RU2085584C1 - Method of preparing recombinant tularemia microorganisms - producers of virulence factors, recombinant strain of francisella tularensis subspecies, holarctica r5s - producer of virulence factor francisella tularensis nearctica shu, recombinant strain of francisella tularensis holarctica rn4 - a producer of virulence factor pseudomonas tularensis subspecies holarctica r1a - a producer of virulence factor francisella tularensis nearctica b 399 a cole - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, recombinant microorganisms. SUBSTANCE: method involves insertion of the foreign chromosomal or plasmid DNA in the parent microorganism by methods of genetic engineering followed by selection by sowing recombinant microorganism on nutrient media. The parent microorganism is the strain Francisella tularensis subspecies holarctica R, VKPM B-6854. Method ensures to obtain virulence factors of protein and polysaccharide nature. By this method the following recombinant strains were obtained: Francisella tularensis subsp. holarctica R55 VKPM B-6853 - a producer of virulence factor Fr. tularensis nearctica Shu, Fr. tularensis subsp. holarctica RN4, VKPM B-6855 - producer of virulence factor Ps. pseudomallei C-141, Fr. tularensis subsp. holarctica R1A VKPM B-6852 - a producer of virulence factor Fr. tularensis nearctica B-399 A Cole. Strains can be used for production of highly specific diagnostic, sera, chemical vaccines, immunomodulators and other preparations. EFFECT: improved method of preparing.

Description

 The invention relates to biotechnology, and more specifically, to a method for producing recombinant microorganisms of producers of virulence factors and to new strains of Francisella tularenssis subsp. holarctica R5S, RN4 and RIA obtained by this method.

 The method may find application for the production of biologically active substances of a protein nature, for example, known immunomodulating proteins, such as interferon, interleukin, colony-stimulating factors, tumor necrosis factors, and others.

 New strains can be used for the production of highly specific diagnosticums and serums, chemical vaccines, immunomodulators used in medicine and veterinary medicine.

 Known methods for the transformation of chromosomal or plasmid DNA for each specific microorganism have their own individual characteristics, which ultimately allow you to select genetically modified variants of various microorganisms, which received obvious selective signs due to foreign genes of chromosomal DNA or plasmids. Therefore, of the many described methods for the transformation of chromosomal and plasmid DNA, only one or two are most optimal for each specific microorganism, and the rest either require significant refinement or are ineffective. This limiting factor in the transfer of foreign genes into the microorganism is primarily associated with the recipient strain.

 The problem of finding a new recipient strain to expand the possibilities of obtaining both known and new genetically engineered products is relevant.

 The known strain of Francisella tularensis subsp. holarctica R (known as the R-form of strain 15 of the Research Institute of EM, or the R-form of LVS strain Fr.tularensis subsp. holarctica, or pop S-mutant Fr. tularensis subsp.holarctica), found as concomitant cells in the vaccine strain against tularemia ( Olsufiev N.G. Taxonomy, microbiology and laboratory diagnostics of the causative agent of tularemia, M, 1975, p.200; Meshryakova I.S. Journal of Hygiene, Epidemiology, Microbiology, Immunology (Czechoslovakia), 1970, v.14, p.420-420 ; Kudelina R.I. Journal of Microbiology, 1973, N 12, pp. 98-101).

 The use of this strain in the literature is not described.

Known microorganisms of the Escherichia coli family and pseudomonads to a greater or lesser extent have a number of negative characteristics, which include the following:
the presence of a pronounced cell wall, which primarily affects the technology for obtaining the final product, since on the one hand it determines the use of expensive reagents to destroy the cell wall or the use of powerful ultrasound systems (which is harmful for personnel), on the other hand, the bacterial cell wall has high pyrogenicity and immunogenicity, which makes it difficult to completely clean the final product from unwanted impurities;
high biochemical activity, usually associated with protease activity, reduces the amount of pure product, since when biomass is obtained, bacteria are destroyed, and protein is isolated, protease enzymes destroy the protein foreign to the cell produced by recombinant plasmid DNA introduced into the host strain;
dissociation of microorganisms into a form that does not allow them to be superproducers of genetically engineered proteins, for example for E. coli, it has been shown that the presence of an own restoration-modification system in the host cell significantly affects the stability of the recombinant plasmid and the expression of genetically engineered proteins;
the presence of their own phages, which in the industrial production of biomass of microorganisms can nullify all the results of lysis of microbial cells, which occurs either due to the introduction of virulent phages from the external environment, or due to the activation of a moderate phage inside the cell;
pathogenicity for animals and humans, which greatly impedes their use.

 All of these characteristics cause an increase in the cost of the final genetic engineering product and interfere with the study of the physicochemical structure of the cloned biologically active compound.

 These strains are new and are not described in the literature.

 The basis of the invention is the task of selecting a new recipient strain of a foreign chromosomal or plasmid DNA to develop a method that allows you to create new recombinant strains producing biologically active substances of both protein nature and polysaccharide, expand the ability to obtain and use genetically engineered products, as well as the possibility studying the nature of the virulence of various microorganisms, including pathogens of dangerous and especially dangerous infectious diseases.

