The bacterial strain bacillus licheniformis, has antiviral and antibacterial activity
(57) Abstract:The invention relates to medical Microbiology and comes to obtain strains of probiotics. Cells of the strain Bacillus licheniformis 31 transform the plasmid rvmv 105 and get a new recombinant strain of Bacillus licheniformis 2336/105. This strain is harmless to warm-blooded, able, along with the suppression of pathogenic and conditionally pathogenic microflora to produce alpha - 2-interferon in micro-aerophilic conditions 102 IU/mg. Constructed strain allows to form the basis for a new probiotic anti-infective drug. 6 table. The invention relates to the field of biotechnology, medicine, veterinary medicine, and is concerned with the obtaining strains of probiotics.Probiotics - the biological basis of live bacteria, representatives of normal microflora, which are used in medical practice for correction of violations of the microflora of the digestive and urogenital tract.It is known that aerobic spore-forming bacteria are characterized by a high antagonistic activity against pathogenic and conditionally pathogenic bacteria that enables them to be used in the composition of probiotics. Among probiotics, Shiroki which are bacteria of the genus Bacillus.In medicine have already found wide application of probiotics from bacilli: this Yugoslavian drug baktisubtil, caribien (China), flanigin (France), enterogermina (Italy) and so on [1-5]. Known and domestic drug biosporin based on live microbial cultures of Bacillus sybtilis 3 and Bacillus licheniformis 31 for the prevention and treatment of gastrointestinal diseases  . However, all of the above probiotics do not have a pronounced antiviral activity.The closest analogue in this area is a strain of Bacillus subtilis VKPM-475  , which is obtained by transforming the original strain by plasmid pBMB 105 . The resulting recombinant acquired antiviral activity through the production of interferon, call the plasmid, while maintaining the antibacterial activity of the parent strain . However, the bacteria Bacillus subtilis are obligate aerobes and can't make their livelihoods in conditions with low oxygen content. In these conditions, there is lysis of the bacteria. It is known that many sections of the inner cavities of the body are different in oxygen content, which limits the activity of Bacillus subtilis with the introduction of macro as part of the preparation of the probiotic.Known bacterial strain Bacillus licheniformis 31 , characterized by antagonistic activity against several pathogenic and conditionally pathogenic bacteria are facultative aerobe. Because of this he is able to grow and show antimicrobial properties in conditions with low oxygen content, but does not have a pronounced antiviral activity.The problem is solved by transforming strain of Bacillus licheniformis 31 plasmid pBMB 105, coding the synthesis of alpha-2-interferon person.Obtaining competent cells and transformation of plasmid DNA carried out by a known method . Transformants plated on plates with selective medium containing kanamycin (10 μg/ml) and grown at 37oC for 24 hours. Engineered strain of Bacillus licheniformis 2336/105 characterized by the following features:
Morphological features.Cells are straight, rod-shaped, with a length of 2-3 microns mobile. Form of aerobic spores oval, which are located in the cell Central. Gram stain - gram.Cultural characteristics.The strain grows well on meat-peptone agar (MPA), sudestada folded colony: white color with uneven edges. On meat-peptone broth (BCH) culture forms a uniform suspension. Optimum temperature for growth is 37oC, a pH of 7.0. The culture of support for MPA and potato agar with the addition of kanamycin (50 μg/ml) with periodic passages at least 1 time per month, and keep on MPA and potato agar with the addition of 50 μg/ml kanamycin.Physiological and biochemical characteristics.The strain grows in aerobic conditions, not hydrolyzes urea. Gives a positive reaction Voges-Proskauer. Grows in the presence of 10% NaCl. Hydrolyzes starch and casein, thins gelatine. With the growth in the BCH forms ammonia, does not form a hydrogen sulfide and indole. Ferments lactose, glucose, arabinose, xylose with the formation of acid without gas. Reduces nitrates, provides methylene blue. Coagulase, hyaluronidase, lecithinase activity is not. Has lipase activity.For cultivation of Bac. licheniformis 2336/105 used the medium of the following composition:
K2HPO43H2O - 18,3 g/l
KH2PO4(b/C) - 6.0 g/l
(NH4)2SO4- 2.0 g/l
Na3C6H5O75,5 H2O - 1.2 g/l,
a pH of 7.0(0,15)
