TRYPANOSOMA CRUZI RECOMBINANT ANTIGENS AND SYNTHETIC PEPTIDES FOR UTILIZATION IN THE IMMUNOLOGICAL DIAGNOSIS OF CHAGAS DISEASE

Abstract: This patent deals with the use of Trypanosoma cruzi antigens obtained using recombinant DNA techniques, or synthetic peptides derived from these antigens, and their use in the immunological diagnosis of Chagas Disease using either ELISA, or western blot, or dot-blot, or hemagglutination, or agglutination, or monoclonal antibodies, or radioimmunoassay. The antigens or synthetic peptides are characterized by displaying the following aminoacid sequences: Ag1: valine (or alanine), alanine, glutamic acid, alanine (orthreonine), glutamic acid, lysine, glutamine, lysine (or arginine), alanine, alanine, glutamic acid, alanine (or serine), threonine (or methionine or alanine) and lysine. Ag2: methionine, glutamic acid, glutamine, glutamic acid arginine, arginine, glutamine, leucine, leucine, glutamic acid, lysine, aspartic acid, proline, arginine, arginine, asparagine, alanine, lysine, glutamic acid, isoleucine alanine, alanine, leucine, glutamic acid, glutamic acid, serine, methionine, asparagine, alanine, arginine, alanine, glutamine, glutamic acid, leucine, alanine, arginine, glutamic acid, lysine, lysine, leucine, arginine, aspartic acid, arginine, alanine, phenylalanine, leucine, aspartic acid, glutamine, lysine, proline, glutamic acid, arginine, valine, proline, leucine, alanine, aspartic acid, valine, proline, leucine, aspartic acid, aspartic acid, aspartic acid, serine, aspartic acid, phenylalanine, valine and alanine.

Description: Icueanfl≤ojaa cruzi. RECOMBINANT ANTIGENS AND SYNTHETIC PEPTIDES FOR UTILIZATION IN THE IMMUNOLOGICAL DIAGNOSIS OF CHAGAS DISEASE. BACKGROUND OF THE INVENTION 05 T is patent request refers to the use of Trypanosoma cruzi repeti ive recomb inant an igens, as well as the corresponding synthetic peptides,, in the immunolog i cal detec ion of Chagas Disease and its ethiological agent, the parasite Trypanosoma cruzi. 10 The immunolog i cal diagnosis of Chagas Disease is in general based on the use of crude or sem i-pur i fi d parasite extracts for the detection of an ibo i s, in different tests such as hemagglu ination, ELISA, radioimmunoassay and Western blot analysis. However, it is 15 generally observed the cross-reactivity with patients bearing other diseases such as isceral and t gumentar 1e i sh an ias i s, toxop1as os is, syph i 1 i s and sch i stosomiasi s.. Furthermore, the use of parasite extracts involves some rises for its preparation and is relatively 2. expensi e. However, wi h the development of genetic engeneering techniques, it became possible to express he erol gou genes in bacte ia with low cost and high spe ifi ity. Then, the cloning and expression of Trypanosoma cruzi 25 genes in E.col i was aimed for the obtention of parasite recomb i nant antigens that might be used for the diagnosis of Chagas Disease., DESCRIPTION OF THE INVENTION T.cruzi DNA was cloned in E.col i using the expression vector lambda gtll, and the recomb inant clones were screened ith a pool of human chaga ic sera. Twelve recomb inant clones were purified to homogeneity and from these two were selected on the basis of the spe ificity of their reactivity with chagasic sera. Partial nucleotide sequence of these clones showed that one of them <Ag#i) encodes an antigen composed of at least 23 repetitions of a 14 aminoa ids motif whereas the other (Ag#2) encodes an antigen containing at least 14 repeti ions of a 68 aminoacids repe itive epitope. The recomb inant ant igems Agtti and Ag#2, as well as a synthe ic peptide corresponding to Agttl, were tested in the immunolo i al diagnosis of Chagas Disease, using the methods of ELISA, dot-blot, western blot and agglutination. The results obtained showed that the aforementioned antigens pro ide an adequate and specific d iagnos is for Cha as Disease„

Claims: CLAIMS 1' Trypanosoma cruzi ant igen, obtained by recomb inant DNA technique and expressed in bacteria, compr ising the following aminoacid sequence ; : Val ine (or alanint), 05 alanine, glutamic 3cιd, alanine (or t reon i ne> , glutamic acid, lysine. gluta ine, lysine (or arginine), aϊanine- alanine, gluta ic ac i cl alanine (or serine), h eonin (or methionine or alanine) and lysine. D Trypanosoma cruzi antigen, obtained by recom inant DNA -, fl > thecnic, according to claim 1, w ei't in thι^= an* igen include crude or semi -pur i i ed and recomb inant ant igens, for u iliza i n in the i munolog i a1 diagnosis of Chagas Di sease.. 3) Syn he ic pe ide comp ising the same aminoacid l- sequence as l i ed in cl im 1„ 4) Synthe ic pept ide, according -~ u claim 3- for ut i J izri ion i the inimunϋlogical d i -πrιo=> i • => of Chana= D i ease., 5"> Trypanosoma cruzi antigen, obtained by recomb inant- DNA 0 thecnic and expressed in bacter ia, comprising the following aminoacid sequences Methionine, gluta i', acu glu a ine, lutami acid, ar inine*, a inine, gluta ine, I'-u ine leucine glut-am ic acid, lysine, aspart ι>- ,ιcιd proline, arginine, arginine, asp&rag i ne alanine, lysine, 5 glutamic acidv isoiencine, alanine al÷hine, leucine glutamic ac i cl . glutamic acid, -ser ine,, methionine , asμarag i ne , alanine ar inine ala ine,- •- I n*- am i n>. glutamic acid, leucine, alanine, arginine glutamic acid lysine, lysine, leucine, ar inine, aspart. i ■„ ^ci ar inine, alanine, phenylala ine, leucine, aspartic acid, glutamine, lysine, proline, glutamic acid, arginine, valine, proline, leucine, alanine, aspartic acid, valine, proline, leucine, aspartic acid, aspar ic aci , aspartic - - acid, serine, aspartic acid, phen l l nine, valine and alan ine. 6 ) Trypanosoma cruzi antigen, obtained by recomb inant DNA thecnic, according to claim 5, wherein this antigen include crude or se i-pur i ied and recomb inant antigens, iø for utilization in the immunolog ical diagnosis of Chagas Disease,, 7) Synthetic peptide comprising the same aminoacid sequence as claimed in claim 5« 8) Synthetic peptide, according to claim 7, for 15 u ilization in the i munolog ical diagnosis of Chagas Disease.,

Inventors: CERQUEIRA DE ALMEIDA ELZA CARM, GOLDENBERG SAMUEL, KRIEGER MARCO AURELIO, CERQUEIRA DE ALMEIDA, ELZA, CARMEN, GOLDENBERG, SAMUEL, KRIEGER, MARCO, AURELIO
Applicants: FUNDAC O OSWALDO CRUZ FIOCRUZ, FUNDACO OSWALDO CRUZ (FIOCRUZ)-SUPERINTENDENCIA D
Application year: 1991
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15