RU2542483C2 - RECOMBINANT POLYPEPTIDE StV POSSESSING PROTECTIVE PROPERTIES IN RELATION TO Streptococcus agalactiae, RECOMBINANT DNA StV, ITS CODING AND RECOMBINANT STRAIN Escherichia coli M15-StV CONTAINING RECOMBINANT PLASMID PQE-StV - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to microbiology and molecular genetics, and concerns recombinant polypeptide StV, recombinant DNA stV coding the above recombinant polypeptide StV, recombinant plasmid DNA PQE-stV representing plasmid DNA PQE-30 carrying the above recombinant DNA stV, and the producing strain Escherichia coli M15-StV producing recombinant polypeptide StV.

EFFECT: presented inventions can be used in medical industry in producing Streptococcus agalactiae, eg Streptococci B vaccines.

4 cl, 6 dwg, 2 tbl, 5 ex

Description

The invention relates to the field of microbiology and molecular genetics and can be used in the medical industry in the production of vaccines against Streptococcus agalactiae, i.e. group B streptococci (SGV). In the present invention, recombinant DNA, recombinant plasmid DNA, which are involved in obtaining a new strain of Escherichia coli, E. coli M15, a producer of a recombinant polypeptide that induces the synthesis of antibodies with protective properties against HBV, is created. The use of a recombinant polypeptide in the experiment as a vaccine preparation gave a positive result.

The fight against streptococcal infection is an urgent problem. Group B streptococci are dangerous because they cause various diseases, especially in newborns and in the elderly. Colonization of a woman’s body with a strain of HBV can adversely affect the course of pregnancy, childbirth and fetal development. HBV is a serious threat to newborns, causing generalized forms of infection with a severe clinical course of the disease: meningitis, sepsis, etc.

In modern clinical practice, drug prophylaxis of GBV infections is widely used. This primarily concerns the sanitation of pregnant women who are carriers of infections in order to prevent the occurrence of infections in children. Due to insufficiently debugged rapid diagnosis of pathogens of HBV infection, there are difficulties in preventing the disease in pregnant women. Disease prevention in the elderly is not carried out either in Russia or abroad.

One way to prevent HBV infection is to vaccinate the population. Conjugated polysaccharide vaccines have been tested in clinical trials. A serious drawback of polysaccharide-based vaccines is that they belong to T-independent antigens, as a result of which immunization does not cause the formation of immunological memory (Streptococci group B in human pathology / A.A. Totolyan, A.N. Suvorov, A.V. . Dmitriev // Under the general editorship of A.A. Totolyan. - St. Petersburg: Man, 2009 .-- 212 p .; ill.).

It is known that polypeptide recombinant vaccines based on various surface HBV proteins are being developed. But this group of vaccine preparations is still far from the stage of clinical trials (Streptococci group B in human pathology / A.A. Totolyan, A.N. Suvorov, A.V.Dmitriev // Under the general editorship of A.A. Totolyan. - St. Petersburg: Man, 2009 .-- 212 p .; ill.).

Currently, there is no effective vaccine against group B streptococci, and the creation of a protein vaccine against group B streptococci is an urgent problem.

The prototype of the invention is RF patent No. 2378374, where the recombinant C5a peptidase polypeptide was chosen as the antigen, but the C5a peptidase gene is found in all strains of group A and B streptococci, regardless of the degree of their virulence, so a search was made for another polypeptide, the gene which is found only in the most virulent strains of group B streptococci.

The objective of the invention is to obtain a recombinant polypeptide of a different origin, having protective properties against Streptococcus agalactiae, with the aim of using it then as a component of an anti-streptococcal vaccine. The creation of a producer strain of such a recombinant polypeptide is a necessary condition in the development of a vaccine.

The search for proteins, probable candidates for vaccines, led to the idea of using a different virulence factor of HBV than in analogues.

