RU2037520C1 - Recombinant plasmid dna pzat1 used for preparing dna-probe which is used for identification of toxigenic strains of anthrax pathogen, method of construction of plasmid dna pzat1 and a strain of bacterium escherichia coli used for preparing recombinant plasmid dna pzat1 and dna-probe which is used for identification of toxigenic strains of anthrax pathogen - Google Patents

Abstract

FIELD: medicinal biotechnology. SUBSTANCE: method involves construction of recombinant plasmid DNA carrying species-specific fragment of anthrax plasmid pXOI which is used for DNA-probe. Synthesis level of recombinant plasmid in cells of strain is 1.5-2 mg per 1 l nutrient medium. DNA-probe prepared on the basis of plasmid DNA pZAT1 is hybridized only with toxigenic strains of anthrax pathogen that ensures to differentiate their from other bacteria including closely related bacilli and avirulent strains Bacillus anthracis no containing plasmid pXOI. EFFECT: construction of plasmid DNA indicated above. 4 cl, 1 dwg, 1 tbl

Description

 The invention relates to medical microbiology and biotechnology, in particular to genetic engineering, and can be used to identify toxigenic strains of anthrax pathogen by molecular hybridization.

 Known methods for identifying the causative agent of anthrax, based on the identification of phenotypic characteristics of the species (antigenic structure, biochemical properties, sensitivity to bacteriophages).

 The disadvantages of these methods are associated with the instability of the phenotypic properties of microorganisms, which requires identification of the type of study of the complex of phenotypic characters. In contrast to the variable phenotype, the genotype is inherently the primary basis of species affiliation, that is, it is more stable and, therefore, specific.

 To develop genotypic identification methods, one proceeds from the presence of conservative nucleotide sequences in the genome, in which genomic rearrangements lead to loss of species affiliation. When searching for a DNA probe, tactics boil down to cloning the gene or its part that determines the most important species trait.

 The anthrax plasmid pX01 determines the synthesis of a three-component toxin, and its presence is necessary for the implementation of the pathogenesis of the disease. However, the native plasmid pX01 cannot be used as a DNA probe, as it contains DNA fragments homologous to closely related B. cereus, B. thuringiensis, B. megaterium (own observation).

 The objective of the invention is the construction of recombinant plasmid DNA carrying a species-specific fragment of anthrax plasmid pX01, and the creation of a strain of Escherichia coli, a producer of recombinant plasmid DNA and a DNA probe used to identify toxigenic strains of the anthrax pathogen by molecular hybridization.

 To solve this problem, restriction and hybridization analysis of fragments of anthrax plasmid pX01 is carried out. Plasmid DNA is digested with BamHI restriction enzyme and the specificity of hybridization binding of various fragments is studied. BamHI Restrictions of 14 kbp providing hybridization only with pX01 plasmid DNA, they are cloned by the shot-gun method after complete hydrolysis with the HindIII enzyme in the pBR322 vector DNA. The E. coli strain HB101 is transformed with a ligase mixture, a clone containing a recombinant plasmid with an insert of 0.9 kb is selected. and the plasmid pZAT 1 is isolated from it.

 The drawing shows a physical map of the recombinant plasmid pZAT 1.

 PRI me R 1. Construction of recombinant plasmids pZAT 1.

 Isolation of plasmid DNA pX01.

300 ml of a culture of Bacillus anthracis Sterne 34F ₂ strain were grown in BH1 medium for 14 hours. Cells were washed with 0.05 M Tris-HCl, 0.01 M EDTA, 0.05 M NaCl, pH 8.0. Resuspendable in 10 ml of 0.05 M Tris-HCl, 0.02 M EDTA, pH 8.0. Add 10 mg / ml lysozyme and incubated for 90 min at 37 ^C. The slurry was slowly added into 200 ml of lysis solution: 20 ml of 0.5M Tris-HCl, pH 8.0; 16 ml 0.25M EDTA, pH 8.0; 20 ml 10% SDS; 120 ml of water. The pH was adjusted to 12.45 with a fresh solution of 2M NaOH and water was added to 200 ml. The lysis is carried out in a beaker with a volume of 1 liter on a magnetic stirrer with a rotation speed of 50 rpm for 10 minutes By titration with 2M Tris-Hcl (pH 7.0), the pH of the lysate was adjusted to 8.6. Subsequently, 1/10 volume of 5M NaCl, 200 ml of phenol balanced with 0.05M Tris-Hcl, 0.02M EDTA, 0.5M NaCl (pH 8.0) was added. After phase separation, the aqueous phase is transferred to a clean beaker and, under similar conditions, treated with an equal volume of a mixture of chloroform and isoamyl alcohol (24: 1).

