Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens

a technology of eukaryotic amino acids and gene encoding genes, which is applied in the field of cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasites and viral mitogens. it can solve the problems of i>trypanosoma cruzi , no effective treatment or vaccine against i>trypanosoma cruzi , the difficulty in the detection

Inactive Publication Date: 2005-06-16
CENT NAT DE LA RECHERCHE SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Attempts to provide effective immunity to parasites are limited by poor specific immune responses to parasite antigenic molecules in early phases of infection.
To date, there is no effective treatment or vaccine against Trypanosoma cruzi infection and Chagas disease pathology.
Research on D-amino acids in living organisms has been hampered by their difficult detection.

Method used

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  • Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens
  • Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens
  • Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens

Examples

Experimental program
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Effect test

example 1

Mice and Parasites

[0217]Trypanosoma cruzi clone CL Brener was used throughout this work. Epimastigotes were maintained by weekly passage in liver infusion tryptose medium. In vitro metacyclogenesis was performed in a protein free defined medium at 27° C., as previously described11. Male euthymic or athymic BALB / c mice 8 weeks of age were purchased from Charles River Laboratories (Saint Aubin les Elbeuf, France). Male C3H / HeI mice 8 weeks of age from our animal facilities were also used.

example 2

Protein Fractionation

[0218] 40 liters of culture supernatants from metacyclic forms, maintained for an additional 96 h at 37° C., were concentrated by vacuum dialysis and dialyzed against buffer A. HPLC was performed using a weak anion exchanger column POROS HQ-10 (Perspective Biosystems) at a flow rate of 1 ml / min according to the following program: a) 10 min with buffer A; b) 30 min linear gradient from buffer A to B; c) 5 min linear gradient from buffer B to C; and d) 5 min with buffer C. One ml fractions were collected, frozen at −80° C., lyophilized and reconstituted in H2O or in non-supplemented RPMI medium for in vitro proliferation assays. (Buffers used: A: 5 mM NH4-acetate, pH 8. B: 1M NH4-acetate, pH 8. C: 1M NaCl / 1M NH4-acetate, pH 8). Fractions 1 ml in volume were collected, frozen at −80° C., lyophilized and reconstituted in water or in non-supplemented RPMI medium for in vitro proliferation assays. SDS-PAGE analysis used standard techniques.

example 3

Generation of Peptides and Amino Acid Sequence Analysis

[0219] HPLC fractions 22 and 23 were pooled and fractionated by 8% SDS-PAGE. After amino black staining, the 45 kDa protein band was cut out, in-gel digested with trypsin, and submitted to reverse phase HPLC to separate peptides. Automated Edman degradation sequence analysis was performed in the Laboratoire de Microséquençage de Protéines of the Pasteur Institut.

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Abstract

A method of preventing or inhibiting infection by a parasite or virus in vivo comprises administering to a human in need thereof a parasite or virus mitogen in a sub-mitogenic amount sufficient to induce a protective immune response against the parasite or virus in the human.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is based on and claims the benefit of each of the following applications: U.S. Provisional Application Ser. No. 60 / 168,631, filed Dec. 3, 1999 (attorney docket no. 03495.6044); U.S. Provisional Application Ser. No. 60 / 220,207, filed Jul. 24, 2000 (attorney docket no. 3495.6054); and U.S. Provisional Application Ser. No. 60 / 221,117, filed Jul. 27, 2000 (attorney docket no. 3495.6055). The entire disclosure of each of these applications is relied upon and incorporated by reference herein.BACKGROUND OF THE INVENTION [0002] This invention relates to the discovery of a new gene, which is the first isolated and cloned, that encodes an amino acid eukaryotic racemase. The invention covers, particularly, the Tc45 gene encoding a Trypanosoma cruzi-derived B-cell mitogen. The encoded protein also is a eukaryotic proline racemase. The invention also relates to a process of production of D-amino acid using an eukaryotic amino acid r...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K39/00C12N9/90C12P13/04
CPCA61K38/00A61K39/00C07K14/44G01N33/56905C12P13/04C12Q1/6893C12Q1/701C12N9/90C12Q2600/156C12Q2600/158Y02A50/30
Inventor MINOPRIO, PAOLAARALA-CHAVES, MARIOARALA, RUI MARIOCOUTINHO, ANTONIOMARTIN, BERNARDOROUGEOT, CATHERINEDEGRAVE, WIMCOSSON, ALAIN
Owner CENT NAT DE LA RECHERCHE SCI
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