medium, destruction of microorganism cells, isolation of inclusion bodies, removal of impurities soluble in 8 M urea , dissolving the recombinant FlicC protein in a buffer solution with 6 Μ guanidine chloride and transferring the recombinant FlicC protein to native conditions using dialysis.

The technical result of the claimed invention is as follows. Our studies have shown that the recombinant FlicC protein is highly hydrophobic and sparingly soluble in buffers containing 8 M urea. Such properties are associated with the features of the amino acid sequence that forms this protein. In our research, we have developed a method for purifying this protein, taking into account its properties. This method eliminates the use of chromatography, which uses expensive nickel-activated sorbents, and the proposed procedure itself is faster.

Brief description of explanatory materials

In FIG. 1 is a diagram of the pPA-FlicC plasmid.

In FIG. 2 shows polyacrylamide gel analysis of protein products (Coomassie R-250 stain) resulting from expression in E. coli of the flicC gene located in the plasmid pPA-FlicC and purification of the recombinant FlicC protein.

Tracks: 1 - proteins of the non-induced producer of the recombinant protein; 2 - proteins of the recombinant protein producer obtained after induction of expression with IPTG; 3 - inclusion bodies containing the recombinant FlicC protein; 4 - purified recombinant FlicC protein; 5 - protein marker (molecular weights of marker fragments: 60, 50, 40, 30, 25 and 20).

In FIG. 3 shows the analysis of blood sera from mice immunized with the recombinant FlicC protein.

Histogram bars: 1 - sera of non-immunized mice (control); 2 - sera of mice immunized with recombinant FlicC protein adsorbed on aluminum hydroxide in a ratio of 1:3; 3 - sera of mice immunized with the recombinant FlicC protein adsorbed on aluminum hydroxide in a ratio of 1:1; 4 - sera of mice immunized only with the recombinant FlicC protein.

In FIG. 4 shows an analysis of the effect of antisera on the motility of P. aeruginosa bacteria in semisolid agar.

Wells of a 12-well plate filled with semi-liquid agar and inoculated with a culture of P. aeruginosa strain PA-103 after cultivation.

Wells: 1st control medium; 2 - with a medium containing the serum of mice immunized with the recombinant FlicC protein adsorbed on aluminum hydroxide in a ratio of 1:3; 3 - with a medium containing the serum of mice immunized with the recombinant FlicC protein adsorbed on aluminum hydroxide in a ratio of 1:1; 4 - with the medium containing the serum of mice immunized only with the recombinant FlicC protein.

Implementation of the invention

Plasmid pPA-FlicC has 4584 base pairs and is characterized by the presence of a DNA fragment cut at the restriction sites ВatHI and HindIII 1161 base pairs, containing the sequence of the P. aeruginosa flicC gene. The plasmid contains the sequences

a modified bacteriophage T5 promoter and two lactose operons; ribosome landing site; start ATG codon; a sequence encoding six histidines; lambda t ₀ transcription stop region; transcription stop region; site of initiation of replication of the plasmid ColE1; ampicillin resistance gene.

Plasmid pPA-FlicC has the nucleotide sequence of SEQ IDNO: 1.

The claimed recombinant FlicC protein encoded in the pPA-FlicC plasmid has the amino acid sequence of SEQ ID NO: 2.

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

The E. coli strain PA-FlicC is a producer of the recombinant FlicC protein.

The E. coli PA-FlicC strain is characterized by the following features:

Cultural and morphological features: Cells are small, straight, thickened rod-shaped, gram-negative, non-spore-bearing. Cells grow well on simple nutrient media. When growing on 1% LB agar medium, the colonies are round, smooth, convex, cloudy, shiny, gray, with smooth edges. When growing in liquid media (on a minimal medium with glucose or LB-medium), they form an intense even turbidity.

Physico-biological features: Aerob. The temperature range of growth is 4-42°C at an optimum pH of 6.5-7.5. Both mineral salts in ammonium and nitrate forms and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc. are used as a nitrogen source. Amino acids, glycerol, carbohydrates are used as a carbon source.

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

Method, conditions and composition of the medium for storing the strain: LB medium with 10% glycerol and antibiotics at -70°C in ampoules and in a lyophilized state in ampoules at 4°C.

