Method to produce adjuvant

FIELD: biotechnologies.

SUBSTANCE: method includes cultivation of a fungus of Fusarium sambucinum type on nutrient medium containing a source of carbon 3-4%, nitrogen 0.2-0.3%, phosphorus 0.2-0.3% and microelements 0.07-0.08%, under sterile conditions at temperature of 26-30C with mixing and aeration within 36-72 hours with subsequent separation of the cultural liquid from fungus biomass. Besides, the cultural fluid is centrifuged at room temperature for 15-20 min. at 800-1500 rpm, and the target product is made from supernatant by separation of peptides with molecular mass from 3,000-60,000 D. Also before centrifugation the cultural fluid is heated up to 110-115C and pressure of 1.3-1.5 atm. with rate of 1.5-2 degrees/min. with the following exposure for 1.5-2.5 hours.

EFFECT: adjuvant prepared in accordance with the invention has higher potency that stimulates immunity, is harmless within vaccines and other products of immunological purpose, areactogenic, may be applied both independently and in combination with oil adjuvants or adjuvants-sorbents on the basis of aluminium hydroxide gel and other substances.

2 cl, 3 tbl, 9 ex

 

The invention relates to veterinary medicine and can be used in the development, production and application of means of specific prophylaxis (treatment) of diseases in the composition of inactivated or live vaccines for prophylaxis (treatment) of diseases, as well as in the mixture of antigen when receiving hyperimmune (immunospecificity) sera and drugs, and in particular to obtain antiviral vaccines, proteobacteria vaccines, receiving highly active hyperimmune sera including low antigens from different backgrounds to create a diagnostic enzyme immunoassay systems, as well as in other compositions for normalization of immune status of the animal.

A method of obtaining biologically active product hepatoprotective activity, comprising culturing a fungus of the species Fusarium sambucinum on a nutrient medium containing a carbon source 3-4%, nitrogen of 0.2-0.3%, phosphorus 0.2 to 0.3% and trace elements 0,07-0,08%, in sterile conditions at a temperature of 26-30C With stirring and aeration within 48-72 hours, followed by the separation of biomass and getting the culture fluid, and for cultivation using seed material obtained by pre-cultivation of the fungus species Fusarium sambucinum in the presence of ethanol, and under cultivation as a factor is silane production of metabolites hepatoprotective activity in culture medium was added to 0.5-1.5 vol.% ethanol (RF Patent No. 2289957, IPC A23L 1/30, BI No. 36, 2006).

However, the known biological product does not possess adjuvant (Adjuvant /adjuvant/ - a substance or complex of substances used to enhance the immune response when introduced simultaneously with immunogen).

A method of obtaining adjuvant in the interaction of aluminum salts with aqueous solutions of alkali (adjuvant/sorbent-based gel of aluminium hydroxide (Yarylo A. A., fundamentals of immunology: a Textbook. - M.: Medicine, 1999. - S. - ISBN 5-225-02755-5).

Also known is a method of obtaining adjuvant ('s adjuvant) by culturing living cells with subsequent separation of the product of their activity (discharge from Mycobacterium tuberculosis lipopolysaccharides, complex fatty acids /derivatives of lanolin/). Product life mixed with oil and an emulsifier to obtain a stable emulsion of the type water/oil, oil/water or water/oil/water (Great medical encyclopedia. Volume 1 /editor-in-Chief academician CH; publishing house "Soviet encyclopedia", Moscow, 1974. - 576 S.).

Known methods for producing adjuvant based on the execution of complex technological procedures that require a long time, also known adjuvants are not effective enough, show low quality target product detected in low-grade stimulation of immunogenicity, high reacto the items, the toxicity.

The objective of the invention is to improve the quality of the target product by increasing the immunogenic activity (eliminate and/or significantly reduce reactogenicity and toxicity of the final product, which increases the antigenic and immunogenic activity, reduces nonspecific stress on the immune system and reduces the reactogenicity).