 The problem is solved in that in the method for producing recombinant microorganisms producing biologically active substances, which involves introducing foreign chromosomal or plasmid DNA into the original microorganism by genetic engineering, followed by selective selection by plating the nutrient medium of the recombinant microorganism according to the invention, the initial microorganism is used strain Fracisella tularensis subspecis holarctica R, VKPM B-6854.

 This method allows you to create new recombinant strains - producers of virulence factors.

 The unique ability of the strain Francisella tularensis subspecies holarctica R, VKPM B-6854 allows it to clone and achieve high expression of not only proteins, but also specific polysaccharides. And since for many causative agents of dangerous infections one of the components of virulence is a polysaccharide, the study of its expression in the avirulent strain will not only understand the physical nature of the virulence of many microorganisms, but also on the basis of recombinant microorganisms to obtain new highly effective chemical vaccines, highly specific diagnostics and serums, necessary for veterinary medicine and medicine.

 The problem is also solved by the fact that according to the invention, a new recombinant strain of Francisella tularensis supspecies holarctica R5S is proposed - the producer of virulence factor Francisella tularensis nearctica Shu, deposited on 08.08.94 at the Russian National Collection of Industrial Microorganisms of the State Scientific Research Institute of Genetics and registered for N VKP B -6853, and the recombinant strain Francisella tularensis subspecies holarctica RN4 producer of virulence factor Ps.pseudomallei C-141, deposited 08.08.94 in the Russian national collection of industrial microorganisms shock research institute of genetics and registered for N VKPM B-6855, as well as the recombinant strain Francisella tularensis supspecies holarctica RIA producer of virulence factor Francisella tularensis nearctica B-399A Cole deposited O8.08.94 in the Russian National Collection of Industrial Microorganisms of the State Scientific Research Institute genetics and registered for N VKPM B-6852.

 The use of these strains as producers of virulence factor Fr. tularensis nearctica Shu, virulence factor of Ps. pseudomallei C-141 and virulence factor Fr. tularensis nearctica B-399A Sole opens up the possibility of studying their physico-chemical basis, which until now remains unknown. Isolation and purification of the virulence factor from these recombinant strains will provide highly effective chemical vaccines, highly specific diagnostics and serums, as well as immunomodulators that will be widely used.

 The method is as follows.

A chromosomal or plasmid DNA is isolated containing the necessary Fr. for transmission to the original strain. tularensis subsp. holarctica R, VKPM B-6854 gene (s). The initial strain is plated on a solid nutrient medium and placed in a thermostat. After growing, the culture is washed with saline or buffer solution with gelatin and centrifuged. The supernatant is drained and the microbial cell pellet is suspended to a cell density of 10 10 cells / ml. To the resulting cell suspension add chromosomal or plasmid DNA in an amount of 1.0-5.0 μg, mix and incubate at O ^o C or at room temperature. After incubation, this mixture is either heated at a temperature of 42 ^o C for one minute (heat shock) and placed in a thermostat at 37 ^o C and after 1 h seeded on solid nutrient media (in the case of the introduction of chromosomal DNA), or placed in kelvinator (-55 ^o C), then thawed and seeded on a solid nutrient medium (in the case of the introduction of plasmid DNA). Sown solid nutrient medium is placed in a thermostat. After 18 hours, the grown culture is washed off and a series of inoculations is again carried out with the selection of colonies of a recombinant microorganism (the cells of the initial strain form a capsule upon acquisition of chromosomal or plasmid DNA, which leads to a sharp change in the appearance of the formed colonies). The results are taken into account for 3-5 days as the colonies of recombinant microorganisms grow. As a control, cells of the original strain are used, passing them through all stages, except for the addition of chromosomal or plasmid DNA.

 The possibility of implementing the method is based on the knowledge of the features of the strain Francisella tularensis holaretica R, VKPM B-6854, in which this microorganism acquires the ability to perceive foreign chromosomal or plasmid DNA through transformation, to stably store and express the necessary biologically active substances.

Cultural and morphological properties of the original strain of Francisella tularensis holarctica R, VKPM B-6854 (R form strain 15 of the Research Institute of EM). Bacteria are small cocciform and rod-shaped cells 0.2-0.7 μm in size 2-2.5 times larger than cells of a virulent or vaccine strain, immobile, gram-negative, the capsule is not expressed. this aerobic strain does not grow on ordinary media, auxotroph is cultivated at 37 ^o C in vitamins rich in vitamins, for example, McCoy vitelline medium, agar containing cysteine (0.05-0.1%) and 1% glucose with the addition of 5 -10% defibrinated blood (black albumin or hemoglobin). Of liquid media, Sheger media are used. Sowing culture on nutrient medium withstand from two to five days. The avirulent form gives a delay in cell growth in time (per day) in comparison with the virulent form, which is most noticeable after prolonged storage (growth delay can be 2-5 days). You can grow the R-form on media with brain, splenic, hepatic tissues, heart extracts.