Antiviral activity of the new designed stamnia conditions in comparison with the antiviral activity of the parent strain Bacillus licheniformis 31 and prototype recombinant Bacillus subtilis, VCPI 475.The amount of antiviral activity in the samples is determined by comparing with the antivirus activity of the reference drug, which drug use reaferona and expressed in international units (ME).The results presented in table 1, indicate that the samples of the culture fluid of the strain Bacillus licheniformis 2336/105 when grown in microaerophilic conditions have antiviral activity in contrast to strains analogues.The engineered strain is also characterized by high antagonistic activity against pathogenic and conditionally pathogenic microorganisms, as the original culture of Bacillus licheniformis 31 (PL. 2).The harmlessness of a new strain of Bacillus licheniformis study by standard methods of evaluating the toxicity of culture filtrate (results are presented in table 3.1. ), virulence (table. 3.2), toxicity (table. 3.3) in comparison with control (table. 3.4) and observing the animals.The introduction of a suspension of living cells 24-hour culture orally in doses of 1-10 billion microbial cells and intraperitoneally in doses of 1-10 billion microbial cells/mouse does not cause the animal any signs of disease. The study DNAs is 2">Administration to mice of the leachate medium growth (after cultivation of the strain Bacillus licheniformis 2336/105 on meat-peptone broth) does not detect animals pathological process, registered clinically or by macroscopic examination of internal organs of animals.Thus, the obtained data give reason to include a constructed strain to the 4-th class according to the classification given in the guidance the USSR Ministry of health, chief sanitary-epidemiological control "Formulation of research to substantiate the maximum allowable concentration of production strains on the basis of ready-made forms of drugs...", /M, 1983/, i.e., to microorganisms, with virtually no allergenic and General toxic effect.Stability of recombinant plasmids pBMB 105 in the strain Bacillus licheniformis 2336/105 determined as follows: cells of Bacillus licheniformis 2336/105 grown in 5 ml of medium 2xLB with starch to the stationary phase; then 0.5 ml of the grown culture is transferred into a 4.5 ml of fresh medium 2xLB and again grown to stationary phase; spend 5 re-seeding and reseeding after each conduct seeding on MPA without antibiotic. Growing up isolated colonies dig up the Cup with MPA with and without kanamycin, Antia MPA with antibiotic without antibiotic. Thus to determine the number of clones that have lost the recombinant plasmid after each subculture. The absence of the plasmid confirmed by the method of selection of plasmid DNA.The safety of the interferon gene of human type alpha-2 in recombinant plasmids pBMB 105 after re-seeding is confirmed by the method of hybridization, the nitrocellulose filters with prints colonies after subcultures are incubated with the labeled fragment containing the gene of interferon alpha-2, isolated from the plasmid pBMB 105. All the clones, not lost recombinant plasmids after transfers, hybridize with labeled fragment that demonstrates the safety of interferon gene in the plasmid pBMB 105. In addition, from randomly selected clones secrete plasmid DNA and analyze it using restriction endonucleases. These experiments established that the recombinant plasmid DNA rvmv 105 is maintained and inherited within 5 transfers to IPA without selective pressure.Thus, for the first time received the bacterial strain Bacillus licheniformis 2336/105 with antiviral and antibacterial activity in microaerophilic conditions, which can serve as the basis for the creation of drugs used for treatment is illustrated by the following specific examples:
Example 1. Obtaining a strain of Bacillus licheniformis 2336/105. Transformation of the strain Bacillus licheniformis 31 plasmid pMB 105 was performed by standard method . For isolation of plasmids from strains of bacilli known technique  was modified. Changes were introduced on the basis of experiments to study the effect of different formulations lyse buffer time and processing cells bacilli to achieve a more complete lysis and maximum yield of plasmid DNA. The period of incubation with lysozyme was increased from 20 minutes to 40 minutesExample 2. Determination of the antiviral activity of the strain Bacillus licheniformis 2336/105.To determine the antiviral activity of the strain, which is determined by interferon, prepare test samples of the culture fluid.For comparison and as a control using the strains analogues: a parent Bacillus licheniformis 31 and recombinant Bacillus subtilis VKPM-475.For preparation of test samples on three randomly selected clone is grown in 450 ml of liquid nutrient medium 2xLB with starch and with kanamycin (yeast extract, 10 g/l; lactotropes, 20 g/l; soluble starch, 10 g/l; ammonium sulphate, 1 g/l; potassium phosphate-disubstituted acid, 7 g/l; potassium phosphoric acid one-deputizing, 2 g/l; to aerophilic conditions. Cells are centrifuged at 800 rpm at 4oC. To 400 ml of the supernatant add 25 ml of 50% solution of trichloroacetic acid (THU), placed in ice for 2.5-3 hours. Then centrifuged at 10 thousand rpm for 20 min at 4oC. the Precipitate is dried in air for 15 min and dissolved in 5 ml of HN buffer (110 mM Hepes, 