Genomic analysis of HBV in strain NEM316 revealed the gbs1356 gene, sspB1, which supposedly encodes a surface protein belonging to type V secretion system (Suvorov A.N., Ferretti J.J., J. Basic Microbiol, 2004, p. 64-74). The type V secretion system includes a group of proteins called motor transporters. Autotransporters are secreted (exported) from the cytoplasm through the Sec-system with cleavage of the amino-terminal signal sequence. Some of them can remain anchored in the cell wall, while others are exported directly to the extracellular space. The protein encoded by the gbs1356 gene is a potential virulence factor and is found in virulent SGV strains (Suvorov A.N., Savicheva A.M. et al. J. Obstetrics and Female Diseases, 2005, p. 50-56).

The ability to use it as a component of the vaccine has been tested. A recombinant polypeptide was obtained - the result of the claimed invention. The authors designated it as StV.

The essence of the invention is the creation of a unique recombinant DNA (designated as stV) encoding a recombinant StV polypeptide having the properties required for a HBV vaccine. Recombinant stV DNA was obtained by polymerase chain reaction (PCR) using the chromosomal DNA of strain 090R 1a of the SGV serotype and created unique for and rev primers followed by cloning using the expression plasmid pQE-30 (The QIAexpress System, Qiagen, USA). Also essential features of the invention are the creation of recombinant plasmid DNA PQE-stV carrying recombinant stV DNA, and a producer strain Escherichia coli M15-StV, allowing under certain conditions to express the recombinant StV polypeptide. The strain is included in the collection of FSBI "NIIEM" SZO RAMS under No. 242.

To create a producer strain of the recombinant StV polypeptide, E. coli M15 strain (The QIAexpress System, Qiagen, USA) was selected. As a result of its modification using plasmid DNA, the strain acquired new properties: the ability to express the recombinant StV polypeptide.

A fragment of a 3684 bp gene encoding the StV polypeptide of strain 090R Ia of the SGV serotype, designated as stV, was obtained by polymerase chain reaction using for and rev primers and the chromosomal DNA of strain 090R Ia of the SVG serotype (Figure 1, Table 1). The gene sequence of strain 090R was deposited in GenBank NCBI with accession number JF436927.

Cloning of stV was performed using the expression plasmid PQE-30 and subsequent transformation of the resulting recombinant plasmid into the heterologous system of E. coli M15. The created producer strain of the recombinant StV polypeptide is designated as E. coli M15 - StV. Cells of E. coli strain M15-StV express the desired recombinant StV polypeptide, which is subjected to subsequent single-stage affinity purification using 2 ml of Ni-Sepharose (Amersham, USA).

The studied recombinant StV polypeptide has a pronounced immunogenicity: with its subcutaneous administration to mammals (mice and rabbits), a humoral immune response develops. The figure 5 presents the dynamics of changes in the titer of antibodies to the recombinant protein StV after immunization of mice. The maximum titer of antibodies to the StV polypeptide was observed on day 40 from the start of immunization of mice and amounted to 1.6 · 10 ^-4 , i.e. this is a fairly high quantitative indicator of antibody formation for the elimination of group B streptococci.

The protective properties of StV-specific antibodies were studied in model experiments in vitro (opsonophagocytic test) and in vivo (studying the dynamics of the development of generalized HBV infection in mice previously immunized with the recombinant StV protein).

In the opsonophagocytic test using mouse peritoneal macrophages and HBV, it was shown that preliminary contact of HBV with a specific immune serum enhances the immobilization of bacteria on the surface of macrophages compared to normal serum. The results of the opsonophagocytic test are presented in table 2. The multiplicity of the increase in the infection index was from 3.1 to 14.8 when treating streptococci with immune rabbit serum compared with the infection index when treating streptococci with normal rabbit serum. Thus, it was found that StV-specific antibodies are able to opsonize group B streptococci, providing their accelerated elimination, and therefore, the recombinant StV polypeptide as an antigen acts as a vaccine. In vivo control of the effect of the StV polypeptide was carried out. Figure 6 "Dynamics of the streptococcal focus in the mouse spleen" shows the content of streptococci in the spleen of experimental animals at various stages of the infection process. As can be seen from the data presented, in non-immunized animals there were two peaks of the maximum content of group B streptococci: the first at 2.5 hours and the second at the end of the experiment (18 hours). In the case of animals immunized with StV protein, there was a gradual increase in the number of streptococci by the 5th hour of the experiment, followed by stabilization of the infection process with a tendency to a gradual decrease in the number of detected streptococci. The data obtained indicate that the recombinant StV polypeptide is capable of stimulating a protective humoral immune response.

