SU1040794A1 - Bordetella bronchiseptica-4994 strain - producer of site-specific endonuclease - Google Patents

Abstract

Strain Bordetella bronchiseptica-4994 (collection of the Scientific Research Order of the Red Banner of Labor Institute named after academician I.F. Gamalei, registration number 15) is a producer of site-specific endonuclease. (L with its

Description

The invention relates to the field of microbiology and genetic engineering, in particular to the production of a site-specific restriction endonuclease BbrI, an analogue of the restriction enzyme Hind III.

Microorganisms that produce Hind III type endonucleases from the genus Haeraophilus D,2 are known.

A strain of Bordetella pertussis is also known that produces the restriction enzyme Bpel, which hydrolyzes the DNA of phages A 989, 641, adenovirus, human type 2 and the plasmid pJDB219, as well as Hind III C3.

However, the known states are pathogenic for humans, demanding for enriched nutrient media and produce other types of endonucleases, which complicates the isolation and production of pure enzyme.

The aim of the invention is to identify a strain-producer that is non-pathogenic to humans, undemanding to nutrient media and selectively produces one site-specific endonuclease of the Hind III type.

The Bordetella bronchiseptica4994 strain was isolated from a rabbit, cloned for resistance to phages of the pertussis and bronchosepticosis microbes and is stored in the collection of the N.F. Gamaleya Research Institute of Epidemiology and Microbiology under registration number 15.

Morphological features. Small, motile, ovoid-shaped coccobacilli measuring 0.3–0.8–0.2 µm. Electron microscopic examination revealed lateral flagella.

Cultural characteristics. Bordet-Gengou medium (potato glycerol agar). Colonies are grey, shiny, with smooth edges, reaching 1.0 mm in diameter after 24 hours. Hemolysis was not detected.

KUA medium (casein-charcoal agar) Colonies are grayish-cream in color, of a viscous consistency, with smooth edges, after 24 hours they reach 1.0 mm in diameter, Pigment is not detected

Difco peptone agar. Colonies are cream-colored, of a thick consistency, reaching 1.5-2.0 mm in diameter after 24 hours.

Liquid nutrient medium of the Cohen-Wheeler type. Aerobic growing conditions. Grows with shaking. Medium

does not stain. Microbial suspension is grayish-white.

Physiological and biochemical characteristics. Assimilates organic compounds. Optimum growth temperature is 33-36 C. Amino acids are used as sources of nitrogen, carbon and energy. Citrates are used. Nitrates are reduced to nitrites. A rapid positive reaction to urease is characteristic (within 30 minutes at room temperature).

Contains water-specific bronchoseptic agglutinogen 12. The culture is stored in lyophilized form.

Bordetella bronchiseptica4994 strain contains the endonuclease BbrI, an analogue of Hind III.

Example. Lyophilized strain B. bronchiseptica-4994 was plated on KUA medium with 10% human blood, followed by a single subculture in test tubes with the same medium, but without the addition of blood. After 24 h of incubation at 35C, they were subcultured on matrices with KUA medium. The microbial cells were washed off with 0.85% sodium chloride solution. The microbial suspension was centrifuged at 7000 g for 20 min and washed with phosphocellulose (PC) buffer (10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 7 mM mercaptoethanol). Then 10 g of biomass was suspended in 10 ml of PC buffer containing 0.4 M NaCl and 100 μg/ml lysozyme and destroyed by ultrasound. Bacterial debris was sedimented by centrifugation (100,000 g, 90 min at 2-4 C). The resulting liquid was diluted with PC buffer 8-fold and applied to a Blue Sepharose column (Blue Sepharose Pharmacia, Sweden), 1.6-10 cm in size, equilibrated with PC buffer with 0.2 M NaCl. The column was washed with the same solution until the UV-absorbing material disappeared in the eluate. The adsorbed material was eluted with a 100 ml gradient of 0.2 M NaCl - 2.0 M NaCl with 30% glycerol in PC buffer. The volume of each fraction was 5 ml. An aliquot from each fraction (2 μl) was incubated for 1 h with 1 μg of phage A DNA and analyzed by electrophoresis in an agarose gel. Fractions that cleave phage A DNA into Hind Ill-specific fragments were combined and analyzed

against PC buffer and applied to a phosphocellulose column (PII Whatmanj England), equilibrated with PC buffer with 0.05 M NaCl, and eluted with a 100 ml gradient of 0.05-1.0 M NaCl in PC buffer. The volume of each fraction was 5 ml. Fractions containing BbrI were determined in the same way as in chromatography on blue sepharose, the enzyme was eluted in the range of 0.2-0.25 M NaCl. Active fractions were pooled and dialyzed against PC buffer containing 0.2 M NaCl and 50% glycerol. The resulting preparation (3 ml) had a specific activity of 2000 units/ml. The minimum amount of enzyme required for the complete cleavage of 1 μg of phage 7 in 1 hour was taken as a unit of activity.

To identify the sequence recognized by BbrJ, hydrolysis of the DNA of phage A, plasmids pBR322 and pSGI (carrying the complete SVAO genome) was performed. In parallel, hydrolysis of the indicated test DNAs was performed.

endonuclease Hind III. Electrophoresis of the hydrolysis products in agarose and polyacrylamide gels showed that BbrI hydrolyzes each of the test DNAs into fragments completely identical to the Hind III-specific fragments. Consequently, the nucleotide sequence recognized by BbrI is identical to the nucleotide sequence recognized by Hind III.

As a result of the treatment of Bbrl DNA fragments with T4 DNA digase, as in the case of Hind Ill DNA fragments, approximately 90% of the material was converted into a high-molecular form. Therefore, as in the case of Hind Ill DNA fragments, Bbrl DNA fragments have single-stranded sticky ends.

Thus, the BbrI restriction enzyme has an AAG CTT recognition site identical to the recognition site of the Hind III prototype and can completely replace the prototype in all genetic engineering experiments.