The selected aqueous phase is diluted 2 times with water and extraction is carried out three times with diethyl ether. Alternately dissolved in purified lysate, 75 g of polyethylene glycol 6000 and 27 g of NaCl and allowed to stand overnight at ⁴ ° C Centrifuge at 9500 rev / min (Ja-14) for 10 min. The precipitate was dried and dissolved in 16 ml of 0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0 and centrifuged in a gradient of cesium chloride in the presence of ethidium bromide (a solution of 1.585 density), at 40,000 rev / min, 15 ^C, 24 h (rotor 70.1.Ti). The supercoiled form of the plasmid is selected (lower band) and after removal of the ethidium bromide by extraction with 1-butanol, the purified plasmid DNA preparation is reprecipitated with 2.5 volumes of ethanol in the presence of 0.3 M Na acetate (pH 7.0).

 Isolation and cloning of a species-specific fragment of plasmid DNA pX01.

The DNA of plasmid pX01 in an amount of 50 μg was subjected to hydrolysis with BamHI restrictase and preparative electrophoresis was carried out in 0.6% low-melting agarose (1.5 V / cm, 16 h). After elution of fragments of size 14 TPN Phenol treatment and ethanol reprecipitation 5 ug drug Selections hydrolyzed with restriction enzyme HindIII (10 units.) for 1 h at ³⁷ ° C in 30 ul buffer containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl _2, 100 mM NaCl, 1 mM dithiothreitol. The completeness of restriction is monitored by electrophoresis of 1/10 of the reaction volume in a 0.8% agarose gel. 1 microgram of pBR322 vector DNA is hydrolyzed with restriction enzymes BamHI (5 units.) And HindIII (5 U.) For 1 h at ³⁷ ° C in 20 ul of the above buffer. The completeness of restriction is controlled by electrophoresis of 1/10 of the reaction mixture in 0.8% agarose.

The fragment and vector restriction preparations were purified by phenolic extraction followed by ethanol reprecipitation in the presence of 0.3 M sodium acetate and resuspended in 50 μl ligase buffer (66 mM Tris-Hcl, pH 7.6, 5 mM MgCl ₂ , 5 mM dithiothreitol, 1 mM ATP), add T4 phage ligase (5 units). The ligation reaction was performed at 15 ^{° C} overnight. Control by electrophoresis 1/10 of the volume of the ligase mixture in 0.8% agarose.

The transformation of E. coli HB101 cells is carried out by the following method: 0.1 ml of a suspension of E. coli HB101 cells is added to 10 ml of L-broth and grown to a culture density of OD / 600 = 0.5. After cooling on ice for 10 min, cells were pelleted by centrifugation (5000g, 10 min, 4 ° ^C), suspended in 10 ml of a 0.1 M solution of CaCl ₂ and incubated 1 hour on ice. Reprecipitated cells and resuspend in 0.5 ml of 0.1 M CaCl ₂ and stored at 0 ° ^C. 200 .mu.l of competent E. coli HB101 cells were mixed with 1/10 volume of the ligation mixture is incubated for 1 h on ice and performed heat shock at 42 ^about With for 2 minutes Add 1 ml of L-broth, incubate for 1 h at 37 ^about C and seeded on L-agar with ampicillin (50 μg / ml). Crops were incubated at ³⁷ ° C overnight, grown colonies is transferred on L-agar with tetracycline and selecting recombinant clones (Ap-r, Tc-s ).

 Analysis of recombinant clones and obtaining a producer strain.

To determine the insert size of the recombinant cell clones were grown in L-broth overnight at ³⁷ ° C for Shuthelah, pelleted by centrifugation in 1.5 mL microfuge culture. Cells are resuspended in 350 μl of lysis solution (8% sucrose, 1% SDS, 50 mM EDTA, 50 mM Tris-Hcl, pH 8.0).

It is aged 5 min at room temperature and placed in a boiling water bath for 1 min. It is centrifuged on a microfuge for 15 minutes, the supernatant is poured into clean tubes and an equal volume of isopropanol is added. It is kept at room temperature for 10 minutes and centrifuged on a microfuge for 15 minutes. The precipitate was washed with ⁷⁰ ethanol, dried and resuspended in 30 ul TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). 10 μl of each preparation is digested with restriction enzymes BamHI and HidIII and the insert size is monitored by electrophoresis on a 0.8% agarose gel in the presence of molecular weight markers (PstI fragments of lambda phage).