 On dense opaque media, R-shaped cells of strain 15 of the Research Institute of EM form flat transparent gray colonies. With further reseeding, the culture stably retains the acquired characteristics.

 The cell wall, characteristic of all other gram-negative microorganisms, is absent, and therefore the cells of the R-form of strain 15 of the Research Institute of EM are sensitive not only to low concentrations of various detergents (sodium dodecyl sulfate, X-100 triton, sodium deoxycholate), but even to distilled water with pH is less than 6 or greater than 10, which partially lyses a suspension of the microorganism during the day. A lytic effect on tween-80 and bile R-cells was also found. However, when the culture is grown in large fermenters (where the pH of the medium and other parameters are strictly controlled), lysis does not occur, and the concentration of cells in the nutrient broth can reach large values (over one hundred billion cells / ml) in comparison with other known microorganisms.

 The ultra-thin structure of R-shaped cells is characterized by the presence of an extensive osmiefil capsule-like cover connected with the cell and not lost in ultrathin sections. The cell cytoplasm is granular, DNA is located in its central part.

 Biochemical properties. The cells of the R-form of strain 15 of the Research Institute of EM have low biochemical and protease activity. They break down proteins with the release of hydrogen sulfide, ferment glucose and maltose on the Downs medium, do not ferment glycerol, sucrose and lactose, intermittently ferment dextrose and fructose, and do not have citrullinureidase activity. The biochemical properties of R-form cells are unstable and depend not so much on the properties of the strain itself as on the composition of the nutrient media and the ability of the microorganism to ferment proteins whose degradation products mask the acids formed simultaneously with them. The main way of assimilation of amino acids is oxidative deamination. The R-form contains the enzyme oxidoreductase.

 Serological and immunochemical properties. Living cells of the R-form of strain 15 of the Research Institute of EM with the introduction of laboratory animals (mice, hamsters, guinea pigs, rabbits and others) do not cause an immune response of macroorganisms, therefore, it is impossible to obtain specific sera. The R-form contains a depleted composition of glycolipids, therefore it is not able to enter into serological reactions with the erythrocytic diagnosticum Fr.tularensis subsp. holarctica (due to the lack of species-specific antigens). In serological reactions with specific sera obtained on vaccine or virulent strains, a suspension of R-form cells forms fine-grained agglutination (all other cells of the Francisella tularensis family form coarse cotton agglutination). In immunoelectrophoresis, the lysate of the R-form of strain 15 of the Research Institute of EM does not give a specific immune profile characteristic of the polysaccharide protein complex of the parent microorganism. R-shaped cells are highly sensitive to the bactericidal action of the blood serum of warm-blooded animals.

 Plasmids and phages of this species. The R-form of strain 15 of the SRI EM, like the Francisella family, does not have its own plasmids and phages.

 Genetic features, markers. R-shaped cells are stable and under no natural or artificial manipulations with them do not acquire the signs of the parent strain 15 of the Research Institute of EM or any other, that is, they are unchanged in phenotypic and genetic characteristics. The R-form is naturally resistant to polymyxin, penicillins, macrolides (erythromycin, oleandomycin), highly sensitive to tetracycline antibiotics, aminoglycosides, chloramphenicol, rifampicin, nalidixic acid.

 Pathogenicity. One of the features of living R-cell cells, unlike the parent strain 15 of the Research Institute of EM, is that when they are introduced to laboratory animals (mice, hamsters, guinea pigs, rabbits) at a concentration of at least one billion cells / ml, they do not cause toxic reactions, do not possess pyrogenicity and do not form specific immunity.

 The activity and productivity of the strain. The presence of specific polysaccharides that determine the characteristic differences of this microorganism from other gram-negative bacteria, as well as the expression of exo- and endotoxin in the R-form of strain 15 of the Research Institute of EM, were not detected.

Conditions and composition for storage and maintenance. Museum culture of R-shaped cells of strain 15 of the Research Institute of EM is stored at a temperature of + 4 ^o C on the jambs of McCoy's medium for up to 6 months, in a freeze-dried state for up to 5 years.

 Cultivation medium. Dense medium with black albumin or hydrolyzed hemoglobin, Scherer's liquid medium.

 Technological features during cultivation. There are no technological features during cultivation. The R-form of strain 15 of the Scientific Research Institute of EM is cultivated as a regular vaccine of the 15th Scientific Research Institute of EM or a virulent strain of the tularemia pathogen Fr. Tularensis subsp. holarctica.

 Resistant to disinfectants. R-shaped cells are killed within 2-5 min by a 2-3% solution of lysol, 3-5% solution of carbolic acid, 3% solution of hydrogen peroxide, 1: 1000 solution of mercuric chloride, are inactivated by ultraviolet radiation for several minutes, and are easily destroyed when exposure to ultrasound.