Thus, in experiments on the active defense of StV-immune mice, it was shown that with an intraperitoneal administration of a sublethal dose of HBV in immune mice in the early stages of infection, accelerated excretion of the infectious agent is observed compared to non-immune animals.

The presented new recombinant StV polypeptide induces the synthesis of StV-specific antibodies with protective properties against Streptococcus agalactiae. The recombinant StV polypeptide may be useful as a vaccine component for the prevention and treatment of bacterial infection caused by Streptococcus agalactiae. The recombinant StV polypeptide can be used both in the form of a single vaccine, and as one of the components of a multicomponent vaccine against Streptococcus agalactiae.

Example 1. Cloning

PCR was used to clone the stV gene fragment. The primers were designed:

TCG GTA CCA AGG CAA AGA CCG AAG GAA T - for

AGG CAA GCT TST GAC AAA TTT CCC GTT CTG - rev

Using PCR, using primers on the matrix of strain 090R, a DNA fragment was amplified.

The polymerase chain reaction was set according to the standard scheme, including total denaturation, annealing of the primers, and elongation of the fragment between the primers.

The temperature profile of PCR was selected as follows:

1) 94 ° C, 1 min - total DNA denaturation

2) 94 ° C, 30 sec - DNA denaturation

3) 60 ° С (610С), 1 min - annealing of primers for 30 cycles

4) 72 ° C, 1 min - elongation

5) 72 ° C, 1 min - completion

6) 4 ° С - storage

The sample volume for PCR was 25 μl. The composition of the mixture for PCR:

1) H ₂ O - 16 μl

2) Buffer with Mg2 + (5x) - 5 μl

3) Direct primer - 0.5 μl

4) Reverse primer - 0.5 μl

5) dNTP - 1 μl

6) Taq polymerase - 0.2 μl

7) DNA under investigation - 2 μl.

DNA analysis in gel electrophoresis showed that the size of the amplification was 3684 N. p. The nucleotide sequence is shown in Figure 1. The amplified fragment was isolated from the gel using the QIAquick Gel Extraction Kit (Qiagen, USA).

To clone this fragment, the vector PQE-30 was used.

The vector and the amplified fragment were digested with KpnI and HindIII enzymes. Restriction products were separated by gel electrophoresis and then isolated using the QIAquick Gel Extraction Kit (Qiagen, USA). During cloning, 2 μl of the PQE-30 DNA fragment obtained after purification in a ratio of 5: 1, 5 μl of ten-fold ligase buffer and 1 μl of T4 phage ligase was added to 30 μl of stV DNA fragment. The volume was adjusted with distilled water to 50 μl. The mixture was incubated at 10 ° C for 12 hours. Then, the calcium transformation of E. coli M15 culture was carried out:

E. coli M15 strain was seeded in 20 ml of BHI from a plate, then it was incubated overnight on a shaker at 37 ° C. The next day, 1% E. coli was seeded on a new medium heated at 37 ° C. Then the strain was grown at 37 ° C on a shaker to an OD of 0.2-0.4. The measurement was carried out at a wavelength of 600 nm. Then incubated in ice for 5 minutes. Then it was centrifuged at 4 ° C for 10 minutes at 5000 rpm. Next, the precipitate was dissolved in 1/2 volume of sterile cold 50 mM CaCl ₂ , and then incubated for 20 minutes in ice. Then it was centrifuged at 4 ° C for 5 minutes at 5000 rpm. The precipitate was resuspended in 50 mM CaCl ₂ in 1/10 volume to the original volume of the medium.