 Recombinant plasmid DNA consisting of a replicon of the vector plasmid pBR322 and an insert of 0.9 kbp denote pZAT 1, and the clone carrying the hybrid plasmid was selected as a producer strain of E. coli KM-51.

 Like m. of the plasmid pZAT 1 is 3.19 MDa (4.9 kb) and consists of the DNA of the vector pBR 322, 4.0 kb in size. and BamHI-HindIII fragment of anthrax plasmid pX01 size 0.9 TPN (drawing).

 Strain E. coli KM-51 producer of the recombinant plasmid pZAT 1 is characterized by the following features.

 Cultural and morphological.

Cells are straight, rod-shaped, gram-negative, non-spore. It grows well on simple nutrient media at temperatures ranging from 4 to 45 ° ^C under optimum pH from 6.8 to 7.5. On meat and peptone agar forms smooth, gray, round, shiny colonies with a smooth edge. On meat and peptone broth forms a smooth intense turbidity.

 Physiological and biochemical.

 It uses many carbohydrates, organic acids, alcohols as a carbon source. As a source of nitrogen, it uses mineral salts (in ammonium or nitrate form), organic compounds (in the form of peptone, tryptone, amino acids), it needs proline. It shows resistance to ampicillin (50-100 μg / ml).

 The characteristic of a useful product.

 The cells of the strain contain plasmid DNA (4.9 kbp), the presence of which is confirmed by the resistance of the strain to ampicillin (50-100 μg / ml), as well as by isolation of plasmid DNA by any known method.

 The level of synthesis of the recombinant plasmid in the cells of the strain is 1.5-2 mg per 1 liter of culture medium at a culture density of 0.4 (at a wavelength of 600 nm).

 Other signs.

 The strain E. coli KM-51 is a derivative of the strain E. coli HB101, is not able to cause an infectious process.

Genotype: F ^- , hsdS20 (r-, m-), supE44, recA13, ara14, proA2, rpsL20 (str ^r ), xyl-5, mlt-5, supE44, λ- (amp ^r ).

Cells of strain stably maintained the plasmid DNA when cultured on media with ampicillin (50 ug / ml) in the lyophilized state as well as in the cryopreservation in the presence of 15% glycerol at -70 ° ^C.

 PRI me R 2. Obtaining a radioactive probe and its use.

 Cells of strain KM-51 are grown overnight in 15 ml of L-broth with ampicillin (50 μg / ml) and plasmid DNA pZAT 1 is isolated. An aliquot of the preparation containing 10 μg of DNA is hydrolyzed with BamHI and HindIII restriction enzymes and 0.8 preparative electrophoresis is carried out. % fusible agarose. A fragment of 0.9 kbp elute and after phenol extraction and reprecipitation with ethanol in the presence of 0.3 M ammonium acetate, it is used for nick translation.

A 1 μg DNA probe was added to 50 μl of the reaction mixture: 50 mM Tris-Hcl (pH 7.5), 5 mM MgCl ₂ , 10 mM mercaptoethanol, 100 μg / ml BSA, 10 μM dATP and dGTP, 50 each μCi [α 32-P] dTTP and [α 32-P] dCTP. 2 µl of DNAase I (100 ng / ml) was added and incubated for 10 min at 14 ° C. Contribute 2 units. DNA polymerase I, and incubated for 1 h at 14 ° ^C. From the nucleotide is not included are exempt minicolumn chromatography on a Sephadex G-50 followed by c rating for the activity of fractions and determining the specific activity of the probe DNA. Preparation of DNA-probe was denatured for 10 minutes in a boiling water bath and rapidly cooled on ice and stored at ^-20 ° C until use.

Total DNA from the studied strains is isolated as follows. Cells are seeded in 5 ml of BHI broth containing 10% inactivated normal horse serum. Grown at 37 ^about C for 5 hours with vigorous stirring (150 rpm). 0.1 ml of broth culture is seeded on serum BHI mowed agar (10%). Incubated overnight at ³⁷ ° C under anaerobic conditions.

Cells were washed with 5 ml of TES buffer (0.05 M Tris-Hcl, pH 8.0, 0.05 M NaCl, 0.005 M Na ₂ EDTA, pH 8.0) containing lysozyme at a concentration of 10 mg / ml. Kept 90 minutes at 37 C. To ^the 3 ml of cell suspension was added two volumes of lysis solution: 50 mM Tris-OH, 3% SDS, 0,5 M NaOH, pH 12.45. Incubated for 30 min at room temperature until the lysate is completely clarified.