The above properties of the R-form of strain 15 of the Research Institute of EM make this microorganism a unique recipient, on the basis of which it is possible to create recombinant microorganisms of producers of biologically active substances. None of the known strains of producers of biologically active substances has a combination of the following advantages:
complete avirulence of cells for animals and humans, therefore, the safety for the health of personnel who are constantly working with the R-form or producer of biologically active compounds based on it, in industrial production or laboratory conditions;
non-toxicity of lysates for all species of animals and humans, which also means personnel safety when isolating biologically active compounds from producer cells and a lower degree of purification of the final product from bacterial impurities, which will reduce the cost of the resulting preparation;
the inability of the R-form of strain 15 of the Research Institute of EM to exist independently in nature (laboratory microorganism) indicates the environmental safety of the production and use of producers of biologically active compounds based on it;
small cell sizes allow in one volume to receive a larger amount of biomass in comparison with other microorganisms, and hence a greater yield of the final product;
low protease activity of the microorganism can significantly reduce the loss of the final product during its isolation;
the absence of a pronounced cell wall makes it easy to destroy cells without the use of ultrasound (unhealthy personnel) or expensive reagents that destroy the cell wall;
the absence of intrinsic phages in this microorganism eliminates losses during industrial production of cell culture in large volumes, since their ingress into the fermenters from the external environment leads to a halt and loss of the accumulated biomass.

 Knowing the above properties of the strain Francisella tularensis subspecies holaretica R, VKPM B-6854 when producing recombinant strains by transformation method provides a unique opportunity to study the nature of the virulence of very many microorganisms, pathogens of dangerous and especially dangerous infectious diseases (tuberculosis, salmonellosis, dysentery, mellioidosis, tularemia and many others ), as well as create strains producing virulence factors.

 To study the survival, contamination, and identity of this strain, the culture was diluted with saline according to the turbidity standard to 5 billion cells per ml and sequentially triturated to 1000 cells per 1 ml. 0.1 ml of the suspension of cells from each dilution of the microorganisms were seeded on solid nutrient media containing the necessary components for their growth. After culturing the culture on Petri dishes, the following was noted.

 By the ability to stain according to Gram and susceptibility to Safranin, according to the morphology and tinctorial properties of the cells in the smear correspond to Fr.tularensis.

 A visual examination of the cups with the grown culture and smears under microscopy is not contaminated with extraneous microflora.

The survival rate of Francisella tularensis subspecies holarctica R VKPM B-6854 corresponds to a concentration of one hundred million living microbial cells in 1 ml. The residual virulence (pathogenicity) of this strain was studied on outbred white mice with subcutaneous infection. Suspensions of microorganisms in working dilutions from 100 to 5 • 10 ⁹ cells according to the optical turbidity standard were used for infection. In parallel, we determined the living microbial cells in the prepared suspensions by seeding to single colonies on nutrient media containing the necessary components for their growth (Difco medium containing hemoglobin, glucose and cysteine in the required proportions). The compliance of the two methods for determining the concentration of microorganisms was 50%
In the experiment there were nine groups of six animals each. Outbred mice were infused subcutaneously at doses from one hundred cells per mouse to 5 x 10 ⁹ cells per mouse with a tenfold step in a volume of 0.1 ml. As a control, a group of animals was used that was not exposed to infection. The observation period for the experimental and control groups was 21 days.

 As a result of the studies, the absence of residual virulence of the studied strain at a dose of up to 5 billion microbial cells per mouse was established. Thus, the strain Fr. tularensis subsp. holarctica R, VKPM B-6854 can be assigned to the 5th group of microorganisms according to the international classification (complete avirulence).

Obtained by this method, a new recombinant strain of Fr.tularensis subsp. holarctica R5S VKPM B-6853 producer of virulence factor Fr. tularensis nearctica Shu is characterized by the following main properties:
Cultural and morphological properties. Bacteria are small cocciform and rod-shaped cells measuring 0.1-0.5 μm in size 2-2.5 times smaller than Fr. cells tularensis subsp. holarctica VKPM B-6854, motionless, gram-negative, form a pronounced capsule. The indicated R5S strain does not grow on ordinary media, auxotroph is cultivated at 37 ^° C in media rich in vitamins, for example, McCoy vitelline medium, agar containing cysteine (0.05-0.1%) and 1% glucose with the addition of 5-10% defibrinated blood (black albumin or hemoglobin). From liquid media uses Scherer's medium. Culture growth lasts from two to five days. After prolonged storage, the R5S strain gives a delay in cell growth over time. Strain R5S can be grown on media with brain, splenic, hepatic tissue, heart extracts. Museum culture on the doorposts of McCoy’s environment is stored for up to 6 months. On dense opaque media, cells of the indicated strain, after seeding from organs of outbred mice, are in the SR form and form convex translucent colonies of white color with a blue tint. These cells have high immunogenicity and residual virulence for white mice. after 5-6 transfers in solid nutrient media, the recombinant culture reduces virulence for white mice, but the original strain practically does not dissociate. To maintain a certain level of residual virulence of the recombinant strain, they periodically need to infect outbred mice and isolate a more virulent R5S culture from the organs of dead animals (as follows from the instructions for working with the known vaccine strain 15 of the Research Institute of EM).

