SU492094A3 - Method for preparing live typhoid vaccine - Google Patents

Description

(54) METHOD OF OBTAINING A LIVING ABOUT LYNTHYPHOPHIC

VACCINES

after 10 days, the same dose of 10 microbes of the same strain, as amplifiers, which are not activated by hourly heating to 58 ° C.

Determining the number of bacteria in the liver and spleen of these animals showed that the attenuated bacteria of the vaccine strain were completely eliminated after 20 days, while the bacteria of the virulent strain, despite a lower dose even after 4 weeks, were present in mice at concentrations of 10 .

Six weeks after vaccination, 10 live bacteria of the virulent strain Tu 2 were injected intraperitoneally in mice. At this point, all animals were free from vaccine bacteria. By determining the number of bacteria in the liver and spleen of mice, they monitor the development of bacteria pathogens.

The advantages of live vaccine from epimyrase-negative mutants are that a single oral dacha can provide reliable, extremely strong, specific immunity against human pathogens Salmonella typhi.

The virulent strain of Salmonella typhi is grown in 30 ml Brain Heart Infision (Difco) in a 100 ml shaker flask at 37 ° C. After 4 hours, the bacteria cells are separated by centrifugation and suspended in physiological sodium chloride solution so that the suspension contains 10 organisms per ml. This suspension is irradiated with UV light until an 80% kill of bacteria is reached. Bacteria cells are then re-grafted with fresh Brain Heart Infision (Difco) and incubated at 37 ° C on a rocking chair. After 2 h, the culture is infected with smooth-specific bacteriophage type FC-1 (1 bacteriophage / 10 bacterial cells) and incubated for another 3 hours. With this treatment, all smooth bacteria are lysed and only rough-bacteria remain, which are spread onto the agar culture medium and incubated for 14 h at 37 ° C. The resulting colonies are then replicated onto endo-agar, which instead of lactose contains 0.2% galactose. On this nutrient medium, one can easily recognize epimyrase-negative mutants due to their type of growth in colonies: epimyrase-negative mutants, unlike the wild type, do not ferment galactose, and they grow in typical colorless, flat colonies with a narrow external shaft and a concave center. from lysed cells. Isolate 30 of. The above colonies and of them are deletion mutants, which even after mutagenic treatment with K-methyl-L-nitro-M-nitrosoguanidine ((NG) do not show reversion.

For this purpose, isolated mutants are grown, given on Brain Heart Infision, after 6 hours, NG is treated in order to obtain a 99% kill.

The surviving cells after twofold separation by centrifugation and

the washings in Brain Heart Infision are suspended, incubated on a shaker at 37 ° C and after 2 hours they are distributed to fresh Brain Heart Infision, to which 0.1% galactose is added.

Due to a defect in the enzyme IDR-galactose-4 epimerase, epimerase-negative mutants are not able to metabolize the entire galactose. Galactose-1-phosphate and IDR-galactose accumulate, which results in complete lysis of epimerase-negative bacteria after 3–4 hours. Then the culture is incubated for 3 hours, which contributes to the occurrence of rare revertants. Bacteria remaining in the living are distributed to endoagar containing galactose and incubated for 14 hours at 37 ° C. Revertants can be easily recognized on this nutrient medium in the form of dark-red colonies bryvating galactoSU.

The finally selected mutant is grown in Brain Heart Infision on a rocking chair at 37 ° C. After 6 hours, the bacteria are separated by centrifugation in a cooling

A centrifuge without washing in a protective medium consisting of 8% sucrose, 1.5 gelatins and 5% powdered milk powder suspended in 1 ml of this suspension from bacteria is lyophilized into 5 ml ampoules.

Lioampulu obtained a new vacuum strain open and the strain is grown on a nutrient medium from the oblique agar at 37 ° C. Bacteria are obtained from the surface of oblique agar culture and suspended in physiological sodium chloride solution. This cell suspension is grafted onto a 1 liter Erlenmeyer flask. The flask contains 600 ml of nutrient medium, which is obtained by dissolving

28 g caseine hydrolyzate, 10 g yeast extract and 2 g glucose in 1 liter of distilled water and a pH value of 7.2, which is obtained through 1 n. NaOH solution. Erlenmeyer flask is shaken for 6 hours at

37 ° C. The resulting bacterial culture is distributed to 25 liters of the resulting medium. The culture is incubated for 12 hours at 37 ° C, ventilated (5 liters of air / min). The growth of the pre-culture and the main culture is determined by nephelometric method. Cultural samples determine purity. After the cultivation time has elapsed, the bacterial cells are separated by centrifugation in a cooled centrifuge without washing, suspended in 600 ml of protective medium, and in 1 ml portions lyophilized in 5 ml ampoules or into punctured bubbles. It is used as follows: ampoules or peelable bottles are opened, the contents are suspended in 3-5 ml of cold or tepid water or milk and given by mouth.

Claims (1)

Invention Formula The method of obtaining a live typhoid vaccine by growing a virulent 5 strain of Salmonella typhi, separating the culture and lyophilizing it in a protective environment, characterized in that, in order to obtain a highly immunogenic vaccine, the mutant Salmo-5b nella typhi mutant, having a block, is used as a vaccine strain, having a block, a body, a body, a body, a body, a body of a vaccine, a mutant Salmo-5 b and a nella typhi mutant, which has a block of the immunity vaccine. -4-epimerase and low galactic kinase and galacto-1-phosphaturidyl trapsferase activity,