The method of obtaining asporogenic strain of bacillus anthracis (options)

 

(57) Abstract:

The invention relates to the field of Microbiology and genetics and can be used in genetic engineering of strains of Bacillus anthracis - producers of biologically active substances. After repeated passage, leading to the accumulation in the population of clones that form the atypical morphology of colonies obtained culture of B. anthracis plated on solid nutrient medium, incubated at 37oC and then at room temperature until pronounced morphological differences between colonies selected colonies in S-shape, which are tested for the ability to form spores. When used as a medium for seed culture of L-agar with the addition of Congo red, the selection is proposed to carry out simultaneously two criteria: colony morphology and pigmentable. A variant of the method, in which instead of stage passage culture before sowing spend transposon mutagenesis (TA 917 composition thermoluminesence vector plasmids LTV3ts). The invention provides efficient retrieval asporogenic strains on the basis of a broad range of cultures of Bacillus anthracis. 2 S. and 2 C.p. f-crystals.

and the genetic construction of strains of Bacillus anthracis - producers of biologically active substances.

A known method of random selection of asporogenic strains of anthrax - microorganism belonging to the spore-forming species of Bacillus. The sporulation is a functional characteristic, therefore, direct selection of clones with the breach is extremely difficult. The cases highlight asporogenic crops in populations of the original spore-forming strains of Bacillus anthracis by random selection are individual character. Selected asporogenic mikoplazmennyh capsulotomies strain Century anthracis Davis (Uchida I. , Sekizaki T., Hashimoto K. // J. Gen. Environ. - 1985. - V. 131. - P. 363-367). Selected similar asporogenic strain Century anthracis M29Spo-(Eremin, S. , Nikiforov, A. , Kostyukova So, Mikshis N. // Ukr. microbiol. - 1999. - 4 - C. 91-92). Selected asporogenic Besplatniy strain Century anthracis used to construct producer of the anthrax protective antigen (Farchaus, J., Ribot W., Jendrek, S., Little, S. Appl. Environ Environ. - 1998 - V. 64. P. 982-991).

However, the routine procedure of selection is reduced to the random choice among a huge number of clones. It is time-consuming, very expensive and ineffective.

Closest to the claimed method is a method, C is AI (R. Worsham, Sowers M // Canadian Journal of Environ. - 1999. - V 45. - P. 1-8). The authors found a pattern, according to which spontaneously formed asporogenic clones Century anthracis differ in ability to sorption of Congo red pigment. The method consists in seeding until isolated colonies culture of strain Century anthracis delta Sterne-1(RRA) on a solid nutrient medium with the addition of Congo red, incubation and selection characterized by the ability to sorption pigment colonies and then to test their characteristic sporoobrazovanie.

However, the above-described method of obtaining asporogenic strain suitable only for individual strains Century anthracis, repeatedly pereselivshiysya in the laboratory and characterized by heterogeneity of population composition.

The objective of the invention is to devise effective way to get asporogenic strains Century anthracis based on a wide range of cultures of Bacillus anthracis.

The problem is solved in two versions.

In the first embodiment of the essence of the proposed method is as follows: the method of obtaining asporogenic strain, including seeding culture in a nutrient medium to obtain isolated colonies, Incubus which begins before sowing culture repeated passage to accumulation in the population of spontaneous asporogenic clones and selection of colonies on atypical morphology (S-form).

In addition, the invention consists in the introduction of advanced operations when selected colonies with atypical morphology in the S-shaped inoculated in nutrient broth and select clones that differ in the nature of growth in liquid nutrient medium; conducting passage before sowing to the appearance of significant amounts (10 - 30%) atypical colonies and improved modes of incubation (within 24-48 hours in a thermostat at 37oC, then at room temperature for another 24 to 48 hours before the appearance of pronounced differences in colony morphology).

Offers modification of selection: 1) colony morphology, 2) on the morphology of the colonies and pigmentable. To do this, as a medium for seed culture using L-or L agar-agar with the addition of Congo red, respectively.