The transformation was carried out as follows:

- Experience: 100 μl of cells + 50 μl of ligase mixture

- Positive control: 100 μl of cells + 2-3 μl of the original plasmid

- Negative control: 100 μl of cells

Then incubated in ice for 40 minutes. Then incubated in a thermal block at 42 ° C for 2 minutes. Then incubated in ice for 5 minutes. 1 ml of sterile medium was added. Incubated for one hour at 37 ° C. Then it was centrifuged, 700-900 μl were taken, and the pellet was resuspended. 100 μl of cells were seeded per medium containing ampicillin (Ap) (100 μg / ml) and kanamycin (Km) (25 μg / ml).

30 clones resistant to ampicillin and kanamycin were obtained.

In order to identify clones in which plasmid DNA contains an insertion fragment of 3684 bp DNA, plasmids were isolated from 16 clones. Analysis by gel electrophoresis (figure 4) showed that plasmids 10 and 13 contain an insert of the stV gene.

When testing clones for their ability to express the StV polypeptide, it was found that E. coli M15 - StV clones possess this ability.

Example 2. Purification of the recombinant polypeptide

The culture of E. coli strain M15 - StV was cultured in 800 ml of BHI liquid medium (Brain Heart Infusion Broth, Gibco, USA) supplemented with antibiotics (ampicillin (100 μg / ml) and kanamycin (25 μg / ml)) until the late logarithmic growth phase (OD600 = 0.7-0.9). The expression of the recombinant polypeptide was induced by the addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Cells were cultured for 4 hours. Then the cells were precipitated by centrifugation, washed with lysis buffer A (20 mM Na ₂ HPO ₄ , 20 mM NaH ₂ PO ₄ , 500 mM NaCl, 20 mM imidazole, pH 8.0) and suspended in the same buffer in 4 ml, adding a protease inhibitor phenylmethylsulfonyl fluoride (PMSF), to a concentration of 1 mm. For cell lysis, triple ultrasonic treatment was used at 4 ° C for 20 seconds with a break of 40 seconds in an ultrasonic disintegrator (UZDN-1U4.2, England). The cell lysate was centrifuged at 20,000 rpm and 4 ° C for 20 minutes. The supernatant was passed through a 0.45 μm filter (Millipore, USA) and then applied to a column of 2 ml Ni-Sepharose (Qiagen, USA), previously equilibrated with buffer A. The column was then washed with the same buffer until the OD values at a wavelength of 280, the effluent was not greater than 0.01, i.e. until the polypeptide began to wash out. The polypeptide was suirable buffer solution B (20 mm Na ₂ HPO ₄ , 20 mm NaH ₂ PO ₄ , 500 mm NaCl, 250 mm imidazole, pH 8.0). The eluate was collected in fractions of 500 μl and OD280 values were measured therein. The fractions with the highest OD280 values were pooled and dialyzed against distilled water for 12 hours.

The molecular weight of StV was determined using 12% SDS-PAGE, comparing the range of the StV polypeptide with the range of proteins of known molecular weight. The molecular weight of the recombinant StV polypeptide was approximately equal to 130 kDa (figure 3).

Thus, the recombinant StV polypeptide contains the amino acid sequence of a surface polypeptide encoded by the gene of the HBV serotype strain 090R Ia.

A polypeptide with the indicated sequence is covalently linked to sixteen amino acid residues encoded by PQE-30. The resulting amino acid sequence of the StV polypeptide consists of 1228 amino acid residues (Figure 2).

Example 3. Dynamics of changes in the level of StV-specific IgG in the blood of experimental animals after immunization.

