The microscopic strain of the fungus aspergillus fumigatus frezenius 157/32 producing protein antigens for diagnosis mikulenas sensitization and allergies

 

(57) Abstract:

The invention relates to medical Microbiology and represents the strain mikroskopische fungus Aspergillus fumigatus Frezenius 157/32. The strain obtained by the method of two-phase natural selection on the basis of intensive germination of conidia. The new strain can be used in biotechnological process of obtaining protein antigens-allergens for the manufacture of allergenic products for immunodiagnostic mikulenas sensitization and allergies.

The invention relates to medicine, in particular to biotechnology, and is a selected strain of microscopic fungus that is used in the process of obtaining protein antigens - allergens for immunodiagnostic mikulenas sensitization and allergies.

Micronized Aspergillus fumigatus can be a significant etiological factor in such severe bronchopulmonary diseases as bronchial asthma, exogenous allergic alveolitis and allergic bronchopulmonary aspergillosis. Therefore, the development of tools for early immunoassay of these diseases is an important issue. Specific sensitization of the organism, caused by antigens mushrooms, mono with difficulty accumulation of significant quantities of mycelium and culture filtrate liquids.

The original strain of Aspergillus fumigatus 157-F isolated from the patient, has a low degree of germination of conidia in liquid nutrient medium, low productivity antigenantibody substances.

The purpose of the invention is the selection of the microscopic strain of the fungus with a strong reproduction of conidia, their intensive growth in a nutrient medium with a subsequent maximum biomass accumulation and protein antigenicity substances in submerged cultivation.

Due to the need to increase output antigenantibody substances applied the method of two-phase natural selection of the original strain of the fungus. Phase I - selection property of the morphology of the colonies, with the selection of the typical population of the original strain on agar medium of čapek-doksa; phase II - selection of the selected clones from the population of strain at the first stage on the basis of intensive germination of conidia in liquid medium Saburo. The result is a strain of Aspergillus fumigatus 157/32 stable property intensive germination of conidia in liquid nutrient medium and high productivity antigenantibody substances. In liquid medium Saburo with 4% glucose intensity germination of conidia selected strain 157/32 46% higher source is od biomass of the fungus and antigennouu activity of native solution in 1.5 times, what determines its projected profitability.

A strain of Aspergillus fumigatus 157/32 deposited in the Museum of mushroom crops research Institute of Medical Mycology them. B.p. Institute of Saint-Petersburg Medical Academy of postgraduate education and is characterized by the following features.

Cultural and morphological properties

Agar Chapek's medium-doksa with 2% glucose.

Colony after 30 days of cultivation at 28oC major 7-8 cm in diameter with a clear or slight radial folds, surface tomentose-velvety, dark-olive-gray, "bottom" side (reversum) yellow.

Conidial heads typical Kalinkovichi, compact. Conidiophores clustered, short, 300-400 μm DL., 5-8 μm wide., with a smooth cell wall of green shade, gradually widening towards the top. The apical part of canadianese plackovica, 20-30 µm in diameter, the same color as conditorem. Sterigma of a single-layer, are formed only in the center of the apical part, taking no more than 2/3 of its curved surface, densely pressed to each other, are arranged on an axis parallel to the axis of canadianese size 5.7 x 2.5 μm. Conidia globose 2.5 x 3.0 mm, the mass is dark the nom shaking on shuttel machine, in the form of small, smooth, spherical colonies, composed of interwoven strands of mycelium light yellow color. Conidia germinate on 13 hours growing micromycete.

Physiological properties

Relation to carbohydrates. On the environment Pridham-Gottlieb fungus grows well in the presence of glucose, arabinose, xylose, mannitol, ramnose, Inositol, galactose, sorbitol, sucrose.

Biochemical properties

The filtrate of the culture fluid of the fungus on the 41st day of submerged cultivation has the following characteristics: maximum accumulation of protein - 540 µg/mg, carbohydrates - 136 mcg/mg ratio of carbohydrates and proteins 1/4. Relative abundance of proteins with different isoelectric points (pI) is: 0.4 services.ed. for proteins with pI = 3.5 to 4.5; 0.1 srvc.ed. for proteins with pI = 5,0-5,5 0,1 and usl.ed. for proteins with pI = 7.5 to 8.5.

In the process of cultivation of the strain occurred acidification of the culture medium up to pH 4.5 (at initial pH of the medium 6,4) due to accumulation of acidic proteins that prevent possible bacterial contamination of the grown object.

The use of strain is illustrated by the following example.

Example. Aspergillus fumigatus strain 157/32 pre-grown on agresively in the Erlenmeyer flask with a capacity of 750 ml, containing 150 ml of seed medium Saburo with 4% glucose following composition per 1 l of water, g: peptone - 10,0, glucose - 40,0, pH is 6.4.

Seed strain of the fungus was grown in this medium with constant shaking at shuttel machine when the 27oC for 48 days.

In order to accumulation of protein antigens fermentation conducted in the medium of the same composition as for obtaining seed, but to a lesser extent medium - 100 ml For submerged cultivation of a strain of the fungus in a fermentation medium was brought to 5 ml of the culture fluid of the seed flasks. Cultures were also grown with constant shaking at 27oC for 41 days. As in sowing and in a fermentation medium before inoculation of the fungal culture was added to 1 ml of yeast extract at a concentration of 0.5%. By the end of the fermentation, the biomass was separated from the native solution by filtration through filter paper (Whatman No. 1). Molecules in the solution was determined by the accumulation of the protein in protein nitrogen with the help of Nessler's reagent.

Criterion allergenic activity of the native solution of the fungus was the amount of protein nitrogen as the standardization of allergens held in protein nitrogen units (PNU). The accumulation of protein nitrogen in selectionlanguage nitrogen was 3.5 10-2mg/ml, and by the end of fermentation was increased to 7.3 10-2mg/ml

Native solution through 41 d of the deep cultivation of the fungus has allergenic activity and preincubate it with a pool serum of patients allergic bronchopulmonary aspergillosis inhibits the reaction PACT (allergies, sorbency test) by 55%.

Selected microscopic strain of the fungus Aspergillus fumigatus Frezenius 157/32 used in biotechnological process of obtaining protein antigens-allergens, and the manufacture of allergenic medication used in immunodiagnostics mikulenas sensitization and Allegria.

 

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FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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