The method for obtaining and purifying the recombinant FlicC protein includes the following steps: cultivation in a nutrient medium of the strain E. coli PA-FlicC; sedimentation of biomass by centrifugation; isolation of inclusion bodies; destruction of inclusion bodies by dissolving them in a denaturing buffer containing 4 M urea, followed by precipitation of the recombinant P. aeruginosa FlicC protein and removal of the urea-soluble fraction; dissolution of the recombinant P. aeruginosa FlicC protein in a buffer solution containing 6 Μ guanidine hydrochloride and dialysis to transfer the protein preparation to native conditions. After dialysis and conversion to native conditions, the recombinant FlicC protein preparation precipitated and was therefore aliquoted with vigorous mixing. The resulting drug proved to be effective in immunizing animals and stimulated specific immune responses.

The invention is illustrated by the following examples:

Example 1. Obtaining recombinant DNA plasmid pPA-FlicC.

The pPA-FlicC DNA plasmid was based on a plasmid vector, a plasmid vector for the expression of inserted sequences in E. coli cells. QE-30 (Qiagen). The full-length sequence of the flicC gene was synthesized by PCR based on template DNA of P. aeruginosa strain PA 103. Primer design was performed using the full-length DNA sequence of P. aeruginosa strain PA 103 published in the GenBank database. The forward primer (5'-GGA TCC GCC TTG ACC GTC AAC ACC AAC ATC G) had an additional BatHI restriction site, and the reverse primer (5'-AAG CTT AGC GCA GCA GGC TCA GAA CCG AC) had an additional HindIII restriction site. The FlicC gene was inserted at the BATHI and Hindill restriction sites in the pQE-30 plasmid. The result was obtained recombinant plasmid pPA-FlicC (Fig. 1). The calculated full-length nucleotide sequence of the pPA-FlicC plasmid is shown in SEQ ID NO: 1. The amino acid sequence of the recombinant protein resulting from translation of the inserted FlicC gene (performed in the OMIGA computer program) is shown in SEQ ID NO: 2.

Example 2. Preparation of E. coli strain PA-FlicC - producer of recombinant FlicC protein.

Producer strain E. coli PA-FlicC was obtained by transformation of cells of the parent strain E. coli K-12 with recombinant plasmid pPA-FlicC followed by selection of recombinant clones on a medium containing ampicillin (100 μg/ml) and kanamycin (50 μg/ml) at a temperature of 37°C. Primary selection of recombinant plasmids was performed using restrictive analysis and sequencing.

Further, the selection of producers was carried out according to the ability of controlled synthesis of recombinant protein in cells. For this, 3 ml of LB medium containing ampicillin (at a concentration of 100 μg per ml) and kanamycin (at a concentration of 50 μg per ml) were inoculated with a bacterial colony. Grown in a thermostatted shaker with an orbital speed of 170 rpm for 12-16 hours and a temperature of 37°C. Then, 250 μl of the grown culture was added to 5 ml of fresh medium and incubated under the same conditions for 1.5–2 hours until the culture reached the active growth phase (0.6–0.7 optical units at a wavelength of 600 nm). 5 μl of a 1 M aqueous solution of the chemical inducer isopropyl~P-c1-thiogalactopyranoside (hereinafter IPTG) was added. Incubation was continued under the same conditions for 3-4 hours, after which the obtained biomass was precipitated in a microcentrifuge.

The bacterial culture of the producer, incubated under the same conditions, but without the addition of IPTG, was used as a control. When analyzing the expression products in a polyacrylamide gel according to the Laemmli method, a recombinant protein with a size of about 40 kDa was identified, which corresponded to the calculated data (40.7 kDa) (Fig. 2).

Example 3 Preparation of purified recombinant FlicC protein preparation from E. coli strain PA-FlicC.

The bacterial colony was inoculated with 50 ml of LB medium containing ampicillin (at a concentration of 100 μg per ml) and kanamycin (at a concentration of 50 μg per ml). Grown in a thermostatted shaker with an orbital speed of 170 rpm for 12-16 hours and a temperature of 37°C. Then the resulting culture was introduced into a 5 L flask containing 1 L of fresh medium with the same antibiotics. Cultivation was carried out under the same conditions for 1.5-2 hours until the culture reached the active growth phase (0.6-0.7 optical units at a wavelength of 600 nm). 1 ml of 1 M IPTG solution was added. The incubation was continued under the same conditions for 3–4 hours, after which the bacterial biomass was precipitated in a Beckman Avanti J-E centrifuge (JA-14 rotor) at a rotation speed of 4000 rpm for 10 min.

