RU2270867C2 - METHOD FOR PREPARING Pseudomonas aeruginosa ANATOXIN - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing bacterium pyocyanic anatoxin used in specific therapy and prophylaxis of infections caused by Pseudomonas aeruginosa (pyocyanic bacterium). Method involves culturing microorganisms in liquid nutrient medium comprising the following components per 1 liter of distilled water: citric acid, 4.9-5.0; sodium chloride, 0.9-1.0; magnesium sulfate, 4.9-5.0; ferrous sulfate, 0.04-0.05; asparagines, 0.9-1.0; glucose, 0.9-1.0; glycerol, 2.9-3.0 ml, balance, to the final concentration of pyocyanic bacterium, 14-15 billion of microbial bodies in 1 ml. Then the prepared biomass-containing cultural fluid is subjected for sterilization by autoclaving at 0.6-0.7 atm for 45-50 min followed by detoxification with formalin and anatoxin-containing cultural fluid is separated from biomass by filtration and sorbed its on aluminum hydroxide gel. Using the invention provides enhancing antigenic activity of toxic product and to standardize condition for culturing the producer.

EFFECT: improved preparing method.

1 tbl, 1 ex

Description

The invention relates to biotechnology, in particular to a method for producing Pseudomonas aeruginosa toxoid for specific therapy and prevention of infections caused by Pseudomonas aeruginosa.

It is known that in medicine, upon receipt of Pseudomonas aeruginosa toxoid (Pseudomonas aeruginosa), Marten broth (Brodinova H.S., Levdikova G.A., Moroz A.F. G. Microbiology, epidemiology, immunology, 1987, No. 3, pp. 6-17).

The specified method for producing Pseudomonas aeruginosa toxoid involves the cultivation of toxic strains of Pseudomonas aeruginosa on Marten broth containing 1% glycerol and 0.05% sodium glutamate for 18 hours at 32 ° C under aeration. Next, use the supernatant obtained after centrifugation of the culture, saturating it with 60% ammonium sulfate at 60 ° C, followed by gel filtration on a Sephadex column.

The disadvantage of this method of producing toxoid Pseudomonas aeruginosa is: non-standard nutrient medium, the basis of which is meat water and peptone; the need to ensure aeration conditions; relatively small accumulation of biomass requiring concentration by centrifugation; weak antigenic activity of the toxic product, requiring saturation with ammonium sulfate and gel filtration on a Sephadex column.

To eliminate the above disadvantages, a method is proposed that includes growing Pseudomonas aeruginosa on a liquid synthetic nutrient medium, sterilizing biomass, formalin detoxification, and sorption of alumina hydrate on a gel.

New is that when growing Pseudomonas aeruginosa use a nutrient medium containing the following ingredients in g per 1 liter of distilled water: citric acid - 4.9-5.0; sodium chloride - 0.9-1.0; magnesium sulfate - 4.9-5.0; iron sulfate - 0.04-0.05; asparagine - 0.9-1.0; glucose - 0.9-1.0; glycerin - 2.9-3.0 ml. After the growth of microbes is completed, the biomass is subjected to autoclaving at 0.6-0.7 atm for 45-50 minutes, then it is detoxified with 0.3-0.4% formalin, for 7-8 days at a temperature of 44-45 ° С then biomass is separated from the culture fluid by filtration. The resulting toxoid is suitable for external use. For parenteral use, the toxoid is sorbed on an alumina hydrate gel.

The use of the above nutrient medium, after 2-3 microbial passage of microbes on it, with a seeding density of 90-100 million microbial bodies per 1 ml of medium, allows maximum biomass accumulation of Pseudomonas aeruginosa to 14-15 billion microbial bodies in 1 ml of culture fluid.

Autoclaving sterilization, conducted in a gentle mode of 0.6-0.7 atm for 45-50 minutes, allows not only to kill the biomass of Pseudomonas aeruginosa, but also contributes to an increase in the release of toxin into the culture fluid, thereby increasing its biological activity and immunogenic properties of the final vaccine preparation.

To obtain the toxoid, detoxification of the Pseudomonas aeruginosa culture fluid is carried out with formalin, without separating it from the Pseudomonas aeruginosa biomass, which allows extraction of additional toxic substances from the microbial cell. After detoxification, the biomass is separated from the culture fluid by filtration, and the latter is used as anatoxin of Pseudomonas aeruginosa.