The purpose of this technical solution is achieved in the way of receiving adjuvant, characterized in that the fungus species Fusarium sambucinum is cultivated in a nutrient medium containing a carbon source 3-4%, nitrogen of 0.2-0.3%, phosphorus 0.2 to 0.3% and trace elements 0,07-0,08%, in sterile conditions at a temperature of 26-30C With stirring and aeration for 36-72 h followed by separation of the culture liquid from the biomass of the fungus, and the culture fluid centrifuged at room temperature for 15-20 min at 800-1500 rpm, and the target product is obtained from the supernatant separation of peptides and proteins with molecular weight of from 3000 to 60000 Yes.

In addition, in the method of receiving adjuvant before centrifugation, the culture fluid is heated to a temperature of 110-115C and a pressure of 1.3-1.5 bar 1.5-2 deg/min followed by exposure for 1.5-2.5 hours.

In the patent and scientific literature is not well-known technical solutions, provided the signs, similarly claimed, i.e. the proposal meets the criterion of "novelty".

The proposal was feasible in laboratory and industrial conditions, aimed at solving real technical problem, i.e. the sentence "industrially applicable".

For the first time received adjuvant to obtain antiviral vaccines, proteobacteria vaccines, receiving highly active hyperimmune sera from the product of fungus species Fusarium sambucinum, which meets the criterion of "inventive step".

The invention is illustrated by the following examples:

Example 1. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 3% of carbon source /sucrose/, 0.2% nitrogen /ammonium nitrate NH4NO3/, 0.2% phosphorus /potassium phosphate one-deputizing KH2PO4/, 0,07% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 26C With stirring and aeration within 36 hours. Cultivation of the fungus are produced in fermenters (bioreactors) volume 16 m3. After the time of cultivation to produce separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 15 min at 800 rpm, the adjuvant 1 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 2. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 4% of carbon source /sucrose/, 0.3% nitrogen /ammonium nitrate NH4NO3/, 0.3% phosphorus /potassium phosphate one-deputizing KH2PO4/, 0.08% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 30C With stirring and aeration within 72 hours. Cultivation of the fungus are produced in fermenters (bioreactors) with a volume of 5 liters. After the time of cultivation to produce separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 20 min at 1500 rpm, and adjuvant 2 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 3. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 3% of carbon source /sucrose/, 0.2% nitrogen /ammonium nitrate NH4NO3/, 0.2% phosphorus /potassium phosphate one-deputizing KH2PO4/, 0,07% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 26C With AC is sevanam and aeration within 36 hours. Cultivation of the fungus are produced in fermenters (bioreactors) volume 16 m3. Before centrifugation, the culture fluid is heated to a temperature of 110C and a pressure of 1.3 ATM to 1.5 deg/min followed by exposure for 1.5 hours. After the time of heating to produce a separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 15 min at 800 rpm, and 3 adjuvant obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 4. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 4% of carbon source /sucrose/, 0.3% nitrogen /ammonium nitrate NH4NO3/, 0.3% phosphorus /potassium phosphate one-deputizing KH2PO4/, 0.08% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 30C With stirring and aeration within 72 hours. Cultivation of the fungus are produced in fermenters (bioreactors) with a volume of 5 liters. Before centrifugation, the culture fluid is heated to a temperature of 115C and a pressure of 1.5 ATM at 2 deg/min followed by exposure for 2.5 hours. After a time prog is evania produce separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 20 min at 1500 rpm, and adjuvant 4 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 5. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 3% of carbon source /glucose/, 0.2% nitrogen /ammonium sulfate (NH4)2SO4/, 0.2% phosphorus /sodium phosphate one-deputizing NaH2PO4/, 0,07% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, the rest water to 100%, under sterile conditions at a temperature of 26C With stirring and aeration within 36 hours. Cultivation of the fungus are produced in fermenters (bioreactors) volume 16 m3. After the time of cultivation to produce separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 15 min at 800 rpm, and adjuvant 5 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 6. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 4% of carbon source /sec is the goat/, 0.3% nitrogen /ammonium sulfate (NH4)2SO4/, 0.3% phosphorus /sodium phosphate one-deputizing NaH2PO4/, 0.08% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 30C With stirring and aeration within 72 hours. Cultivation of the fungus are produced in fermenters (bioreactors) with a volume of 5 liters. After the time of cultivation to produce separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 20 min at 1500 rpm, and adjuvant 6 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 7. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 3% of carbon source /glucose/, 0.2% nitrogen /ammonium sulfate (NH4)2SO4/, 0.2% phosphorus /sodium phosphate one-deputizing NaH2PO4/, 0,07% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 26C With stirring and aeration within 36 hours. Cultivation of the fungus are produced in fermenters (bioreactors) volume 16 m3. Before centrifugation, cultural the second liquid is heated to a temperature of 110C and a pressure of 1.3 ATM to 1.5 deg/min followed by exposure for 1.5 hours. After the time of heating to produce a separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 15 min at 800 rpm, and adjuvant 7 is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 8. For the preparation of adjuvant use mushroom species Fusarium sambucinum strain Sambucinum Fuckel F-3051D, which are cultivated on a medium containing 4% of carbon source /glucose/, 0.3% nitrogen /ammonium sulfate (NH4)2SO4/, 0.3% phosphorus /sodium phosphate one-deputizing NaH2PO4/, 0.08% trace elements Mg, Mn, Fe, Ca, Zn, taken in equal proportions, and the rest water to 100%, under sterile conditions at a temperature of 30C With stirring and aeration within 72 hours. Cultivation of the fungus are produced in fermenters (bioreactors) with a volume of 5 liters. Before centrifugation, the culture fluid is heated to a temperature of 115C and a pressure of 1.5 ATM at 2 deg/min followed by exposure for 2.5 hours. After the time of heating to produce a separation of the biomass from the culture liquid of one of the known methods. The culture fluid centrifuged at room temperature for 20 min at 1500 rpm, and adjuvant 8 is obtained from the supernatant otdelannaya and proteins with molecular weight from 3000 to 60000 Yes with the help of porous filter.