The humoral immune response to the recombinant StV protein was studied in outbred white mice (males weighing 16-18 g) and rabbits (females weighing 2.5 kg). Mice were immunized twice and subcutaneously with an aluminum hydroxide adjuvant. Mice were injected twice with 0.2 ml of StV with adjuvant in a volume ratio of 1: 1. The concentration of the StV polypeptide was 20 μg per mouse.

Rabbits were immunized twice and subcutaneously with Freund's complete adjuvant. Rabbits were injected subcutaneously twice with 0.5 ml of StV with adjuvant in a volume ratio of 1: 1. The concentration of the polypeptide was 100 μg per rabbit.

StV-specific IgG was determined using enzyme-linked immunosorbent assay (ELISA). 100 μl of recombinant StV polypeptide at a concentration of 2 μg / ml was added to the wells of a polystyrene tablet with a high sorption capacity (Costar, USA). Sorption was carried out in 100 mm bicarbonate buffer (pH = 9.45) for 16-20 hours at 4 ° C. Then the contents were removed from the plate and 150 μl of buffered saline solution, pH = 7.4 (PBS) containing 0.05% Tween-20 (PBST) was added to the wells. Incubation was carried out at 37 ° C for 30 min. The contents of the tablet were removed and washed three times with PBST. Then, immune or normal (as a negative control) serum (with an initial dilution of 1: 100) was introduced into the wells of the tablet with a subsequent dilution step of two. All dilutions were performed in PBST and each sample was duplicated. Incubation was carried out for 1 hour at 37 ° C. Next, the contents of the plate were removed and the PBST plate was washed twice. After that, 100 μl of A-PX conjugate (staphylococcus A protein covalently linked to horseradish peroxidase) at a concentration of 10 ^-7 mol / L was added to the wells. After one hour incubation at 37 ° C, the contents of the plate were removed and the PBST plate and once PBS were washed three times to avoid the inhibitory effect of Tween-20 on the enzymatic activity of peroxidase. Then, to visualize the reaction, 100 μl of the substrate mixture (0.5 μg / ml o-phenylenediamine in 150 mM phosphate-citrate buffer (pH = 5.0), 10 μl of 30% hydrogen peroxide) was prepared immediately before application. The plate was incubated for 30 min in the dark at room temperature and the reaction was stopped by adding 30 μl of 50% concentrated sulfuric acid to the wells. The reaction was recorded at a wavelength of 492 nm using a Titertek Multiskan device (USA).

The titer of specific antibodies was determined at different times from the start of immunization. The results presented in figure 5 "Dynamics of changes in the titer of antibodies to recombinant StV protein after immunization of mice" illustrate the development of a humoral immune response to the introduction of a recombinant StV polypeptide. In the presented experiment, it is seen that the formation of class G immunoglobulins begins on the 20th day of the experiment, and the maximum titer of antibodies to the StV protein was observed on the 40th day from the start of immunization of mice and amounted to 1.6 · 10 ^-4 . Subsequently, a gradual decrease in antibody titer by day 60 was observed. The total duration of antibody circulation in the blood of the observed animals reached 6 months.

Example 4. The study of the protective properties of StV-specific antibodies on a model of HBV infection in mice.

The protective efficacy of StV-specific antibodies in vivo was studied in a mouse model of generalized infection. It is known that under these conditions the opsonophagocytic reaction is the leading defense mechanism.

In in vivo experiments, laboratory mice were injected subcutaneously with the StV polypeptide according to the immunization schedule shown in Example 3. At the peak of the immune response (day 40 from the start of immunization), a suspension of HBV was administered intraperitoneally to the experimental and control groups of mice (LD ₅₀ = 2, 7 × 10 ⁷ CFU / ml). Infection was monitored by seeding streptococci from the splenic suspension of mice on medium with blood agar at different times after the introduction of HBV. Figure 6 "Dynamics of the streptococcal focus in the mouse spleen" shows the content of streptococci in the spleen of experimental animals at various stages of the infection process.

Example 5. The study of the opsonizing efficacy of rabbit anti-StV antibodies.

