Immunogenic activity of an expressed Trypanosoma cruzi Antigen F1 fusion protein

BUA, J; ESTEVA, MI; PORCEL, B; GARCIA, GA; BONTEMPI, EJ; SEGURA, EL; RUIZ, AM

Caxambu, Mina Gerais, Brasil

Congreso; XIX Annual Meeting on Basic Research in Chagas' Disease, VIII Meeting of the Brazilian Society of Protozoology; 1992

Sociedad Brasilera de Protozoologia

Ag F1 is a flagellar fraction antigen of Trypanosoma cruzi with immunprotective capacity in mice and it is an acceptable antigen to detect specific antibodies in human chagasic sera. A T. cruzi genomic lgt11 library was screened with an anti-AgF1 mouse serum and several clones were obtained. The clone with the longest insert was called l(a-AgF1)/9. Other screenings were performed to obtain the 5’ end of the gene. The complete sequence of AgF1 and its translation to amino acids showed that more than half of the length of the sequence is non-coding. The estimated MW of the protein, from the first ATG, base 912, to the end, in base 2130, is 44.9 kDa. Two potential N-glycosylation sites are present in this gene. No internal repeats were found. The calculated isoelectric point is 6.2, very close to the experimentally found with the AgF1: 5.5. Hybridization of the l(a-AgF1)/9 probe to the northern blot of total RNA from epimastigotes of one cloned stock and three strains of T. cruzi highlighted a single band of approximately 2 kb. The copy number of the complete gene was estimated by hybridization and indicated that 26-39 gene copies are present per haploid genome of the parasite. Hybridization to partial digests of Tul2 total DNA showed a ladder-like pattern suggesting a tandem array of genes, the length of the basic unit is 2914 kb. From the ladder it was estimated that the number of copies of the gene in the cluster was at least 10. Hence, the number of  tandems per cell  is around 5-8. Chromosomes from several strains and cloned stocks from T. cruzi were size fractionated by Pulse Field Gel Electrophoresis and the l(a-AgF1)/9 probe hybridized to very large chromosomal bands in all strains and cloned stocks tested. The minimal sizes of these hybridizing chromosomal bands are about 2,000 kpb. A search in the Swiss protein bank revealed homology between AgF1 and aminotransferases. Rat and human tyrosine aminotransferases (TAT) showed an identity at the amino acid level of 39.7 and 39.4 %, respectively, although it increases to 79% when considering conservative substitutions. All of the 12 residues that are known to be essential for activity are present in AgF1. Experiments aimed to established the sub-cellular location and diagnostic-immunoprotective value of this antigen, as well as to confirm the identity with the TAT of T. cruzi are under way.