Коллабораторы: |
Tufts University / Tufts New
England Center Hospital, Boston, MA, USA
Hauptman-Woodward Medical Research Institute, Inc.,
Buffalo, New-York, USA
University of Aarthus, Aarhus, Denmark |
|
Immunoglobulin A (Ig A) is one of the five classes of
antibodies, induced in response to the appearance of foreign substances,
antigens, in an organism. IgA protect mucosas from adhesion and invasion
of pathogenic microorganisms.
IgA are generally resistant to the
effect of protheolytic enzymes. However, it has been found recently that
some parasites secrete the highly-spec. proteases, which break bonds in
in human IgA, (each brealing only one bond). IgA loses thereby its
protective properties; and the cell of the parasite gets the opportunity
to adhere and reproduce in mucosas. The information about Virulent
strains of such microorganisms, for example N.meningitidis differ from
nonvirulent ones by hegira higher concentration of the secreted
proteases. The study of such proteases, structure and properties of
which is still little, can make it possible to find new approaches to
the treatment of the diseases, caused by these microorganisms. For
example, synthetic peptides, which have the sequence of amino acids
similar to the region of cleavage in IgA, could be effective inhibitors
of IgA-protease.
The test-systems, developed on the basis of
IgA-protease, could help to differentiate virulent and nonvirulent
strains.
The Objective of the project
The major objective of the project proposed is:
- to obtain
IgA-protease, secreted by virulent N.meningitidis strain and to study
its properties.
It is important both for the control over the
dangerous disease and for solution of basic problem - the cause of
exclusive specification of IgA-proteases. The choice of the object is
determined by the importance to control meningitis.
Main research
tasks:
- to optimize cultivation of the virulent N.meningitidis
strain;
- to isolate and purify the secreted IgA-protease;
- to study physic-chemical properties of the enzyme;
- to predict
the structure of peptides and to synthesize them as potential inhibitors
or substrates of IgA-protease;
- to develop an immunoenzymatic
test-system to determine IgA-protease;
- to obtain and use monoclonal
antibodies to IgA-protease;
- to isolate and purify the substrate for this enzyme-monoclonal IgA
from sera of patients.
The expected results
The studies, carried
out under the project, should facilitate the understanding of molecular
bases of exclusive specificity of IgA-proteases, which make them
different from serineproteolytic enzymes of chymotrypsin type.
The
immunoenzyme systems on the basis of IgA-protease could help to test
diagnosis of meningitis in patients and carriers. Production of
synthetic inhibitors of the enzyme is a possible way to design an
effective medicinal compound of the new type.
Technical Approach and
Methodology
There is a good methodological basis for the execution of the
project at the State Scientific Center of Applied Microbiology. Thus,
employees of the Center use in their studies the method of computer
simulation of protein steric structure, synthesis of the peptides,
having biological activity, procedures to obtain and purify monoclonal
antibodies and physic-chemical methods to study protein properties,
methods of development of test-systems and isolation and purification of
proteins.
A range of physico-chemical methods will be used:
-
spectrophotometric methods;
- the method of chemical modification of
ammo acid residues;
- adiabatic scanning difference microcalorimetry;
- determination
of specificity, etc.
The pathogenic N.meningitidis strain is
supposed to be cultivated at the State Scientific Center of Applied
Microbiology experienced in cultivation of pathogenic microorganisms.
Thus we have a good methodological base for every stage. Besides, it
should be noted that the participants of this project have broad
experience in investigating properties of proteolytic enzymes.
International cooperation under the project submitted to the ISTC. It is
planned to exchange the information received under the project, to
prepare joint publications, to exchange samples required for research,
to make visits to laboratories of collaborators aiming at carrying out
the work under the project, to organize scientific seminars at the State
Scientific Center of Applied Microbiology with the participation of
collaborators.
This project has positive estimation from Dr. A.
Wlodamer (USA) and Prof. D' Ari (France). They will take part in
proposed project. |
