The strain of shigella sonnei, phase 1, stable producer s-lipopolysaccharide

 

(57) Abstract:

The invention relates to the field of biotechnology, and more particularly to a strain of Shigella sonnei No. 262 and can be used to produce vaccine and diagnostic products. This strain has the ability to consistently produce S-LPS. 1 PL.

The invention relates to the field of biotechnology and can be used to obtain S-lipopolysaccharide (LPS) Shigella sonnei, phase 1, suitable for a vaccine and diagnostic products.

The instability of the production of O-antigen strains of S. sonnei is well known (Kopecko, D. J., et.al., 1980; Hale, T. L., et.al., 1983). Strains growing in laboratory conditions, often spontaneously pass into the R-shaped bacterium, which produces R-LPS, not containing O-specific polysaccharide chain (Gamian A., Romanowska E., 1982). The absence of strain, consistently producing O-antigen, is a serious obstacle for the development dysentery vaccine against Shigella sonnei. A number of developers had to use instead of S. sonnei strain Plesiomonas shigelloides, producing similar serotype S-LPS (D. Taylor N. et.al., 1993).

The aim of the invention is a strain No. 5063 S. sonnei, phase 1, consistently producing S-LPS.

Cultural characteristics: when growing by 1.5% mesopatamia agar (pH 7.4) at a temperature of 36.5-37°With forms small (2-2 .5 mm in diameter) mainly delicate translucent colonies with smooth edges (S-form), can meet and more dense colonies with cut edges (R-form). When grown in a broth of Hottinger (pH 7.4) at a temperature of 36.5-37°C for 16-20 hours gives intense turbidity, the film does not form.

Biochemical characteristics:

- Decompose glucose and maltose without the formation of gas.

Slowly ferments lactose and mannitol.

Not liquefies gelatin.

- Does not produce hydrogen sulfide.

- Does not form indole.

Serological properties: gives a clear positive reaction with standard serum sonnei. Reacts positively with sorbirovannoe serum phase I and negatively with serum phase II

Biological properties: strain No. 5063 has expressed virulently.

When introduced into the conjunctival SAC of Guinea pigs weighing 200-250 g, causing purulent keratoconjunctivitis with subsequent corneal opacity, 100% lethal dose (LD10010what I lethal dose (LD50)no more than 2×108microbial cells.

When introduced into the lungs of young mice (16-hour) broth cultures LD50do not exceed 1.5-2×105microbial cells.

When passirovannye on mesopatamia agar (the length of the passage 18 hours, the temperature 37°C) and mesopatamia broth (the duration of the passage 4 hours, temperature 37°C) strain stably retains the characteristics of an S-shape that distinguishes it from other strains of S. sonnei (see table). The contents of the colonies in the R-form in 3 passage does not exceed 10%.

The strain is stored in a dried state in ampoules at 4-10°C. Mandatory control all properties of the strain are conducted annually.

Example 1. The passage of the strain No. 5063 S. sonnei in a dense environment.

Vials of freeze-dried culture was sterile revealed and made it sterile saline. A drop of the resulting suspension of cells with a sterile pipette, transferred to a Petri dish with mesopartner agar and subcultured on the entire surface of the Cup with a sterile spatula. The same spatula did the seeding for another five cups with a dense nutrient medium. Cups were placed in a thermostat, and incubated at 37 ° °C. after 18 hours was chosen Cup with the growth of BA is kodashim light. To obtain each of the following passage in transmitted light was collected S-colonies, suspended biomass in sterile saline and using a sterile spatula was scattered over the surface of the cups with a dense nutrient medium.

Example 2. Preparation of the specimen for the GEF.

18-hour culture were washed from the agar phosphate buffer (pH 7,2), was added sodium azide to a concentration in the flushing of 0.01% and left at 4-10°C for 3 hours. After this culture, killed sodium azide, were diluted to an optical density equal to 0.4. Samples of 1.5 ml were placed in centrifuge tubes and centrifuged for 1.5 minutes. Adosados was removed, to the precipitate was added to 50 ál lyse buffer (2% sodium dodecyl sulfate, 4% 2-mercaptoethanol, 10% glycerol, 1 M Tris-HCl, pH 6.8). The samples were placed in a boiling bath for 10 minutes After that to each sample lysate of bacterial cells was added 10 ál lyse buffer with 25 µg of proteinase K (Sigma) and incubated for 1 hour at 60°C.

Example 3. Holding the GEF with subsequent staining ammoniacal solution of silver nitrate.

GEF study of the drugs was performed on the instrument for electrophoresis of the company BioRad (Sweden) in Laemli [Laemli, U. K. Cleavage of strucrural prote 3% concentrating and 12.5% separating gels (thickness of the gel 0.8 mm). Sample size when the application was 5-10 μl. The path length of the samples into the separating gel does not exceed 15 see Electrophoresis in concentrating the gel was carried out at an amperage of 15 mA, and dividing - 30 mA. Staining of the gel after electrophoresis was carried out according to the method of Tsai [Tsai,C.-M., C. E. Frasch.E. Rivera,H. D. Hochstein. Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage. // J. Biol. Stand. - 1989. - V. 14. - P. 25].

Comparative analysis of the painting, the GEF has demonstrated that LPS of S. sonnei strain No. 5063 obtained from bacterial cells for 10 passages, is a typical S-LPS, and the number of links it O-polysaccharide chain (which reflects the number of discrete zones in the picture GEF) does not change the result of the passage of culture.

Sources of information

Gamian A., Romanowka E. The core structure ofShigella sonnei lipopolysaccharide and the linkage between the O-specific polysaccharide and the core region. Eur J Biochem 1982,129: 105-109.

Hale T. L., P. Guerry, Seid R. C., C. Kapfer, Wingfield M. E., Reaves C. B., L. S. Baron,

Formal, S. B. Charchteriozation of virulence plasmids and plasmid asosiated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Eshcerichia coli. Infect Immun 1984, 40:340-350.

D. J. Kopecko, Washington O., Formal, S. B. Genetic and phisical evidence for plasmid control of Shigella sonnei form I cell surface antigen. Infect. Immun. 1980, 29:207-214.

Laemli.U.K. Cleavage of strucrural proteins during the assembly of the head of bacteriophage T4. // Nature.l9clinical evaluation of conjugate vaccines composed of the O-specific polysaccharides of Shigella dysenteriae type 1, Shigella flexneri type 2a and Shigella sonnei (Plesiomonas shigelloides) bound to bacterial toxoids. Infect Immun. 1993 Sep;61(9):3678-87.

Tsai, C. - M.,C. E. Frasch.E. Rivera,H. D. Hochstein. Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage. // J. Biol. Stand. - 1989. - V. 14. - P. 25.

The strain of Shigella sonnei gisk named after. L. A. Tarasevich No. 262, phase 1, stable producer S-lipopolysaccharide.



 

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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

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