SU1640165A1 - Method for preparing bacterial pyrogene - Google Patents

Abstract

This invention relates to medical biotechnology, in particular, to the production of bacterial pyrogens used in medical practice to stimulate natural defenses. The aim of the invention is to increase the yield of the target product. The method is as follows. The sources used are experimentally obtained by the method of sexual conjugation using highly efficient donors of the Escherichia group HfrH (/ T) of the Shigella Flexner and Escherichia hybrids. Cell lipids are preliminarily removed, lyophilized, suspension of biomass in distilled H-0, repeated water-phenol extraction (2: 1) using freshly distilled phenol, extraction temperature 65-70 ° C, followed by centrifugation (3000 rpm, 20 min), dialysis for 3 days through a parchment paper membrane with a pore size of 10-20 A, a specific surface area of 1000-1200 mg / g and a thickness of 0.16-0.20 mm previously washed with a chloroform-methanol mixture (2: 1) for degreasing, evaporation at 30 ° C using liquid nitrogen, ultracentrifugation in the mode: 108000 rpm, 1 h, three times, followed by freeze drying of the obtained products. 2 tab. s / s

Description

The invention relates to medical biotechnology, in particular to the production of bacterial pyrogens used in medical practice to stimulate the body's natural defenses.

The aim of the invention is to increase the yield of the target product.

As sources of lipopolyaccharides (LPS), experimentally obtained by the method of sexual conjugation using highly efficient donors of the HfrHW group of the Escherichia group of the Flexer Flexner and Escherichia hybrids, pre-removal of cell lipids, lyophilization, suspension of biomass in distilled water, multiple water, repeated lyophilization, lyophilization, suspension of biomass in distilled water, multiple water, multiple water, and multiple water are used. : 1) using freshly distilled phenol (extraction temperature 65 - 70 ° C) followed by centrifugation at 3000 rpm, 20 min), dialysis for 3 days through the gummed paper

ate

membrane with a pore size of 10–20 A, a specific surface area of 1000–1200 mA / g and a thickness of 0.16–0.20 mm, previously washed with a chloroform-methanol mixture (2: 1) for degreasing, evaporation using liquid nitrogen, ultracentrifugation mode: tOSOOOg, 1 h, three times, followed by freeze drying of the obtained products.

Example. Bacteria are genetically recombined using E. coli donor HfrH (fl), (str5, mon1,), recipient

sh.flexneri No. 737 type 2a (strr, neo). Sexual hybrids (genetic recombinants) of Shigella Flexner and Escherichia HfrHCft are obtained, three strains: R-737-2, R-737-4, and R-737-6, which have the same biological characteristics: gram-negative,

Immobile, resistant to both rtreptomycin9 and antibiotics of the reserve

(strps monr, peo), are active against lactose, which is used together with streptomy resistance as an important selective recognized for the selection of recombinants. Virulence

(genital laboratories follow the recipient culture of Sh. flexneri No. 737 serotype 2a (amounts to 133i t 47 ppm microbial bodies). Since all

Since three strains of recombinants have similar biological characteristics, R-737-4 is selected for further studies, which is characterized by the highest stability of morphotinctual and biochemical parameters.

The biomass of bacterial cultures is grown on standard dense MPA media of one production series. Washing the cultures from the residues of the medium is performed with a solution of sodium chloride with a mass fraction of 0.50% to a negative reaction with ninhydrin. The purity of the grown colonies is checked microscopically. The biomass of bacteria is dried in air. Cell LIPIDS are removed.

The biomass residue is freeze-dried, divided into three equal parts and suspended each in distilled water at 37 ° C, then each batch is treated with phenol-water mixture (1; 2, V / V) using freshly distilled phenol at 65-70 ° C (5 times a) the obtained extracts are combined and centrifuged (3000 rpm), dialyzed through

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parchment paper membrane with a pore size of 10-20 A, a specific surface area of 1000-1200 mg / g and a thickness of 0.16-0.20 mm, previously defatted with chloroform-methanol mixture (2: 1) for 3 days, changes water through every 6 hours, evaporated on a rotary evaporator using liquid nitrogen (), the obtained extracts are centrifuged in the following mode: 108000s, 1 hour, 3 times. The resulting products are lyophilized. The output is calculated according to the weight analysis.

The general characteristics of the composition of APS and their yield in genetic recombinants and initial cultures of the donor and recipient are given in Table. one.

The output of LPS is greatest in genital f hybrids (Table 1). LPS of each of the cultures is hydrolyzed with 1% acetic acid (50 ml, 2 hours, 100 ° C) in sealed ampoules. Lipid A re-precipitation is carried out with a multiple volume of acetone.