The biomass pellet was resuspended in 30 ml of phosphate buffer (pH 7.2) containing 5 mM EDTA, 7 mM β-mercaptoethanol and sonicated (hight, 3 sec, 3 times). The resulting lysate was centrifuged (using a Beckman Avanti JE centrifuge with a JA-20 rotor) at 10,000 rpm for 30 minutes at 4°C. The precipitate was collected and dissolved in 30 ml of the following solution: Tris-HCl (40 mM, pH 8.8), 10 mM NaCl, 6 mM MgCl ₂ , 10 mM CaCl ₂ . 1 μl of RNase A (10 mg/ml) and 2 μl of DNase I were added to the suspension. Repeatedly sonicated and incubated at 37°C for 15 minutes, and then centrifuged at 10,000 rpm for 30 minutes at 4°C. The precipitate was dissolved in 20 ml of phosphate buffer containing 5 mM EDTA, 20% sucrose and 1% Triton X-100 and incubated for 10 minutes at 4°C. It was then centrifuged at 10,000 rpm for 30 minutes at 4°C to collect the pellet containing inclusion bodies.

The resultant inclusion body pellet was dissolved in 40 ml of the following lysis buffer: 4 M urea; 0.1 M NaH ₂ PO ₄ ; 0.01M Tris-HCl; pH - 8.0. Biomass dissolution was carried out in an orbital shaker at 70 rpm for 12 hours at room temperature. Then, the insoluble fraction of Beckman Avanti J-E was precipitated with a JA-20 rotor at 10,000 rpm for 30 minutes at room temperature. The supernatant was removed and the pellet containing the recombinant FliC protein was dissolved in 10 ml of the following buffer solution: 6 M guanidine hydrochloride; 0.1 M NaH ₂ PO ₄ ; 0.01M Tris-HCl; pH - 8.0.

The material was transferred into ten 1.5 ml microtubes, which were placed in a multi-vortex V-32 (Biosan) and shaken vigorously for one hour at room temperature. Then the insoluble components were precipitated in a microcentrifuge at a speed of at least 13,000 rpm and room temperature.

A solution containing the recombinant FliC protein was collected and dialyzed against 0.05 M Tris-HCl; pH - 9.0. Preparations of the recombinant FliC protein after dialysis were packaged with vigorous shaking in aliquots and stored at -20°C.

The analysis of the purified recombinant protein was carried out in polyacrylamide gel according to the method of Laemmli (Fig. 2).

Example 4. Study of the antigenic properties of the recombinant FlicC protein.

The purified recombinant FlicC protein was used to immunize Balb/c mice. At the same time, the recombinant FlicC protein was studied in its pure form and with an adjuvant with aluminum hydroxide in the ratio of protein: adjuvant 1:1 and 1:3. The drugs were administered to animals intraperitoneally twice with a two-week interval at a dose of 50 μg of recombinant FlicC protein per animal. Each group included ten animals. Non-immunized mice of the same batch were used as controls.

Two weeks after the course of immunization, the meshes were totally bled and serum was obtained. Serum preparations were individually analyzed by ELISA, and then the mean value and confidence interval were calculated. As a result, high titers of specific antibodies were found in the sera of all immunized animals. At the same time, the amplifying effect of aluminum hydroxide on the immunizing dose was not revealed.

Example 5 Study of the effect of antisera on the motility of P. aeruginosa bacteria.

The resulting immune mouse sera were tested for motility inhibition analysis of P. aeruginosa bacteria in a semi-liquid nutrient medium. The experiment used LB-medium with the addition of agar at a concentration of 0.3%. The sera were added to a warm agar medium (with a temperature not exceeding 60°C) with various dilutions (1:100, 1:300, 1:1000 relative to the medium). The medium was then poured into 12-well plates with a well radius of 22.6 mm and a working volume of 2.0 ml.

A fresh culture of P. aeruginosa strain PA-103 was diluted to an optical density of 0.2 at OD ₆₀₀ and added to the center of each well of the plate in a volume of 0.2 ml. Phosphate buffered saline was used as a control. Next, incubation was carried out at a temperature of 37°C for 18 hours. As a result of the experiment, it was revealed that the culture of P. aeruginosa in semi-liquid agar spread over the entire surface in the control wells. At the same time, in the wells with the addition of immune sera, the propagation radius was 3-5 mm, which indicated the inhibition of organelles (flagella) of the movement of bacteria.