Example 9. Adjuvant 1-9 obtained in examples 1-9, provides specific immune response to the introduction of haptens in combination (chemical compound) with a macromolecular protein in a significantly higher degree compared with the classical adjuvant - complete adjuvant-blockers.

The impact of adjuvant prepared by the present method, on the immunogenicity of adhesive antigens of Escherichia studied in comparative experiments on rabbits. For the experiments used adhesive antigen of Escherichia vaccine # 1 - control 1), the adhesive antigen in a mixture with aluminum hydroxide in the ratio of 7:3 (vaccine No. 2 control 2), adhesive antigen of Escherichia mixed with adjuvant prepared by the present method, in the ratio of 1:4 (vaccine 3 - experienced with adjuvant 1; vaccine 4 - experienced with adjuvant 2; vaccine 5 - experienced with adjuvant 3; vaccine 6 - experienced with adjuvant 4; vaccine 7 - experienced with adjuvant 5, vaccine 8 - experienced with adjuvant 6, vaccine 9 - experienced with 7 adjuvant, vaccine 10 - experienced with adjuvant 8). For testing were formed six groups (two control and four experimental) rabbits weighing 2.8-3.0 kg, 9 animals in each group. Each rabbit experimental and control groups were injected subcutaneously with 1 cm3vaccine, after 7 days of repeated introduction subcutaneously to rabbits respective groups according to stuudy vaccine at a dose of 3 cm 3.

The data presented in table indicate that adjuvant obtained by the present method, enhances the immune response to adhesive antigens of Escherichia 2-8 times. On the adjuvant properties of the claimed drug significantly exceeds the adjuvant properties of aluminium hydroxide in the composition of the vaccines.

In the production of adjuvant completely eliminates the need for special operations selection, refining mineral oils or cooking gel aluminium hydroxide, almost completely eliminates the need for time-consuming operations.