Lipid A (10 mg) is hydrolyzed with hydrochloric acid (C (1/1 HC1) 4 mol / l; 12 4J 100 ° C) in sealed glass ampoules. Fatty acids are extracted with chloroform, followed by evaporation and methylation with diazomethane. The purity of the methyl zofir fraction is monitored. Gas-liquid chromatography was carried out using a chromatograph with a flame ionization detector under the following conditions: column 0.5 cm x 35 m, column temperature 180 ° C, Sp-UOO phase. Chromato-mass spectrometry of the methyl esters of F are carried out on the instrument (the same column). Interpret mass spectra. Determine the total protein content in LPS, nucleic acids, neutral monosaccharides by the phenol-acid method, find the content of keto-deoxy-aconic acid (KDO), calculate the mass fraction of total phosphorus in LPS. Phenol in Che preparations is found.,

The data obtained is statistically based on the Studen and Fiche distribution.

After the pyrogenic curve has reached the pyrogenal regime, a comparative study of the effect of LPS depending on the source and dose of the preparation is carried out. The data are statistically processed taking into account the Student-Fisher distribution.

The results obtained indicate that sex hybrids are better endoproducts of LPS than the original escherichia (2 times) E. coli HfrH (fT) and shigella. The average temperature of the pyrogenic effect mode in LPS hybrids is 40.5 ° C at 40, OtfC at pyrogenal.

The content of fatty acids and carbohydrates in lipid A of genetic recombinants and initial donor and recipient cultures is given in table. 2

 w Thus, the proposed method of obtaining bacterial pyrogen is new and, based on biotesting, has a temperature and a temporal component of the pyrogenic effect, indicating its enhancement in comparison with pyrogenal.

1640165

F o

v

rmula of invention

The method of obtaining bacterial

pyrogen, which involves extraction of pyrogen from bacteria, followed by dialysis for 3 days by ultracentrifugation and lyophilization of the target product, is different because, in order to increase the yield of the target product, pyrogen is extracted from genetic recombinants Shigella Flexner and Eshe - richia coli HfrH (lp) ЗЗХ-ary water-5 phenolic mixture at 70 ° С after preliminary suspension in distilled water, and dialysis is carried out through degreased parch paper with a pore size of 1020 A, a specific surface area of 1000–120 m2 / g and 0.16-0.20 mm thick with a change of dialysis water after 6 h.

T a 5 l and c a 1

table 2

Claims (1)

Claim The method of obtaining bacterial $ pyrogen, providing for the extraction of pyrogen from bacteria, followed by dialysis for 3 days, ultracentric trifugation and lyophilization of the target product, as indicated by the fact that, in order to increase the yield of the target product, pyrogen is extracted from genetic recombinants Shigella Flexner and EzegchSaHeN Н £ гН (^ ~) 33% water-phenol mixture at 70 ° С after preliminary suspension in distilled water, and dialysis is carried out through degreased parchment paper with a pore size of 10Σ А 20 A, the specific surface area of the pores 1000 · 1200 m ² / g and a thickness of 0.16-0.20 mm with a change of dialysis water after 6 hours. Table 1 Component Content components (M +. Go) % ! Donor .Recipient 8N. £ ex- Gene ty- E. coH H + rH ^ "') • Peg! № 737 type 2 a chesky re-combinant LPS 1.49 + 0.21 1.82 * 0.10 , 46 * 0.13 Lipid A 21.00 * 1.62 36,0011.49 '35.0 * 1.52 Monosacha REEDS 0,0010,00. 0.00 + 0.00 0,0010,00 kdo 0.00 * 0.00 0,0010,00 0,0010,00 Protein 2.79 + 0.31 0.00 + 0.00 one , 20 * 0.02 ν nucleus high acids 0.33 + 0.01 0.23 + 0.00 one , 00 * 0.04 Phosphorus 2.11 * 0.52 1.88 * 0.36 2 23 * 0.50 table 2 The statistical accuracy of decomp. Component Content (M ± t),% I (donor) 11 (recipient (rec. ent) binant) Ι-ΙΙ ^! ^: ¹¹¹ Fatty acids 43.70 * 0.50 37.80 * 0.45 21,2010,40 <0.05 <0.05 <0.05 Carbohydrates 55.80 * 0.25 61.70 * 0.38 72.20 + 1.73 <0.05 <0.05 ^ 0.05 ZON. WITH 46.04 * 2.48 45.96 + 1.58 48.66 + 2.33 <0.001 <0.001 <0.001