The method of producing biomass parasite plasmodium falciparum malaria

 

(57) Abstract:

The invention relates to medical Parasitology, to methods of cultivation of malarial parasite. The cultivation of Plasmodium falciparum in culture medium RPMI - 1640, optionally containing Hepes, whey, sodium carbonate, add antioxidant enzymes - superoxide dismutase (SOD) and catalase. These enzymes are separated from the erythrocytes of human blood. Enzymes enter in the number, excluding the hemolysis of erythrocytes. The method increases the yield of parasites, and biomass accumulation occurs over a short period of time. table 1.

The invention relates to medical Parasitology, to methods of cultivation of malarial parasite and can be used for the preparation of the malaria antigen.

A method of obtaining biomass of the pathogen of malaria P. falciparum (USSR author's certificate N 1563700 A1, A 61 K 39/02 C 12 N 5/00, 1988). The method is based on the method of frequent transfers of fresh washed and passed through a capillary column erythrocytes. Evaluation of results is carried out in thin smears, count of parasites per 1000 erythrocytes and expressed in percent.

However, this method is accompanied by hemolysis of erythrocytes, zadergka way of frequent transfers, however, a positive effect, i.e., stimulation of growth and reproduction of parasites observed in approximately 50% of cases. The erythrocytes that are in preromanticism condition and not suitable for the development of the parasites interfere with the effective cultivation.

The purpose of the invention is the improvement of biomass yield the causative agent of malaria P. falciparum due to the improvement of red blood cells in culture.

This objective is achieved in that the daily subcultures in complete growth medium RPMI - 1640 add inhibitors of oxidative processes is a complex of antioxidant enzymes - superoxide dismutase (SOD) and catalase (patent N 2033169 from 20.04.95,, MKI A 61 K 35/1811, C 12 N 9/00 for the invention "Method of obtaining complex of superoxide dismutase and catalase from human blood cell").

According to the proposed method of preparing a medium for the cultivation of complete RPMI - 1640 medium, Hepes (25 mmol/l) + 10% serum + 5% NaCO3pH of 7.2 + antioxidant enzymes SOD + catalase).

The number of enzymes necessary for optimal growth and reproduction of parasites was determined by adding the full nutrient medium different weight amounts of the complex.

Daily cultural tile is dcost selected, the besieged erythrocytes add an equal volume of prepared nutrient medium and receive a 50% cell suspension. After thorough mixing in a sterile environment to a suspension of red blood cells from the culture add 50% suspension of fresh washed erythrocytes to the number of parasites was 0.3 to 0.5%, then add cooked full nutrient medium with a complex of enzymes. The suspension is thoroughly mixed Pasteur pipette, pour in the culture Cup, 1.5 ml each, and continue to cultivate.

Example. Of the four cultural cups poured the contents into a sterile tube and mixed. Fill in complete nutrient medium to which 100 ml of medium add 5 mg of the complex of enzymes (SOD + catalase), centrifuged, the residue is poured into cultural 4-6 cups, add complete growth medium with complex enzyme, and continue to cultivate (A) (table). When reseeding of these cells with fresh erythrocytes count the number of parasites in a smear stained by Romanovsky-Institute. Added on Wednesday, 7 mg of the enzyme complex in 100 ml of complete medium. The percentage of parasites 5-10 achieved for 48 hours (B). Add in nutritious .

Add enzyme complex 10 mg per 100 ml of medium, which leads to lysis of infected erythrocytes (G).

The results of the study are presented in the table.

Thus the proposed method reduces the time of reproduction of malaria parasites in culture and increases the yield of biomass, which is achieved by adding in a complete nutrient medium of inhibitors of oxidative processes (SOD + catalase) in the amount of 5-10 mg per 100 ml of medium. Further adding to the complex environment is impractical because it leads to the destruction of erythrocytes containing pathogens.

The biomass yield (i.e. the number of parasites per 1000 erythrocytes) is significantly higher (2-4 times) and faster as early as 24 hours with the inclusion of the proposed method in the cultivation process.

The method of producing biomass malaria parasites, Plasmodium falciparum, including getting infected red blood cells, culturing of pathogens in a nutrient medium RPM1-1640, optionally containing Hepes, whey, sodium carbonate, by re-seeding with the addition of fresh red blood cells, wherein the culture medium was added to the complex of antioxidant enzymes - superoxide dismutase and catalase in erythrocytes of human blood is

 

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