Partial Characterization of a gene encoding the putative Rhop protein in Babesia bovis.

BARAVALLE, M.E; THOMPSON, C; TORIONI DE ECHAIDE, S; SUAREZ, C; FLORÍN CHRISTENSEN, M; ECHAIDE, I

Buenos Aires. Argentina

Conferencia; VI International Conference on Ticks and Ticks-Borne Pathogens; 2007

In silico search in the Babesia bovis T2Bo genome for a homologous gene to that encoding for Plasmodium falciparum rhoptry protein PfRhop148 yielded the identification of a 1833 bp sequence, encoding for a hypothetical protein we have named BboRhop (Acc. Number XM001611611). In this work we analyzed the polymorphism of a fraction of rhop gene (bp 159 to 1315) among B. bovis strains and field isolates, and the transcription of the gene in merozoites grown in vitro. Looking for sequence polymorphism, we evaluated DNA from B. bovis strains BboR1A and BboS2P (Argentina), MO7 (México) and T2Bo (USA) multiplied in vitro and from blood of twelve B. bovis naturally-infected cattle. DNA was always extracted with phenol/chloroform/isoamyl alcohol method and the rhop gene was amplified using specific primers. Amplicons from reference strains were cloned into vector pGEM-T easy and three clones from each strain were sequenced and analyzed (Alignments were made with Clustal W (Bioedit) and distance analysis with Mega 4.0.1.). Sequence analysis revealed a high degree of conservation among all strains. A 97.4 % similarity was found between BboS2P and BboR1A. The highest degree of polymorphism (96.9 % identity) was observed between BboS2P and MO7. Alignment of all the sequences showed 45 base substitutions, of which 13 were synonymous. These results suggest that rho could be useful as a new molecular marker to characterize B. bovis strains. Messenger RNA was isolated from BboS2P and BboR1A strains using an oligo(dT) affinity column kit and cDNA transcripts of rhop were obtained by RT-PCR. Products were cloned into pGEM-T easy vector and sequenced. The comparison of sequences of both strains obtained by PCR and RT-PCR showed 100% identity. Our results demonstrate that this gene is transcribed in cultured merozoites and contains no introns. Ongoing work is directed to clone and express the protein to study its subcellular localization and characterize its antigenic pattern. (Acknowledgments: Supported by EC (INCO 0003691 MEDLABAB), ANPCyT (PICT 00054) and TCP INTA EEA Rafaela-Asoc. Coop.426100. We acknowledge to the Biological Sciences Doctorate Program, of the National University of Córdoba Argentina, for the scientific support, and to Dr. WL Goff the B. bovis T2Bo strain).