In the second variant the essence of the proposed method is as follows: the method of obtaining asporogenic strain, including seeding culture in a nutrient medium to obtain isolated colonies, incubation and selection of ancient colonies and then to test whether they have the ability to the formation of spores, as well as the implementation before sowing culture transposon mutagenesis to obtain asporogenic clones is the composition vector plasmids pLTV3ts cells besplodnogo strain Century anthracis method transduction and off characteristic sporoobrazovanie by embedding the transposon in the sequence of the chromosomal gene with simultaneous temperature elimination of vector plasmids.

The first variant of the method of obtaining asporogenic strain Century anthracis is as follows.

The strain of B. anthracis several times passedout on a nutrient medium to accumulate in a population of clones with the broken characteristic sporoobrazovanie.

For this one loop spore culture of any strain Century anthracis seeded into the vial with 25 ml of broth of Hottinger.

Incubated with aeration at shuttel-apparatus (80 rotations/min) at a temperature of 37oWith from 3 to 18 hours.

After a period of incubation, 2.5 ml of the grown culture is transferred into a vial with 25 ml of sterile medium and continue incubation in the same conditions.

So I repeat four or more times. The number of passages is considered sufficient if the seeding 0.1 ml of culture in a Cup with L-agar forms 10 - 30% atypical morphology of the colonies.

After the last passage of the strain B. anthracis make seeding to obtain isolated colonies on solid culture medium (L-or L agar-agar with the addition of Congo Krasnov 24-48 hours before the appearance of pronounced differences in colony morphology.

Grown colonies viewed in reflected light, and selection is performed on colony morphology or on the basis of pigmentable.

Clones in the S-form are tested for the ability to sporulation.

For this colony with crops, inkubirovanija from 2 to 5 days (the time required for spore formation), suspended in 1-2 ml of saline.

Tubes with culture placed in a water bath and heated at a temperature of 65oC for 30 minutes, or when 80oWith 10 minutes.

After warming up doing the seeding of 0.1 ml from each tube on a solid nutrient medium (agar of Hottinger or any environment for growing Bacillus anthracis).

Crops incubated in a thermostat at 37oWith 48 hours and in the absence of growth incubated for another 5 days for inspection. The lack of growth on nutrient medium after heating indicates a violation of the characteristic sporoobrazovanie. As a control, use the original spore-forming strain Century anthracis.

Additionally, the ability to form spores checked by microscopic examination of smears stained with fuchsin so. In a asporogenic strain lacking characteristically coloured spores.

1. After repeated passage culture plated on L-agar and selection is carried out on the morphology of the colonies. Select among dense and rough colonies (R-form) more transparent and smooth, shiny surface (S-form).

2. After repeated passage culture plated on L-agar with the addition of Congo red. Congo red add in the melted and cooled to 45oWith L-agar in the form of sterilized by filtering the aqueous solution to a final concentration in the environment of 25 μg/ml of the Selection carried out simultaneously in two ways: colony morphology and pigmentable. Among pale pink, pink, reddish colonies in the R-form select colonies bright red in the S-form.

In some cases, selected on the above environments atypical colony additionally tested for growth in liquid nutrient medium. One loop of culture from the colonies in the S-shaped inoculated in nutrient broth, poured in 2 to 5 ml in a test tube. Incubated without aeration in thermostat at 37oC. analysis carried out after 48 hours. Selected clones that form during the growth of uniform opacity of the medium (usually the anthrax microbe in a liquid nutrient medium is growing in the bottom osedc="ptx2">

Transposon (TP) in the vector plasmid pLTV3ts injected into cells besplodnogo strain Century anthracis method transduction using phage CP51ts45.

To do this, one loop of culture recipient besplodnogo strain Century anthracis seeded into the vial with the liquid nutrient medium. Incubated with aeration at shuttel-apparatus (80 rotations/min) at a temperature of 37oWith 18 hours.

The grown culture was diluted in 10-fold with sterile medium and continue incubation in the same conditions during 6-7 hours.

Then 0.5 ml of cell suspension is mixed with 0.5 ml of phage, multiplied by the donor culture, and to cause the transduction mixture onto a membrane filter (0.22 μm, "Millipore") located on the plate L-agar with Subselection concentration of erythromycin (0.1 ág/ml). Incubated in the incubator for 4 hours at 30oC.

After 4 hours, the filter is transferred to a plate of L-agar with selective concentrations of erythromycin (1 μg/ml), lincomycin (25 µg/ml) and tetracycline (10 μg/ml) and continue incubation until 36 to 48 hours at a temperature of 30oC.

