Cloning of gene encoding the GP28.5 protein of toxoplasma gondii

Details Cloning of gene encoding the GP28.5 protein of toxoplasma gondii Cloning of gene encoding the GP28.5 protein of toxoplasma gondii

1995-01-30

1998-10-20

Sidberry, Hazel F.

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 221, 536 231, 536 234, 435 691, 435 693, 435 697, 435 911, 4242731, C07H 2102, C12N 1509, C12N 1530, C12P 1204

Patent

active

058247882

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to the cloning of the gene encoding a 28.5 kDa Toxoplasma excretion-secretion antigen, and to the production of peptide fragments representing epitopes thereof, as well as to preparations of said antigen and of its fragments, and to their uses.
2. Description of the Background
Toxoplasmosis is one of the most widespread protozoal infections, both in man and in animals. It is responsible for about 25% of deaths in AIDS patients. The congenital infection, the cause of abortions or severe neonatal malformations in man and domestic animals, could be prevented. Indeed, it is known that the primary infection induces a long-lasting immunity.
In the search for protective antigens permitting the development of a vaccine against toxoplasmosis, various antigens have been studied. The inventors' team was in particular interested in the excretion-secretion antigens (ESA) of the tachyzoites. It has indeed been established, during experiments both in man and in animals, that the ESA antigens were immunogenic. It was also shown that some ESAs possess epitopes in common with antigens of bradyzoites. However, bradyzoites are the resistant form of the parasite.
Various approaches, comprising in particular the production of monoclonal antibodies, colloidal gold labeling, and molecular biology, led the (1990)!. The main one, called Gra2 or GP28.5, is a glycoprotein of 28.5 kDa, and it has been shown that it is a constituent of the matrix of the dense granules of tachyzoites, and that it is associated with the microvilli network of the parasitophorus vacuole of the parasite, after invasion of the host.
A 28 kDa antigen (P28), considered as being similar to the GP28.5 antigen, 55:2137 (1987)!, and a DNA sequence encoding this antigen has been


SUMMARY OF THE INVENTION

The present invention set itself the aim of producing purified preparations of the GP28.5 antigen, as well as of producing this antigen in recombinant form, and its immunological characterization. In particular, the aim of the present invention is the localization and the characterization of specific epitopes of the GP28.5 antigen.
The inventors succeeded in obtaining a purified preparation of the GP28.5 antigen, and demonstrated the protective effect of an immunization by this preparation against Toxoplasma gondii infection in mice.
The inventors also cloned the entire gene encoding the GP28.5 antigen, and located the introns and exons, as well as the 5' and 3' noncoding regions.


DETAILED DESCRIPTION OF THE INVENTION

The subject of the present invention is a nucleic acid fragment, which comprises a sequence encoding the Toxoplasma GP28.5 antigen. A nucleic acid fragment conforming to the invention is represented in the list of sequences in the annex under the number SEQ.ID.NO:1. "Sequence encoding the Toxoplasma GP28.5 antigen" is understood to mean not only the coding sequence identified in the sequence SEQ.ID.NO:1, but also any sequence which, taking into account the degeneracy of the genetic code, encodes the polypeptide represented in the list of sequences in the annex under the number SEQ.ID.NO:2.
The inventors also showed that the GP28.5 antigen contains several major epitopes specific for the B cells; one of them, which is recognized by a 71:117 (1990)! is located at the C-terminal end of the molecule.
The inventors characterized this epitope and showed that it contained at least the 5 C-terminal amino acids of GP28.5. In addition, they showed that this epitope is also a major epitope recognized by human polyclonal antibodies directed against T. gondii.
The invention also encompasses nucleic acid fragments which encode polypeptides representing epitopes of the GP28.5 antigen.
According to a preferred embodiment of the present invention, said nucleic acid fragment encodes a polypeptide comprising at least the 5 C-terminal amino acids of the sequence SEQ.ID.NO:2.
According to another preferred embodiment of the invention, said nucleic acid fragment encodes a polypeptide

REFERENCES:
patent: 5633139 (1997-05-01), Prince et al.
patent: 5665542 (1997-09-01), Prince et al.
patent: 5686575 (1997-11-01), Prince et al.
Prince et al, Molecular and Biochemical Parasitology 34:1-14, 1989.
Murray et al Applied Parasitology 34:235-244, 1993.
Cesbron-Delauw et al Molecular Immunology 29:1375-1382, 1992.

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