33 mM NaCl; pH 7,4) containing 1 mM phenylmethylsulfonyl (PMSF) and 1 mM ethylenediaminetetraacetic acid (EDTA). When dissolved controlling the pH of the solution to 7.4, if necessary, adding a concentrated solution of HCl. The resulting solution cialiswhat against HN - buffer containing EDTA at 4oC for 6 hours at double shift and placed in 5 ml sterile test tubes 0.5 ml, frozen in liquid nitrogen and stored at -20oC.Antiviral activity is determined by the inhibition of the cytopathic effect of EMC virus (encephalomyocarditis mice). Suspension diploid cells human fibroblasts (DFC) DK-58 is brought to a concentration of 2 x 10 cells/ml of growth medium. In each well politiaromana.ro tablet make 100 ál (2 x 10 cells) suspension DFC in the growth medium and incubated at 37oC before the formation of the monolayer of cells. Under the microscope formed a monolayer of cells similar to mosaic, obrazovash truemove sample and 180 μl of growth medium, after careful mixing 100 µl of the first hole is transferred to the second, mixed with 100 μl of growth medium, etc. Cells incubated 24 hours at 37oC and then make 100 tissue cytopathic doses causing loss in half of the samples of EMC virus and incubated at 37oC 24-48 hours. After 1 day spend the first account, after 2 days the second. The amount of antiviral activity in the samples is determined by comparing with the antivirus activity of the reference drug, which drug use reaferona, and expressed in international units (ME). The results presented in table 1, indicate that the samples of the culture fluid of the strain Bacillus licheniformis 2336/105 when grown in microaerophilic conditions have antiviral activity in contrast to strains analogues.Example 3. Determination of antagonistic activity of the strain Bacillus licheniformis 2336/105.Antagonistic activity investigate the radial strokes . 18-hour culture of Bacillus licheniformis 2336/105 sown into the center of the Cup with a diameter of 12 cm on the area delineated by a circle with a diameter of 2.5 see After 3 days incubation at 28oC podshuveyt in the radial strokes culture test on smear-Cup without a culture of B. licheniformis.the results presented in table 2, show the persistence of the antagonistic properties of the recombinant strain compared to the parental strain.Example 4. Study of the safety of the strain Bacillus licheniformis 2336/105.The harmless strains studied by standard methods of evaluating the toxicity of culture filtrate (table. 3.1), virulence (table. 3.2.), toxicity (table. 3.3), comparing with the control (table. 3.4) and observing the animals.Observations of the animals is carried out for 15 days after the course of injections or oral administration. During the observation period all animals were active, well ate food rations, physiological functions have not been violated, behavioral responses were normal, changes from wool cover was not observed. Dynamics of changes in the body weight of animals in the experiment and the control was similar. All experimental and control animals after the end of the observation period were euthanized, dissected and their organs subjected to microscopic examination.The findings showed the following:
The heart is of normal shape and size; in laboratory animals is the same as that of the control.Legalega are separated from each other, adhesions are not marked.Stomach, loops and small intestine - appearance, signs of inflammatory changes are not marked. At the opening of the lumen is visible unchanged (the same as that of the control animals) figure mucous.The liver is dark red, medium blood, not increased. Fraction separated from each other, the surface smooth.Kidney size and shape does not differ between the control and experimental animals, surface smooth, the cut seen a clear picture of the cortical and medullary substance.The spleen is not enlarged, normal consistency. On the cut pulp is moderately full, dark red color.The results of the experiments indicate that the safety of the strain Bacillus licheniformis 2336/105 for warm-blooded.Literature:
1. Smirnov centuries, Reznik, S. R. and others //Microbiol.journal, 1993, T. 55, N 4 C. 92-112.2. RF patent 1839459, CL C 12 N 1/21, 19903. Chang , S., O. Gray, D. Ho et.al. //Molecular Cloning and Gene Regulation in Bacillus. Acad. press, 1982, p. 159-169.4. Egorov N. With the basic teaching about the antibiotics. The graduate school. M., 1969, S. 176.5. Smirnov centuries, Reznik, S. R. and others //Microbiol.journal 1992, T. 54, N 6, S. 82-94.6. A. C. the USSR 1722502, class A 61 K 39/02, 1989.8. Shchelkunov, S. N. , Il'ichev, A. A., Red Century. N. etc. // Biotechnology, 1987, T. 3, N 2. C. 146-150.9. Bacilli. Genetics and biotechnology. //Under the editorship of K. Harwood, M., Mir, 1992, S. 460. Recombinant bacterial strain of Bacillus licheniformis 2336/105 with antiviral and antibacterial activity.
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.
EFFECT: valuable medicinal properties of strain.
5 cl, 8 dwg, 10 ex