Sources of scientific and technical information:

1. Kolesova T.S. The etiological role of Pseudomonas aeruginosa in various opportunistic and nosocomial infections // Modern medicine: topical issues. - 2017. - No. 3. - S. 134-140.

2. Karpunina T.I., Kuznetsova M.V., Nikolaeva N.V. Epidemiological aspects of nosocomial Pseudomonas aeruginosa infection in a multidisciplinary surgical hospital // Siberian Medical Journal. - 2009. - No. 4-1. - T. 24. - S. 70-73.

3. Parkins D-M., Somayaji R., Waters J-V. Epidemiology, Biology, and impact of clonal Pseudomonas aeruginosa, infections in cystic fibrosis // Clin Microbiol. - 2018. - P. 29-31(4).

4. Pang Z., Paudonis R., Glick R-B., lin T-J., Cheng Z. Antibiotic resistanse in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies // Biotechnology Advances. - 2019. - Vol. 37, no. 1. - p. 177-192.

5. Mikhailova H.A., Zimina E.M., Soldatenkova A.V., Kaloshin A.A. Development of a vaccine based on recombinant Pseudomonas aeruginosa antigen // Journal of Microbiology, Epidemiology and Immunobiology. - 2019. - No. 1. - S. 78-80.

6. Hajam I.A., Lee J.H., Dar R.A., Shahnawaz I., Jaume J.C. Bacterial flagellin-A potent immunomodulatory agent // Experimental and Molecular Medicine. - 2017. - Vol. 49, - No. 9.

7. Safronov G.A., Murzina E.V., Bolekhan B.H. Prospective directions of use of preparations based on recombinant flagellin // Medical academic journal. - 2017. - No. 2. - T. 17, - S. 7-20.

8. Dukhovlinov I.V., Bogomolova E.G., Fedorova E.A. Study of the protective activity of a candidate vaccine against rotavirus infection based on the recombinant FLICVP6VP8 protein // Medical Immunology. - 2016. - V. 18, No. 5. - S. 417-424.

9. Baofeng C., Xinsheng L., Yuzhen F., Peng Z., Yongguang Z., Yonglu W. Flagellin as a vaccine adjuvant // Expert review vaccines. - 2018. - Vol. 17, no. 4. - P. 335-349.

10. Goudarzi G., Sattari M., Roudkenar M.H., Montajabi-Niyat M., Zavaran-Hosseini A., Mosavi-Hosseini K. Cloning, expression, purification, and characterization of recombinant flagellin isolated from Pseudomonas aeruginosa. Biotechnol Lett. - 2009. - Vol. 31, no. 1. 9. - P. 1353-1360.

11. Tanom A., Farajnia S., Majidi J. Cloning, expression and characterization of recombinant exotoxin A-Flagellin fusion protein as a new vaccine candidate against Pseudomonas aeruginosa infections // Iran Biomed J. - 2013. - Vol. 17, no. 1. - P. 1-7.

Claims (3)

1. Recombinant plasmid DNA pPA-FlicC encoding the synthesis of recombinant Pseudomonas aeruginosa FlicC protein, 4584 base pairs in size, cleaved by hydrolysis with restriction nucleases VatHI and HindIII into DNA fragments with sizes of 1161 and 3423 base pairs; containing the sequences of the modified bacteriophage T5 promoter and two lactose operons; ribosome landing site; start ATG codon; a sequence encoding six histidines; lambda t ₀ transcription stop region; transcription stop region; site of initiation of replication of the plasmid ColE1; a gene encoding resistance to ampicillin; in this case, as a result of the expression of this construct, the synthesis of a recombinant product with a mass of about 40.7 kDa occurs. 2. Bacterial strain Escherichia coli PA-FlicC is a producer of the recombinant P. aeruginosa FlicC protein with a molecular weight of 40.7 kDa, obtained by transforming the parent bacterial strain E. coli K-12 with recombinant plasmid DNA pPA-FlicC according to claim 1. 3. A method for obtaining a recombinant P. aeruginosa FlicC protein using the E. coli PA-FlicC strain according to claim 2, including its cultivation in a nutrient medium, destruction of microorganism cells, isolation of inclusion bodies, removal of impurities soluble in 8 M urea, dissolution recombinant FlicC protein in a buffer solution with 6 Μ guanidine chloride and transfer of the recombinant FlicC protein to native conditions using dialysis.

Images