The resulting transductant three times subcultured on solid nutrient medium with the addition of antibiotics to prevent secondary infection of the phage and the ins and lincomycin).

The presence in the cells of transductant transposonderived vector data confirm electrophoretic analysis.

At the next stage, the elimination of vector plasmids pLTV3ts with simultaneous integration of the transposon into a sequence of chromosomal genes.

To do this, one loop of culture stable transductant, received in the result of genetic transfer plasmid pLTV3ts, seeded in 25 ml of liquid nutrient medium with the addition of tetracycline in selective concentration (10 μg/ml) and erythromycin in subselections concentration (0.1 ág/ml).

Incubated at shuttel-apparatus (80 rotations/min) at 30oC for 16-20 hours.

Transfer 1 ml of culture in 25 ml of sterile medium with the addition of erythromycin and lincomycin in selective concentrations (1 and 10 µg/ml, respectively). Incubated at 43oC.

Thus, repeat 4 times. The first 2 times the incubation is carried out on shuttel-apparatus (80 rotations. /min) for 4 hours, thermostat 18 hours.

After the fourth passage 1 ml of culture inoculated in 25 ml of liquid nutrient medium without antibiotics and grown overnight at 30oC.

The effectiveness of temperaturesalinity to tetracycline and resistance to erythromycin and lincomycin and confirm data electrophoretic analysis.

The obtained cell suspension is a depot transposants with random transposon insertions in the sequence of the chromosomal gene.

Following the transposition (in terms of not longer than 1 month) shall conduct sampling asporogenic transposon mutants on colony morphology and pigmentable.

To do this, the cellular depot transposants carry out seeding until isolated colonies on solid culture medium (L-or L agar-agar with the addition of Congo red).

Crops incubated in a thermostat at 37oC for 24 to 48 hours, then at room temperature for another 24 to 48 hours before the appearance of pronounced differences in colony morphology.

Grown colonies viewed in reflected light, and selection is performed on colony morphology or on the basis of pigmentable.

Clones with different colony morphology and sorption pigment, are tested for the ability to sporulation by the method described in option 1.

The possibility of practical application of the invention is illustrated by examples.

Example 1 (option 1) - getting asporogenic strain Century anthracis KM89.

For the accumulation of spontaneous mutants in the population of strain Century anthracis Stern temperature 37oC.

Then do the seeding until isolated colonies on L-agar.

Cups with crops incubated in a thermostat at 37oC for 48 hours and then at room temperature for 24 hours.

Grown colonies are viewing in reflected light and select among rough and dense colonies (R-form) more transparent and smooth with shiny surface (S-form).

The selected clones that differ in morphology, are tested for the ability to sporulation.

For this colony with crops, inkubirovanija 48 hours, suspended in 2 ml of physiological solution. Tubes with culture placed in a water bath and heated at a temperature of 65oC for 30 minutes. Then do the sowing of 0.1 ml per Cup with L-agar. The lack of growth after heating indicates a violation of the characteristic sporoobrazovanie.

As a control using spore-forming strain Century anthracis Sterne 34F2. Additionally, the ability to form spores checked by microscopic examination of smears stained with fuchsin so. In experienced a lack of typical colored spores.

Example 2 (option 2) - getting asporogenic strain of B. anthracis KM90.

Recipient cells once with sterile medium and continue incubation in the same conditions for 6 hours.

After an incubation period, 0.5 ml of cell suspension is mixed with 0.5 ml of phage, multiplied by the donor culture. Transduction mixture is applied on a membrane filter, located on the plate L-agar with Subselection concentration of erythromycin (0.1 ág/ml).

Incubated for 4 hours at 30oC. the filter is Then transferred to the plate L-agar with selective concentrations of erythromycin and lincomycin and continue incubation for up to 48 hours at a temperature of 30oC.

Grown resistant to erythromycin and lincomycin colonies are transferred to a nutrient medium containing tetracycline, eritromicin and lincomycin. Selected clones growing on three antibiotics, and then determine plasmid profiles.

Phenotypic expression of resistance to the above three antibiotics and the presence on electrophoregram zones DNA corresponding to the higher mobility of DNA plasmids pLTV3ts, testify to the presence in the cells of transductant transposonderived vector.