The main protective mechanism of a macroorganism against bacterial infection is the process of phagocytosis mediated by specific antibodies (opsonophagocytosis). The opsonizing activity of rabbit anti-StV antibodies in the composition of hyperimmune serum was studied on a monolayer of mouse peritoneal macrophages. Macrophages were induced two days before receiving peritoneal exudate by administering to mice 0.5 ml of 10% peptone intraperitoneally. Peritoneal exudate from 4-5 mice was washed with IMDM nutrient medium (Iscove's Modified Dulbecco's Media, BioloT, Russia), cells were washed, suspended at a concentration of 1 million / ml and plated in a volume of 0.2 ml on 96-well plates for tissue culture, 1.0 ml per 24-well tissue culture plate containing coverslips (Sarstedt, Germany). After 1 hour of incubation, non-adherent cells were washed and macrophages continued to be cultured in IMDM medium with 10% ETS (fetal calf serum, BioloT, Russia) in the presence of streptomycin (100 μg / ml) and penicillin (100 U / ml). After a day, the monolayer was washed from antibiotics and a suspension of HBV was added in IMDM medium at a concentration of 1 × 10 ⁷ CFU / ml. Bacteria were pre-incubated for 30 minutes with normal and immune rabbit serum diluted 50 times in IMDM medium. The contact of bacteria with macrophages lasted 30 min, after which the monolayer was thoroughly washed from streptococci not attached to the surface of the cells. All the steps described above were carried out at 37 ° C in an atmosphere of 5% CO ₂ . Further, the preparations were fixed for 30 min with absolute alcohol and sequentially stained with azide and azure for 30 min. The monolayer was thoroughly washed from paint residues. Microscopy on coverslips examined at least 100 cells in three parallel samples, counting the percentage of macrophages infected with streptococci and the average number of cocci per cell. The degree of infection was assessed by the phagocytic index (PI), which is a product of both indicators.

The results of the opsonophagocytic test are presented in table 2. The multiplicity of the increase in the infection index was from 3.1 to 14.8 when treating streptococci with immune rabbit serum compared with the infection index when treating streptococci with normal rabbit serum, which indicates the anti-StV antibodies are effective against HBV . Strain 7 contains the sspB1 gene, but strains 60/59 and 5/70 do not, which indicates the importance of the presence of the sspB1 gene for the process of opsonization of group B streptococci. The data presented show that StV-specific antibodies are able to opsonize group B streptococci, providing their accelerated elimination.

Table 1 Primer sequence Title Sequence for TCG GTA CCA AGG CAA AGA CCG AAG GAA T rev AGG CAA GCT TCT GAC AAA TTT CCC GTT CTG

table 2 Results of an opsonophagocytic monolayer test of peritoneal mouse macrophages using normal and immune rabbit sera. Strain SGV Serum Indicators of opsonophagocytosis Infected macrophages (%) The average number of cocci per cell FI The ratio of increasing FI 60/59 normal 71 9 639 3,1 anti-stv 90 21.9 1971 5/70 normal 64 6.0 384 7.3 anti-stv 92 30.5 2806 7 normal 45 5.7 257 14.8 anti-stv 97 39.3 3812

Claims (4)

1. Recombinant StV polypeptide with a protective effect against Streptococcus agalactiae, characterized by the amino acid sequence shown in figure 2, intended for the preparation of a vaccine for the immune defense against group B streptococci. 2. Recombinant stV DNA encoding the recombinant StV polypeptide according to claim 1, the nucleotide sequence of which is shown in figure 1. 3. Recombinant plasmid DNA PQE-stV, which is a plasmid DNA PQE-30, carrying the recombinant stV DNA according to claim 2, providing a recombinant StV polypeptide according to claim 1. 4. The producer strain Escherichia coli M15-StV, producing the recombinant StV polypeptide according to claim 1, obtained on the basis of Escherichia coli M15 and containing the recombinant plasmid DNA PQE-stV according to claim 3.

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