 Biochemical properties. R5S strain cells break down proteins with the release of hydrogen sulfide, glucose and maltose are fermented on Down's medium, glycerol, sucrose and lactose are not fermented, dextrose and fructose are constantly fermented. Insensitive to erythromycin, oleandomycin, polymyxin, penicillin, streptomycin, does not have citrullinureidase activity.

 Immunochemical and serological properties. The rabbit experimental serum obtained for the recombinant strain R5S is poor in antibodies compared to the experimental rabbit and commercial sera obtained for strain 15 of the Research Institute of EM. However, this serum has a higher specificity for Fr.tularensis compared to commercial serum.

 Relation to phages of this species. The recombinant strain R5S, like the entire Francisells family, does not have its own phages.

 Pathogenicity for laboratory animals. The recombinant strain R5S has an average residual virulence for outbred mice, is slightly virulent for guinea pigs and not virulent for rabbits.

 A method of obtaining a recombinant strain. Genetic engineering, recipient cells of Fr.tularensis subsp. holarctica R, VKPM B-6854 is transformed with a chromosomal DNA fragment encoding the synthesis of virulence factor from tularemia pathogen Fr.tularensis nearctica Shu.

 Genetic features. Cells of the recombinant strain R5S are naturally resistant to penicillins, polymyxins, macrolides, and the chromosome genes of the virulence factor of the tularemia pathogen are marked with streptomycin resistance.

 Strain productivity. The recombinant strain R5S is a producer of virulence factor Fr.tularensis nearctica Shu, which is determined by immunochemical and biochemical methods as the synthesis of a protein complex and a specific polysaccharide.

The conditions and composition of the media for storage and support of the recombinant strain. The museum culture of strain R5S is stored at a temperature of +4 ^o C on the jambs of McCoy's medium for up to 6 months, in a freeze-dried state for up to 5 years.

 The medium for the cultivation of the strain. Dense medium with black albumin, Scherer's fluid medium.

 The indicated recombinant strain R5S was tested for survival, contamination, identity and residual virulence at the Research Center for Toxicology and Hygienic Regulation of Biological Preparations under the Ministry of Health and Medical Industry of the Russian Federation and the Federal Administration for Biomedical and Extreme Problems. The tests were carried out according to the method similar to that described for the original strain.

 The test results of the specified strain R5S showed the following.

 By the ability to stain according to Gram and susceptibility to Safranin, according to the morphology and tinctorial properties of the cells in the smear correspond to Fr.tularensis.

 A visual examination of the cups with the grown culture and smears under microscopy is not contaminated with extraneous microflora.

 Survival corresponds to a concentration of one hundred million living microbial cells in 1 ml.

As a result of the studies established the presence of residual virulence of strain R5S. LD ₅₀ for white outbred mice was 25 thousand microbial cells. The specificity of the death of mice is confirmed by the presence of the growth of the culture Fr.tularensis subsp. holaretica R5 from organ prints on culture media. The death of animals in the control group was absent.

 Thus, in accordance with the established residual virulence, the R5S strain can be assigned to the 4th group of microorganisms according to the international classification.

 Recombinant Fr. strain tularensis subps. holarctica RIA, VKPM B-6852 is characterized by the following basic properties.

Cultural and morphological properties. Bacteria are small cocciform and rod-shaped cells 0.2-0.7 microns in size, motionless, gram-negative, form a capsule. The recombinant RIA strain does not grow on ordinary media, auxotroph is cultivated at 37 ^° C in media rich in vitamins, for example, McCoy vitelline medium, agar containing cysteine (0.05-0.1%) and 1% glucose with 5- 10% defibrinated blood (black albumin or hemoglobin). From liquid media, Scherer's medium is used. Culture growth on nutrient media lasts from two to five days. After prolonged storage, this strain gives a delay in cell growth over time. RIA strain can be grown on media with brain, splenic, hepatic tissue, heart extracts. Museum culture on the shoals of McCoy’s environment is stored for up to 6 months. On dense opaque media, cells of the indicated strain, after plating from organs of outbred mice, are in the SR form and form white colonies with a blue tint. These cells have high immunogenicity and residual virulence for white mice. After 5-6 passages on solid nutrient media, the recombinant culture reduces virulence for white mice, but practically does not dissociate into the original strain. To maintain a certain level of residual virulence of the recombinant strain, they periodically need to infect outbred mice and isolate a more virulent RIA culture from the organs of dead animals (in accordance with the instructions for working with the known vaccine strain 15 of the Research Institute of EM).

 Biochemical properties. RIA strain cells break down proteins, maltose, do not ferment glycerin, sucrose and lactose, and dextrose and fructose are not constantly fermented. Not sensitive to erythromycin, oleandomycin, polymyxin, penicillin, streptomycin, do not have citrulline urease activity.

 Immunochemical and serological properties. The rabbit experimental serum obtained for the recombinant RIA strain is poor in antibodies compared to the experimental rabbit and commercial serum obtained for strain 15 of the Research Institute of EM. However, this serum has a higher specificity for Fr.tularensis compared to commercial serum. In reactions with specific sera on glass, a suspension of RIA cells forms coarse cotton agglutination.