An example of the method

To obtain Pseudomonas aeruginosa toxoid, the pathogenic strain Pseudomonas aeruginosa was used, the cultivation of which was carried out in standard 2 liter bio-shoes with a volume of nutrient medium of 1 liter. The composition of the medium included the following ingredients in g per 10 liters of distilled water: citric acid 49-50.0; phosphoric acid potassium bisubstituted 49-50,0; magnesium sulfate 49-50.0; sodium chloride 9-10.0; iron sulfate 0.4-0.5; asparagine 9-10.0; glucose 9-10.0; glycerin 29.0-30.0 ml. The initial pH was adjusted within 7.0. The sowing density of Pseudomonas aeruginosa was 90-100 million microbial bodies per 1 ml of medium. Pseudomonas aeruginosa was grown at 37 ° C to a final concentration of 14-15 billion microbial bodies in 1 ml. The grown biomass of Pseudomonas aeruginosa was autoclaved in a 0.6-0.7 atmosphere mode for 45-50 minutes, after which it was detoxified with 0.4% formalin at a temperature of 44-45 ° C for 7 days, shaking the contents daily. The biomass was separated from the culture fluid by filtration. The resulting native Pseudomonas aeruginosa toxoid was subjected to sorption on an alumina hydrate gel at a rate of 3 mg per 1 ml.

Evaluation of the immunogenic properties of the toxoid was carried out by the protective effect in the experiment on white mice weighing 18-20 g. For infection of mice, homologous and heterologous vaccine strains of Pseudomonas aeruginosa were used. The infectious dose of microbes was 1 billion microbial bodies, providing 100% death of control mice for 1-2 days after their intraperitoneal infection.

The results of studying the protective properties of the toxoid Pseudomonas aeruginosa for clarity are shown in the table.

Table Evaluation of the protective effect of Pseudomonas aeruginosa toxoid upon infection of white mice with homologous vaccine and heterogeneous strains of Pseudomonas aeruginosa No. p / p (n = 7) The procedure for the use of Pseudomonas aeruginosa toxoid Pseudomonas aeruginosa strain taken for infection The survival of mice at different times of infection after immunization (survived / died) In 7 days In 21 days one. Once, at a dose of 0.5 ml subcutaneously Homologous 8/0 - 2. Also Homologous - 6/2 3. Also Heterologous 6/2 - four. Also Heterologous - 5/3 5. Double interval of 7 days at a dose of 0.3 + 0.3 ml Homologous 8/0 - 6. Also Homologous - 7/1 7. Also Heterologous 7/1 - 8. Also Heterologous - 6/2

The observation period is 10 days.

Control weighing of animals on the 5th and 7th day after immunization did not reveal a tendency to reduce their weight, which indicated the harmlessness of the toxoid. With regard to the protective effect, the following data were obtained: for a single immunization and subsequent infection on the 7th and 21st day of infection with the white mouse homologous vaccine strain Pseudomonas aeruginosa, the protective effect was 100 (group 1) and 75% (group 2), respectively; and with double immunization - 100 (group 5) and 87.5% (group 6).

Somewhat worse, as one would expect, there were results of the protective effect of Pseudomonas aeruginosa toxoid when infected with the heterologous vaccine strain Pseudomonas aeruginosa. Nevertheless, the protective effect was: with a single immunization and infection on day 7 - 75% (group 3), versus 87.5% (group 7) with double immunization; when infected on day 21 after a single immunization, the protective effect was 82.5 (group 4), versus 75% (group 8) after a double immunization.

The conducted experiments indicate the formation of a pronounced protective effect against both homologous and heterologous strains of Pseudomonas aeruginosa. Moreover, the protective effect is largely determined by the fragmentation of the toxoid. The presence of a degree of protection on average of more than 70% - meets the requirements for killed vaccines and toxoids.

Claims (1)

A method of producing Pseudomonas aeruginosa toxoid, including the cultivation of Pseudomonas aeruginosa in a liquid nutrient medium, formalin detoxification, filtration and isolation of the target product, characterized in that the cultivation is carried out in a nutrient medium containing citric acid 4.9-5.0 g in 1 liter of distilled water , sodium chloride 0.9-1.0 g, magnesium sulfate 4.9-5.0 g, ferrous sulfate 0.04-0.05 g, asparagine 0.9-1.0 g, glucose 0.9-1 , 0 g, glycerol 2.9-3.0 ml, to a final concentration of Pseudomonas aeruginosa 14-15 billion microbial bodies in 1 ml, the resulting culture fluid awn containing biomass is subjected to sterilization by autoclaving at 0.6-0.7 atm for 45-50 minutes, and then subjected to formalin detoxification separated culture liquid containing toxoid, and sorbed by the biomass in the aluminum hydroxide gel.