Adjuvant prepared according to the invention, when more productive and technological method of production, is more stimulating immunity potency, harmless in the composition of vaccines and other immunological products destination, reactogenic, can be used both independently and in combination with oil adjuvants or adjuvants-based sorbents gel aluminium hydroxide and other substances.

td align="center" morerows="1"> Immunogen
Table 1
Titers of rabbit sera obtained with and without the use of adjuvant prepared by the present method
Titers of sera
With the use of the inventive adjuvantWith adjuvant-blockers
KLH-CP-AOZ30000-400005000-10000
KLH-NCA30000-500004000-8000
KLH-QSA30000-500002000-7000

Table 2
The results of the test adjuvant activity, prepared by the present method, the recombinant proteins
The immunizing composition of the mixtureDose infectionLD50IE*
oprF + aluminium hydroxidePA 10320057,431,77
100
50
25
12,5
oprF + adjuvant 1PA 10320091,382,83
100
50
25
12,5
oprF + adjuvant 2PA 103200101,033,12
100
50
25
12,5
oprF + adjuvant 3PA 10320096,222,97
100
50
25
12,5
oprF + adjuvant 4PA 10320091,412,82
100
50
25
12,5
AB + aluminium hydroxidePA 10320061,561,9
100
50
25
12,5
AB + adjuvant 1RA 10320072,462,22
100
50
25
12,5
AB + adjuvant 2PA 10320076,272,36
100
50
25
12,5
AB + adjuvant 3PA 10320080,082,47
100
50
25
12,5
AB + adjuvant 4PA 10320072,42,24
100
50
25
12,5
ControlPA 10320032,42
100
50
25
12,5
6,25
* where IE is the index of the effectiveness of vaccination.

Table 3
The titer of antibodies to adhesion antigens of Escherichia in samples of blood serum of rabbits experimental and control groups
Name of vaccineThe titer of antibodies in RA
Through 7 days after the second vaccinationAfter 14 days the after the second vaccination After 21 days after second vaccination
Adhesive antigen (control No. 1)1:101:201:50
Adhesive antigen + aluminium hydroxide (control 2)1:201:1001:200
Adhesive antigen + beta-blockers (control 3)1:201:1001:200
Adhesive antigen + ADJUVANT 11:1001:2001:400
Adhesive antigen + ADJUVANT 21:1001:2001:400
Adhesive antigen + ADJUVANT 31:1001:4001:800
Adhesive antigen + ADJUVANT 41:1001:4001:800
Adhesive antigen + ADJUVANT 51:1001:200 1:400
Adhesive antigen + ADJUVANT 61:1001:2001:400
Adhesive antigen + ADJUVANT 71:1001:4001:800
Adhesive antigen + ADJUVANT 81:1001:4001:800

1. The method of receiving adjuvant, characterized in that the fungus species Fusarium sambucinum is cultivated in a nutrient medium containing a carbon source 3-4%, nitrogen of 0.2-0.3%, phosphorus 0.2 to 0.3% and trace elements 0,07-0,08%, in sterile conditions at a temperature of 26-30C With stirring and aeration for 36-72 h followed by separation of the culture liquid from the biomass of the fungus, and the culture fluid centrifuged at room temperature for 15-20 min at 800-1500 rpm, and the target product is obtained from the supernatant separation of peptides and proteins with molecular weight from 3000 to 60000 Yes.

2. The method according to claim 1, where before centrifugation, the culture fluid is heated to a temperature of 110-115C and a pressure of 1.3-1.5 bar 1.5-2 deg/min followed by exposure for 1.5 to 2.5 hours



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to method of obtaining immunotropic preparation. Method of obtaining immunotropic preparation by extraction of air-dry leaves of Lophanthus anisatus Benth with aqueous ethyl alcohol, heating, cooling, filtering, solvent evaporation, residue dissolving and extraction with chloroform, filtering and drying of sediment, under specified conditions.

EFFECT: method makes it possible to obtain efficient immunotropic medication.

3 tbl, 5 ex

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30oC or with ozone at temperature 15-25oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

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