 Relation to phages of this species. The recombinant RIA strain, like the entire Francisella family, does not have its own phages.

 Pathogenicity for laboratory animals. The recombinant RIA strain is highly virulent for outbred mice, slightly virulent for guinea pigs, and avirulent for rabbits.

 A method of obtaining a recombinant strain. Genetic engineering. Fr. tularensis subsp. holarctica R, VKPM B-6854 with a chromosomal DNA fragment introduced by transformation from Fr. tularensis nearctica B-399 A Cole.

 Genetic features. Cells of the recombinant strain RIA are naturally resistant to penicillins, polymyxins, macrolides, chromosomal virulence factor genes are marked with streptomycin resistance.

 Strain productivity. Recombinant PIA strain is a producer of virulence factor Fr. tularensis nearctica B-399 A Sole, which is determined by immunochemical and biochemical methods as the synthesis of a complex of protein and a specific polysaccharide.

The conditions and composition of the media for storage and support of the recombinant strain. The museum culture of the RIA strain is stored at a temperature of +4 ^o C on the jambs of McCoy's medium for up to 6 months, in freeze-dried state for up to 5 years.

 The medium for the cultivation of the strain. Dense medium with black albumin, Scherer's fluid medium.

Tests of the indicated strain for survival, contamination and identity, conducted similarly to the above, showed that for the RIA strain:
according to the ability to stain according to Gram and susceptibility to Safranin, according to the morphology and tinctorial properties of the cells in the smear correspond to Fr.tularensis;
a visual examination of the cups with the grown culture and smears under microscopy is not contaminated with extraneous microflora;
survival corresponds to a concentration of fifty million living microbial cells in 1 ml.

As a result of the studies established the presence of residual virulence of the specified strain. LD ₅₀ for white outbred mice was 30 thousand microbial cells. The specificity of the death of mice was confirmed by the presence of the growth of the Fr.tularensis holarctica RIA culture from organ prints on culture media. The death of animals in the control group was absent. Thus, in accordance with the established residual virulence, this RIA strain can be assigned to the 4th group of microorganisms according to the International classification.

 Recombinant Fr. strain tularensis subsp. holarctica RN4, VKPM B-6852 is characterized by the following basic properties.

Cultural and morphological properties. Bacteria are cocciform and rod-shaped cells 0.3-0.8 μm in size 3-4 times larger than Fr.tularensis subsp cells. holarctica R, VKPM B-6854 are motionless, gram-negative, form a capsule. The indicated recombinant strain RN4 does not grow on ordinary media; auxotroph is cultivated at 37 ° C in media rich in vitamins, for example, McCoy vitelline medium, agar containing cysteine
0.05-0.1% glucose 1% and 5-10% defibrinated blood (black albumin or hemoglobin). From liquid media, Scherer's medium is used. Culture growth on nutrient media lasts from two to five days. After prolonged storage, the indicated strain RN4 gives a delay in cell growth over time. Strain RN4 can be grown on media with brain, splenic, hepatic tissue, heart extracts. Museum culture on the doorposts of McCoy’s environment is stored for up to 6 months. On dense opaque media, the cells of the specified strain RN4 after seeding from organs of outbred mice are in the SR-form and form convex translucent colonies of white color. These cells have high immunogenicity and residual virulence for white mice and golden hamsters. After 5-6 passages on solid nutrient media, the recombinant culture reduces virulence for mice and golden hamsters, however, EMR practically does not dissociate into the R-form of strain 15. To maintain a certain level of residual virulence of the recombinant strain, they periodically need to infect outbred mice and isolate a more virulent RN4 culture from the organs of dead animals (in accordance with the instructions for working with the known vaccine strain 15 of the Research Institute of EM).

 Biochemical properties. Cells of the specified strain RN4 break down proteins with the release of hydrogen sulfide. glucose and maltose are fermented on Downs medium, glycerol, sucrose and lactose are not fermented, dextrose and fructose are constantly fermented. Not sensitive to erythromycin, oleandomycin, polymyxin, penicillin, streptomycin, do not have citrullinureidase activity.

 Immunochemical and serological properties. The rabbit experimental serum obtained for the recombinant strain RN4 is poor in antibodies compared to the experimental rabbit and commercial sera obtained for strain 15 of the Research Institute of EM and has high specificity for Fr.tularensis compared to commercial serum.

 Relation to phages of this species. The recombinant strain RN4, like the entire Francisella family, does not have its own phages.

 Pathogenicity for laboratory animals. The recombinant strain RN4 has a high residual virulence for mice and golden hamsters, is slightly virulent for guinea pigs, and avirulent for rabbits.

 A method of obtaining a recombinant strain. Genetic engineering. Recipient cells Fr. tularensis subsp. holarctica R, VKPM B-6854 is transformed with a chromosomal DNA fragment on a vector plasmid encoding the synthesis of virulence factor from the causative agents of the mellioidosis Ps.pseudomallei C-141.