 Add 0.8 ml of 2 M Tris-Hcl (pH 7.0), 1 ml of 5 M NaCl and an equal volume of freshly distilled buffered phenol, mix and incubate overnight at room temperature. Centrifuged at 6 thousand g, 10 min. The aqueous phase is taken, mixed with an equal volume of chloroform, centrifuged in the indicated mode and the aqueous phase is taken. 1/5 volume of 10 M ammonium acetate and an equal volume of isopropanol are added. It is kept for 10 minutes at room temperature and centrifuged at 12 thousand g, 30 minutes. The supernatant is drained, the precipitate is dried at room temperature and resuspended in 0.5 ml of TE buffer. The concentration of the obtained preparations is determined on a DNA fluorometer or electrophoretically.

To an aliquot of solutions containing 2 ug of total DNA (based on one spot of the filter) was added 1/10 volume of 3 M sodium acetate and two volumes of ethanol, incubated for 30 min at ^-20 ° C, centrifuged at 12 tys.g, 20 min . The supernatant is drained, the precipitate is dried and dissolved in 10 μl of 0.3 M NaOH. Place in a boiling water bath for 10 minutes. Cool on ice.

Nitrocellulose (SC) filters are soaked sequentially in 2xSSC and 20xSSC (SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0). Dry at room temperature. Denatured DNA preparations were applied to the filters in a volume of 10 μl per spot. NC-filters were placed for 10 minutes on filter paper moistened with a solution of 2M ammonium acetate, dried at room temperature and heated in vacuo at ⁸⁰ ° C for 2 hours.

Prehybridization processing of NC filters is carried out. To do this, they are soaked for 5 min in 5xSSC and transferred to 10 ml of prehybridization solution: 5xSSPE (0.8 M NaCl; 50 mM NaH ₂ PO ₄ , pH 7.4; 5 mm EDTA, pH 7.4), 50% formamide, 5x Denharda solution (0.1% Ficoll 400; 0.1% BSA;. 0.1% polyvinylpyrrolidone (PVP), 0,1% SDS, 100 ug / ml denatured thymus DNA was incubated at 42 ^{° C} for 4 h. The labeled probe was added and hybridization was carried out overnight at 42 ° ^C. Filters were washed 3-4 times for 10 minutes at room temperature 300 ml of 2 × SSC, 0,1% SDS and doubly at 68 ^{° C} for 1 h in 500 ml 1xSSC, 0.1% SDS and dried at room temperature.

After hybridization, the filters were exposed to X-ray film cassette with RM-B "Tasma" at ^-20 ° C for 2 days. The presence of hybridization binding of the probe is evaluated by the exposure of the film at the spot of application of the stain of the corresponding preparation of total DNA of the studied strain.

 The results of DNA-DNA hybridization using a DNA probe are shown in the table. As can be seen from the table, the DNA probe proposed as an invention hybridizes only with toxigenic strains of the anthrax pathogen, differentiating them from representatives of other bacteria, including closely related bacilli and avirulent B.anthracis strains that do not contain pX01 plasmid.

 Thus, the obtained E. coli KM-51 producer strain carrying the recombinant plasmid pZAT 1 can be used to isolate this plasmid and generate a DNA probe. The recombinant plasmid pZAT 1 is multi-copy, has a selective marker of ampicillin resistance, amplifies in the presence of chloramphenicol. The fragment proposed as a DNA probe is easily isolated by hydrolysis of plasmid DNA with restriction enzymes BamHI and HindIII, provides high species specificity of hybridization binding with toxigenic strains of B.anthracis, which allows the invention to be used to improve identification of anthrax pathogen using molecular hybridization.

Claims (3)

 1. Recombinant plasmid DNA pZAT1, used to obtain a DNA probe, with the help of which toxigenic strains of the anthrax causative agent are identified, sizes 4.9 bp containing the plot of the plasmid pBR 322 size 4 T. p. Bam H I Hind III fragment of anthrax plasmid pX 01 with a size of 0.9 kb genetic marker: bla-gene for the synthesis of β-lactamase.  2. The method of constructing recombinant plasmid DNA pZAt1, which consists in the fact that the anthrax plasmid pX01, which determines the synthesis of the three-component toxin Bacillus anthracis, is hydrolyzed with restriction enzyme Bam H I, fragments of 14 kb are isolated. they are treated with the restriction enzyme Hind III, the obtained fragments are cloned using the dipmetod method in plasmid pBR 322, transforming Escherichia coli cells of strain HB101, a clone containing a recombinant plasmid with an insert of 0.9 kb is selected. and the plasmid pZAT1 is isolated from it.  3. The bacterial strain Escherichia coli KM-51, used to obtain recombinant plasmid DNA pZAT1 and a DNA probe, with which toxigenic strains of the causative agent of anthrax are identified.

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