Transductant seeded in 25 ml of nutrient broth with the addition of tetracycline in selective and erythromycin in subselections inducing transposition, concentrations.

Grown on shuttel-apparei 43o(2 times in 4 hours with aeration and 2 times 18 hours without aeration). For this, each time transfer 1 ml of culture in 25 ml of sterile nutrient broth with the addition of erythromycin and lincomycin in selective concentrations.

After the 4th passage 1 ml of culture inoculated in 25 ml of broth VN without antibiotics and grown overnight at 30oC.

thermal efficiency of elimination of plasmids pLTV3ts and transposition TP in a chromosome is determined by the phenotypic expression of sensitivity to tetracycline and resistance to erythromycin and lincomycin and confirm data electrophoretic analysis.

Cellular depot transposants carry out seeding until isolated colonies on plates with L-agar with the addition of Congo red.

Crops incubated in a thermostat at 37oC for 48 hours, then at room temperature for another 48 hours before the appearance of pronounced differences in morphology and color of the colonies.

Grown colonies viewed in reflected light. Selected colonies are bright red in S-form.

Clones that differ in morphology and sorption pigment, are tested for the ability to sporulation analogously to example 1.

Premilitary use Besplatniy derived natural strain Century anthracis 81/1. Getting asporogenic strain is carried out according to example 1, but the seed culture after passage of the produce on L-agar with the addition of Congo red, and selected colonies with both atypical morphology (S-form) and sorption pigment (bright red).

Similarly obtained asporogenic strains on the basis of the following crops Bacillus anthracis B. anthracis STITH, B. anthracis KM34, B. anthracis KM35.

Example 4 (option option 1 - obtaining an asporogenic strain Century anthracis KM92.

As the original culture used vaccine strain Century anthracis 55. Getting asporogenic strain is carried out according to example 1, but after the selection of atypical clones on L-agar with the addition of Congo red by a single loop of culture from the clones in the S-shaped inoculated in nutrient broth, poured in 2 to 5 ml in a test tube. Incubated without aeration in thermostat at 37oC. analysis carried out after 48 hours. Selected clones that form during the growth of uniform opacity environment.

Similarly obtained asporogenic strain Century anthracis CM(9) on the basis of monoplotting toxinogenesis derived natural strain Century anthracis 81/1.

Thus, the inventive method allows the wire is n cultures of Bacillus anthracis is practically unlimited. Multiple passage leads to the accumulation in the population of strain Century anthracis spontaneous asporogenic clones in sufficient quantity to their accidental discovery. Using transposon mutagenesis allows you to get asporogenic strains on the basis of cultures of Bacillus anthracis, where the selection of spontaneous asporogenic clones difficult. The procedure for the selection of strains with impaired functional characteristic sporoobrazovanie on concomitant change of sign, which can be visual assessment (colony morphology, the ability to sorption of pigment, the nature of growth in liquid nutrient medium), cheap and effective.

1. The method of obtaining asporogenic strain of Bacillus anthracis, including seeding culture in a nutrient medium to obtain isolated colonies, incubation and selection of atypical colonies and then to test whether they have the ability to the formation of a dispute, characterized in that before seeding culture conduct repeated passage to the accumulation of 10-30% of the clones forming on a solid nutrient medium atypical morphology of the colony, incubation of culture on solid nutrient medium is carried out at 37oC for 24-48 h, and the selection of clones is carried out on the basis of education is that the selected colonies with atypical morphology in the S-form is additionally inoculated in nutrient broth and selected clones forming with the growth of uniform opacity environment.

3. The method of obtaining asporogenic strain of B. anthracis under item 1, characterized in that as the medium for seed culture and subsequent selection using L-or L agar-agar with the addition of Congo red, and in the latter case, the selection is performed simultaneously in two ways: colony morphology and pigmentable.

4. The method of obtaining asporogenic strain of B. anthracis, including seeding culture in a nutrient medium to obtain isolated colonies, incubation and selection of atypical colonies and then to test whether they have the ability to the formation of a dispute, characterized in that before seeding culture perform transposon mutagenesis, incubation of culture on solid nutrient medium is carried out at 37oC for 24-48 h, and the selection of clones is performed according to the indication of the formation of colonies in the S-form.

 

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