 Genetic features. Cells of the indicated recombinant strain RN4 are naturally resistant to penicillins, polymyxins, macrolides, chromosomal genes of the virulence factors of the causative agent of mellioidosis are marked by streptomycin resistance.

 Strain productivity. The indicated recombinant strain RN4 is a producer of the virulence factor of the causative agent of the mellioidosis Ps.pseudomallei C-141, which is determined by immunochemical and biochemical methods as the synthesis of a protein complex and a specific polysaccharide.

The conditions and composition of the media for storage and support of the recombinant strain. Museum culture is stored at a temperature of + 4 ^o C on the doorposts of McCoy's medium for up to 6 months, in freeze-dried state for up to 5 years.

 The medium for the cultivation of the strain. Dense medium with black albumin or hydrolyzed hemoglobin, Scherer's liquid medium.

Tests of the indicated strain RN4 for survival, contamination and identity, carried out similarly to the above, showed that for strain RN4:
according to the ability to stain according to Gram and susceptibility to Safranin, according to the morphology and tinctorial properties of the cells in the smear correspond to Fr.tularensis;
a visual examination of the cups with the grown culture and smears under microscopy is not contaminated with extraneous microflora;
survival corresponds to a concentration of fifty million living microbial cells. As a result of the studies established the presence of residual virulence of the specified strain RN4. LD ₅₀ for white outbred mice amounted to 35 thousand microbial cells. The specificity of the death of mice is confirmed by the presence of the growth of the culture Fr.tularensis subsp. holarctica RN4 from organ prints on culture media. The death of animals in the control group was absent. Thus, in accordance with the established residual virulence, the indicated recombinant strain RN4 can be assigned to the 4th group of microorganisms according to the International classification.

 The use of recombinant strains as producers of these virulence factors will allow us to study their physicochemical basis. This is fundamentally important, since the pathogens of infectious diseases belong to the category of dangerous or especially dangerous infections against which there is no specific protection in the form of a live or chemical vaccine. For genetic engineering, cloning in the avirulent recipient Fr.tularensis subsp. holarctica R, VKPM B-6854, a group of genes encoding the synthesis of a complex of polysaccharide and protein (proteins), responsible for the virulence of a specific pathogen, is a new direction in this area, not previously studied. Isolation and purification of the virulence factor from the recombinant strain will make it possible to obtain highly effective chemical vaccines, highly specific diagnostics and serums, as well as immunomodulators, which will be widely used in medicine and veterinary medicine.

 Example 1. Obtaining a recombinant strain of Fr.tularensis subsp. holarctica R5S, VKPM B-6853 producer of virulence factors Fr. tularensis nearctica Shu.

Microbial cells of the strain Francisella tularensis subspecies holarctica R, VKPM B-6854 are seeded on a solid nutrient medium (meat-peptone agar) containing 5% hemoglobin, 1% cysteine, 1% glucose, and incubated at 37 ^° C for 24 hours. The grown culture is washed with GS-buffer (1% gelatin, 0.85% sodium chloride) and centrifuged at + 4 ^° C and 6000 rpm for 10 minutes, the supernatant is drained, and the microbial cell pellet is suspended in the same buffer and washed with microbial cells by repeating the procedure twice. After washing, the precipitate is suspended in TM buffer (per 100 ml, g: sodium chloride 1.6 calcium chloride 0.6 magnesium sulfate 0.6; Tris-HCl 0.6; threonine 0.5; lysine 0.06; proline 0 , 4; cysteine 0.03; serine 0.02; spermine 0.004; arginine 0.04; aspartic acid 0.04; histidine 0.04; pH 6.2) to a cell density of 10 cells / ml, poured into 300 tubes μl and add 50 μl of chromosomal DNA containing virulence factor genes Fr.Tularensis nearctica Shu in an amount of 5 μg. This mixture was stirred and kept at 0 ^{° C.} for 30 minutes. After incubation of the mixture, it is heated at 42 ^o C for 1 min (heat shock) and kept in an incubator at 37 ^o C for 1 h. Then the mixture is sown on a solid nutrient medium of the same composition. A dense nutrient medium with a seeded suspension of cells is placed in a thermostat at 37 ^° C for 18 hours. After 18 hours, the grown cells are washed with GS-buffer, a suspension of 10 ¹¹ cells / ml is prepared and a series of inoculations are performed on solid nutrient media with selection of colonies of a recombinant microorganism ( upon acquisition of chromosomal DNA, the cells of the original strain form a capsule, which leads to a sharp change in the appearance of the colonies. The resulting colonies are convex, translucent, white). The results are taken into account for 3-5 days as the colonies of the recombinant strain grow. A recombinant strain of Fr.tularensis subsp. holarctica R5S producer of virulence factor Fr.tularensis nearctica Shu.

 As a control, the same microorganism is used, passing it through all stages except the addition of chromosomal DNA. On selective media, the cells of the original strain do not give colonies. Colonies of the obtained recombinant strain grown on selective media are checked for virulence, toxicity and immunogenicity by a known method.

