RU2051969C1 - Method of bacterial lipopolysaccharide preparing - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves preparing bacterial mass, hot aqueous-phenolic extraction and further purification by phase distribution using nonionic detergent Triton-114. The use of Triton-114 ensures decrease preparation purification period from nucleic acids impurity, eliminate nonfavorable effect of lyophilic drying repeated procedures. EFFECT: enhanced quality, simplified method, decreased cost and time for purification.

Description

 The invention relates to medicine, namely to the production of drugs, and can be used to design diagnostic test systems, chemical vaccines and immunomodulators.

Known methods for obtaining varying degrees of purification of lipopolysaccharides (LPS): extraction of cells with trichloroacetic acid, water-butanol, water-ether mixtures, followed by purification of LPS from impurities of nucleic acids and proteins by ultracentrifugation, treatment with proteases and nucleases [1,2,3]
The disadvantage of these methods is the low degree of purification of LPS, the use in some cases of volatile, toxic to humans substances, the duration and multi-stage cleaning procedure, the use of expensive equipment and chemicals.

Most common is the water-phenol method for producing LPS [4] The method comprises isolating a microbial mass, treating it with a water-phenol mixture at 68 ^{° C} for 15 min, separation by centrifugation and collecting the aqueous phase. The resulting aqueous phase containing LPS and nucleic acids is lyophilized. The separation of LPS from nucleic acids is carried out using multiple ultracentrifugation (105000xg for 4 hours). The precipitate formed as a result of ultracentrifugation is dissolved in water and subjected to repeated ultracentrifugation. The precipitate was dissolved in water and lyophilized.

 The final purification of LPS from nucleic acids is carried out by precipitating the latter with a cationic detergent with cetyltrimethylammonium bromide (cetavlon), LPS purified by ultracentrifugation is dissolved in water and a certain amount of a 2% aqueous solution of cetavlon is added to the stirred solution. The turbid solution is centrifuged, the precipitate is discarded, and the supernatant is lyophilized. The dry preparation is dissolved in water and freed from impurities of cetavlon, precipitating LPS with acidified ethanol or acetone. The LPS precipitate was separated by centrifugation, washed thoroughly with 96% alcohol or acetone, dissolved in water and the solution was lyophilized. LPS purified by this method does not contain nucleic acids.

 The disadvantages of the method are the complexity and duration of the process of purification of the extracted LPS from impurities of nucleic acids (6 days), the use of expensive equipment (ultracentrifuge), the loss of LPS at the stages of ultracentrifugation and treatment with cetavlon, the presence of several lyophilization procedures, which can significantly violate the native properties of LPS (in first of all, the solubility of the target product deteriorates).

 The aim of the invention is to simplify and reduce the time to obtain LPS, avoid losses and improve the quality of LPS.

The goal is achieved in that when carrying out the method of producing LPS by culturing bacterial cells on a solid nutrient medium, followed by separation of the microbial mass, extracting LPS from it with a hot water-phenolic mixture, purification of LPS from impurities of nucleic acids is carried out by treating the aqueous phase with a non-ionic detergent Triton X-114 in an ice bath. The resulting solution is heated at 37 ^about C and centrifuged to form two phases: aqueous and detergent. This treatment allows one to separate LPS from impurities of nucleic acids: LPS is concentrated in the lower detergent phase, and impurities of nucleic acids remain in water. LPS is isolated from the detergent phase by precipitation with cold acetone. The resulting LPS precipitate was collected by centrifugation, washed with acetone to completely remove detergent impurities, dissolved in water and lyophilized.

 The claimed technical solution differs from the prototype in that the non-ionic detergent Triton X-114 is used at the cleaning stage.

 The method is as follows.

As starting material for the LPS of Vibrio cholerae using slurry microbial pathogen Vibrio cholerae cholera strain 569 C. The culture was grown on casein agar, pH 7.6 for 18 hours at ³⁷ ° C, rinsed with sterile 0.9% sodium chloride pH 7.2. The resulting slurry was added freshly distilled phenol to a final concentration of 1% and incubated for 18 hours at ³⁷ C. The obtained microbial mass checked for sterility and the absence of a specific microbial cell growth was separated by centrifugation, washed three times with sterile 0.9% sodium chloride, pH 7 , 2, then with distilled water and lyophilized.

Further microbial mass was suspended in equal volumes of water and phenol at 68 ^{° C} for 15 minutes, the emulsion was centrifuged 30 min at 3000 rev / min, after cooling to 10 ^C. The aqueous layer was collected and the phenol layer and the insoluble residue was treated a second time with a new portion water as described above. Combine aqueous extracts, dialyze against water to remove phenol. The dialysate, if necessary, freed from insolubles by low speed centrifugation and added into it at 0-4 ^{° C} and constant stirring, the detergent Triton X-114 to a final concentration of 6% The resulting solution was heated to ³⁷ ° C water bath to separate phases and centrifuged for 5 minutes at 6000-8000 rev / min and at a temperature above 25 ° ^C the detergent phase forms an oily layer at the bottom of the tube and, after removal of the aqueous phase detergent layer was mixed with 1% aqueous solution of Triton X-114 at 0-4 ^C. in a ratio of 1: 3 and repeat n procedure for the complete removal of impurities of nucleic acids. LPS from the detergent phase is precipitated with six volumes of acidified acetone, the resulting precipitate is collected by centrifugation, washed with acetone, dissolved in water and lyophilized. The yield of the drug is 18-25% of the crude aqueous extract (2-3% of the dry weight of bacteria). The resulting preparation is a lipopolysaccharide that does not contain nucleic acid impurities; it contains 29% glucose equivalents (reaction with phenol and concentrated sulfuric acid). The drug causes a shift in the absorption maximum of the Stainsall carbocyanin dye (Serva, Germany) to the short-wavelength part of the spectrum, which is typical for LPS of gram-negative bacteria. The drug is immunochemically identical to LPS isolated by the prototype method.

 The advantages of the proposed method: improving the quality of the drug (increased solubility in water) by reducing several lyophilization procedures; the time for cleaning the drug from impurities of nucleic acids is reduced from 6 days to the 1st day; the cost of the drug is reduced.

Claims (1)

Process for the preparation of bacterial lipopolysaccharide, comprising culturing a microbial mass, the separation of the bacterial cells, treating them with water-phenol mixture, collecting the aqueous phase by centrifugation and purification of the desired product from the impurities of nucleic acids, characterized in that the aqueous phase at 0-4 ^o C is treated with the detergent Triton X -114 at a final concentration of 6%, the aqueous and detergent phases are separated at 37 ^° C, the detergent phase is collected, the target product is precipitated with acetone, the precipitate is determined by centrifugation, washed with acetone, p dissolve in distilled water and lyophilize.