The way to prevent sapa

 

(57) Abstract:

The use of medicine to prevent disease low glanders in sensitive to P mallei mammals. The inventive protein with a molecular mass of 37 - 30 KD, vydelennaya from the outer cell wall spnego microbe and presumably perform the function of steam formation, injected subcutaneously sensitive to Sapa mammals at the rate of 10 to 30,000 mg per kg of body weight. After 12 - 27 d immunization repeat. The method ensures the survival of 83% Golden hamsters infected subcutaneously with 50 - 100 lethal doses agar culture of the highly virulent strain. table 2.

The invention relates to medicine, in particular to methods of prevention of infectious diseases, and can be used to prevent infection low glanders in sensitive P. mallei mammals.

Against Sapa not developed methods of specific prophylaxis. Working with this pathogen is carried out under the conditions required mechanical protection of the eye, hand and respiratory tract. Infected animals usually kill, burn corpses.

Closest to the claimed is the method lies in the effects of antibacterial drugs (sulfanilyl).

The method is used exclusively for personal prophylaxis of the individuals involved in the accident [1]

The disadvantage of this method is nespecificnomu steps used to prevent Sapa medicines. In the result, preventive measures are not purposeful in nature and can be implemented only after human contact with the causative agent of glanders, when it became known representatives of the medical service. Due to their non-specificity of the method cannot be used for large-scale prevention of glanders in animals.

The aim of the invention is the creation of a mammal-specific resistance to infection with P. mallei.

The goal is achieved due to the impact on sensitive agent of glanders mammalian proteins with a molecular mass 37-40 KD isolated from the cell wall of the pathogen Sapa.

The invention is based on the first detected the ability of this protein, the causative agent of glanders to have a protective effect against sapnay infection.

The essence of the proposed method is as follows.

A protein with a molecular mass 37-40 KD, purified from cytoplasmic proteins and peptidoglycan the ssy. Through 12-27 day immunization repeat. To determine the protective effect perform subcutaneous or airborne contamination of animals virulent culture of P. mallei in quantities sufficient to cause their death. Within 30 days daily basis, record the number of dead animals. Protective effect is defined as the ratio of the LD 50 virulent strain for vaccinated animals to the LD 50 for intact (resistance index). While infecting a culture of a virulent strain of P. mallei index of resistance depends on the dose and frequency of drug administration. The method ensured the survival of 83% Golden hamsters infected subcutaneously 50-100 deadly infective doses agar culture a highly virulent strain of P. mallei C-5.

The essential feature of the claimed invention is that a protein with a molecular mass 37-40 KD isolated from the outer membrane of the cell wall of the pathogen Sapa, has a strong immunoprotective effect, which is related to the fact that the main function of this protein in the cell wall is the formation of pores for low molecular weight substances. Between the essential feature of the claimed object and achievable technical result there is a causal relationship, the proteins, porins, forming on the surface of the bacterial wall channels for the passage of solutes, leading to disruption of normal metabolism of bacteria that conical end and is the cause of their death.

Another possible mechanism for the prevention of infectious process can be stimulating specific cellular immunity by facilitating recognition by the immune system (cells, performing killer function) spnego microbe. Important in clarifying the mechanisms of development of a protective effect achieved in this way is the fact that some bacterial porins play the role of one of the pathogenic factors, facilitating their penetration into mammalian cells. The causative agent of glanders is an intracellular parasite, so any interaction of the antibody with its surface proteins and porins especially, leads to disruption of adhesion of this agent on the surfaces of target cells.

Preparations of membrane protein with a molecular mass 37-40 KD does not have a toxic effect against Golden hamsters at doses of 30 mg/kg weight of the animal. The detection ability of this protein to induce specific immunity is. delanie purinovogo protein from a culture of a strain of P. mallei C-5.

To highlight purinovogo protein was used the method developed Novikova O. D. for Yersinia pseudotuberculosis microbe (Novikova O. D. a pore-forming protein from the outer membrane of Yersinia pseudotuberculosis. Chemical characterization and biological activity: Dis.Kida.Biol.Sciences. Vladivostok, 1986). The method consists in the following. To 6 g of acetone-dried cells of the pathogen Sapa add 600 ml of chilled saline solution and within 2 hours the cells are suspended on a magnetic stirrer at a temperature of 42aboutC. and Then cooled by centrifugation for 45 min at 600 g. The residue is suspended in 200 ml of 0.03 M Tris-Hcl buffer (pH 7,2) (hereinafter buffer A), containing 2% Triton X 100, and extracted at a temperature of 42aboutC for 2 h under vigorous stirring. The precipitate was separated by centrifugation at 20000 g for 15 min, washed with buffer And suspended in 200 ml of buffer, add 2 ml of 1M solution of MgCl and 10 mg dnaase (Reachim NGO Biochemical) and incubated for 12 h

The precipitate was separated by centrifugation at 20000 g for 30 min, washed with buffer And suspended in 150 ml of buffer a containing 2% sodium dodecyl sulfate, and extracted at temperatures

The precipitate is washed with water, suspended it in 100 ml of buffer A, containing 0.5% sodium dodecyl sulfate and 0.5 M NaCl, incubated at a temperature of 370,5aboutC for 30 minutes and Then the precipitate was separated by centrifugation at 100000 g for 30 minutes Suspended it in 50 ml of buffer a containing 2% sodium dodecyl sulfate, and heated in a water bath for 10 minutes

The precipitate was separated by centrifugation at 150000 g for 1 h Then cleaning are gelfiltration in sephacryl S-200 or other media. The selected drug is a hydrophobic protein with a molecular mass 37-40 kilodaltons. In reaction diffusion precipitation diagnostic sera to the agent of glanders forms a clear line.

For membership in the selected protein to proteins of class Parinov says the following: the allocation Novikova O. D. can only be used to highlight a pore-forming protein, a protein localized in the outer membrane and is closely related to lipopolysaccharide; exhibits a pronounced hydrophobic properties and insoluble in water; it has the ability to induce a pronounced species-specific immunity; molecular mass of the monomer 37-40 KD that membrane proteins typical for Parinov.

O p s t 1. To determine the specific protective effect of animals were vaccinated subcutaneously twice with an interval of 21 days. The dose for each injection of 25 mg of antigen (protein) animal (animal weight 100 g). After 18 days after the second vaccination, animals were infected subcutaneously two-day agar culture of the strain P. mallei 5584. As control was used Intertie animals. The results of the experiment are given in table.1.

The definition of a protective effect derived the claimed invention in experimental Sapa Golden hamsters

O p s t 2. To study the possibility of increasing the protective action porina immunization was carried out with different doses and concentrations of drug.

The conditions of the experiment and the results are shown in table. 2. Animal infections were performed as described in experiment No. 1.

The WAY to PREVENT SAPA, including effects on the body medicamental means, characterized in that the impact of implementing isolated from the cell wall spnego microbe proteins mol. m 37 - 40 KD, which perform the role of species-specific antigen and induce the immunization in animals species-specific immunity to the pathogen sa is

 

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