 Example 2. Obtaining a recombinant strain of Fr.tularensis subsp. holarc tica RIA, VKPM B-6852 producer of the virulence factor Fr.tularensis nearctica B-399 A Cole.

Microbial cells of the strain Francisella subspecies holarctica R, VKPM B-6854 are seeded on a solid nutrient medium analogous to example 1 of the composition and incubated in a thermostat at a temperature of 37 ^o C for a day. The grown culture is washed with GS-buffer (1% gelatin, 0.85% sodium chloride) and centrifuged at + 4 ^° C and 6000 rpm for 10 minutes, the supernatant is drained, and the microbial cell pellet is suspended in the same buffer and washed microbial cells, repeating the procedure twice. Then the precipitate is suspended in TM buffer (composition similar to Example 1) to a cell density of 10 ¹⁰ cells / ml, poured into test tubes of 300 μl and 50 μl of chromosomal DNA containing virulence genes Fr.tularensis nearctica B-399A Cole in an amount of 3 μg is added . This mixture was stirred and kept at 0 ^{° C.} for 30 minutes. After incubation, the mixture is heated at a temperature of 42 ^o C for 1 min and kept in an incubator at 37 ^o C for 1 h. Then the mixture is seeded on a solid nutrient medium of a similar composition and placed in a thermostat at 37 ^o C for 18 h. After 18 h the grown cells are washed with GS-buffer, a suspension of 10 ¹⁰ cells / ml is prepared and a series of inoculations are carried out on solid nutrient media with selection of colonies of the recombinant strain. The resulting colonies of the recombinant strain are translucent, white with a blue tint.

 The results are taken into account for 3-5 days as the colonies of the recombinant strain grow. A recombinant strain of Fr.tularensis subsp. holarctica RIA producer of virulence factor Fr.tularensis nearctica B-399 A Sole. The control of the experiment is carried out analogously to example 1. Colonies of the recombinant strain grown on selective media are checked according to a known method for virulence, toxicity and immunogenicity.

 Example 3. Obtaining a recombinant strain of Fr.tularensis subsp. holarctica RN4, VKPM B-6855 producer of virulence factor Ps.pseudomallei C-141.

Microbial cells of the strain Francisella tularensis subspecies holarctica R, VKPM B-6854 are seeded on a solid nutrient medium analogous to example 1 of the composition and kept in an incubator at 37 ^o C for a day. The grown culture is washed off with a buffer solution of the composition analogous to Example 1 and centrifuged at room temperature and 6000 rpm for 10 minutes, the supernatant is drained, and the microbial cell pellet is suspended in 0.2 M potassium chloride to a cell density of 10 ¹⁰ cells / ml. The resulting cell suspension is poured into 25 μl and add an equal volume of saline buffer (200 mm magnesium sulfate, 10 mm Tris-acetate, 10% polyethylene glycol 600, pH 7.5). 1.5 μg of plasmid DNA containing virulence factor genes Ps.pseudomallei C-141, in an amount of 1.5 μg, was added to 50 μl of the suspension, mixed and incubated at room temperature for 10 minutes. This mixture is placed in a kelvinator (-55 ^° C) for 15-20 minutes, then thawed in a water bath at 37 ^° C for 5 minutes and plated on a solid nutrient medium similar to Example 1 with a spot without rubbing over the entire surface. A dense nutrient medium with a sown suspension of cells is placed in a thermostat at 37 ^° C for 18 hours. After 18 hours, the grown culture is washed off with 0.2 M potassium chloride and a suspension of 10 ¹⁰ cells / ml is prepared and a series of colonies are seeded on solid nutrient media. recombinant microorganism. The resulting colonies of the recombinant strain are convex, translucent, white.

 The results are taken into account for 3-5 days as the colonies of the recombinant microorganism grow. A recombinant strain of Fr.tularensis supsp is obtained. holarctica RN4 producer of virulence factor Ps.pseudomallei C-141.

 The control of the experiment is carried out analogously to example 1. Colonies of the recombinant strain grown on selective media are checked for virulence, immunogenicity, and toxicity.

Claims (4)

 1. The method of producing recombinant tularemia microorganisms - producers of virulence factors, which consists in the fact that cells of the strain Francisella tularensis subspecies holarctica R, UKRM B-6854 are incubated in a liquid medium in the presence of foreign chromosomal or plasmid DNA, then the grown culture is transplanted onto a solid nutrient medium then recombinant microorganisms are selectively selected.  2. Recombinant strain of Francisella tularensis subspecies holarctica R5S, VKPM B-6853 producer of virulence factor Francisella tularensis nearctica Shu.  3. Recombinant strain of Francisella tularensis holarctica RN4, UKRM B-6855 producer of virulence factor Pseudomonas pseudomallei C141.  4. Recombinant strain of Francisella tularensis subspecies holarctica RIA, UKRM B-6852 producer of virulence factor Francisella tularensis nearctica B399 A Cole.