Process for preparing major histocompatibility antigen class...

Details Process for preparing major histocompatibility antigen class... Process for preparing major histocompatibility antigen class...

1997-09-11

2003-10-07

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C210S679000, C210S691000, C424S140100, C435S007320, C435S007800, C436S501000, C436S005000, C530S413000, C604S005010, C604S005020, C604S005040

Reexamination Certificate

active

06630315

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a process for producing major histocompatibility antigen class II protein (hereinafter referred to as “MHC class II” for short) occurring on the surface of antigen-presenting cells and the like, and MHC class II-bound materials in which MHC class II, &agr; and/or &bgr; subunit of MHC class II, or a part thereof is bound to a carrier such as beads, fibers and hollow fibers via covalent bond, as well as a module for removing superantigens using the same.
This invention also relates to a method for detecting or quantifying superantigens using MHC class II or a part thereof having an affinity to superantigens, as well as an assay kit therefor.
PRIOR ART
MHC class II occurs on the cell surfaces of B cells, macrophages, endothelium cells in blood vessels and the like. MHC class II is a glycoprotein used for distinguishing self from others. Recently, it was found that MHC class II is a binding protein which binds to a group of proteins called superantigens such as toxins of bacteria, and the subtypes of MHC class II in patients suffering from autoimmune diseases are peculiarly distributed, so that it is now regarded as importance in field of medicine and immunology.
Superantigens are a group of proteins which bind to MHC class II on antigen-presenting cells without being processed in the antigen-presenting cells unlike conventional antigens, and form complexes with MHC class II so as to stimulate T cells having a specific V region, thereby abnormally activate immune system.
Superantigens hitherto confirmed include
Staphylococcus aureus
toxin, Streptococcus toxin, Yersinia toxin, some kinds of virus proteins and heat shock proteins. Superantigens may also be identified in the future.
At present, to isolate and obtain MHC class II, it is necessary to introduce MHC class II gene in mammalian cells or insect cells and express the gene, or it is necessary to purify naturally occurring MHC class II from cell membranes of B cells, macrophages, endothelium cells in blood vessels and the like.
However, for obtaining naturally occurring MHC class II from cell membranes in a large scale, a large number of cells are necessary because the amount of MHC class II on the surface of the membranes is small, so that this method takes a long time and a large cost even if cultured cells are employed.
Similarly, for producing recombinant MHC class II by mammalian cells or insect cells by a genetic recombination technique, a large cost and a long time are required for culturing the cells.
Although system for expressing major histocompatibility antigen class I protein by using
Escherichia coli
has been reported (K. C. Parker et al., Molecular Immunology, Vol. 29, 371 (1992)), no such an expression system is known for MHC class II.
Conventional materials to which MHC class II is bound include those in which whole cells expressing MHC class II on the cell surfaces are adsorbed on a plate, and those in which chemically synthesized amino acid sequence that is a part of a subunit of MHC class II is adsorbed on a material (J. K. Russel et al., Biochemical and Biophysical Research Communications, Vol. 168, 696 (1990)).
As for a material for adsorbing superantigens, a material to which antibodies specific to superantigens are immobilized has been reported and the material is used for immunoassays of the superantigens.
The present invention provides a process for producing MHC class II by using bacteria, thereby promoting productivity and ease of operation.
Unlike insect cells and mammalian cells, bacterial cells grow fast, no expensive components such as fetal calf serum and growth factors are necessary to be contained in culture medium, and operations such as subculturing and replacement of culture medium are not necessary.
However, in many cases, proteins originating from mammals are not expressed in bacteria such as
E. coli
because of the problems such as the toxicity to bacteria and the like.
The present inventors studied to improve various expression vectors and succeeded in expressing MHC class II in bacterial cells such as
E. coli
, yeasts and
Bacillus subtilis
, thereby made it possible to produce the protein efficiently in a large scale.
The present invention provides, in the medical field, a blood-purificatlon system for removing pathogenic substances including superantigens, which have affinities to MHC class II, and in the field of immunology, the present invention provides a material capable of binding the pathogenic substances including superantigens, which have affinities to MHC class II, that is used for isolating the pathogenic substances and for immunoassays for assaying the pathogenic substances.
If the whole cells are immobilized on a carrier, since proteins other than MHC class II are also immobilized, the material may have an unknown activity. Further, the freedom of optionally selecting the-density of immobilized MHC class II is limited. As for the material on which a chemically synthesized partial amino acid sequence of the MHC class II subunit is adsorbed, although it can be used for detecting superantigens contained in a high concentration in a buffer, it cannot be used for removing superantigens from the blood because the adsorbed ligand may be liberated from the carrier.
Further, the binding ability of the partial sequence to superantigens is small, so that sufficient performance for detecting and quantifying superantigens cannot be obtained. With the material on which an antibody is immobilized, it is necessary to immobilize different antibodies each of which is specific to each type of superantigens respectively.
DISCLOSURE OF THE INVENTION
The present invention aims at overcoming the above-mentioned problems in the prior art and has the following constitution. That is, the present invention provides
(1) A process for producing major histocompatibility antigen class II protein, comprising the step of transforming a microorganism with a gene encoding said major histocompatibility antigen class II protein;
(2) A material in which major histocompatibility antigen class II protein or a part thereof is bound via covalent bond;
(3) A material in which &agr; subunit of major histocompatibility antigen class II protein or a part thereof is bound via covalent bond;
(4) A material in which &bgr; subunit of major histocompatibility antigen class II protein or a part thereof is bound via covalent bond;
(5) A module for removing superantigens, comprising said material of any one of (2)-(4);
(6) A method for detecting a superantigen by using major histocompatibility antigen class II protein oria part thereof having a length of not less than 40 amino acid residues, which part has an affinity to said superantigen; and
(7) A method for quantifying a superantigen by using major histocompatibility antigen class II protein or a part thereof having a length of not less than 40 amino acid residues, which part has an affinity to said superantigen.


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J. D. Altman et al, Proc. Natl. Acad. Sci. USA, 90, 10330-10334, 1993.*
K. C. Parker et al, Molec. Immunol., 29, 371-378, 1992.*
J. K. Russell et al, Biochem. Biophys. Res. Commun., 168, 696-701, 1990.*
H. M. Johnson et al, Scientific American, Apr. 1992, pp. 92-101.*
Letters to Nature vol. 346, 8/90 pp 474-476 Karp et at The &agr;1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1.

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