Julio V. Figueroa
National Research Center for Veterinary Parasitology, Mexico
Title: Cloning and sequencing of the rap-1α1 gene from Mexican isolates of Babesia bigemina
Biography
Biography: Julio V. Figueroa
Abstract
The Babesia bigemina RAP-1 antigen is a 58 kDa protein located in the merozoite roptries of the protozoan parasite, which participates in the invasion to the bovine erythrocyte. rap-1 sequences of B. bigemina isolates from Mexico, Brazil, Argentina, Uruguay and Puerto Rico are known. However, the degree of conservation of rap-1 sequence in different geographic isolates of Mexico is not known. The aim of this study was to analyze rap-1α1 sequences in 8 different isolates of B. bigemina from Mexico. The methodology included the extraction of genomic DNA from B. bigemina-infected erythrocytes; PCR amplification of rap-1α1 using rap-1F and rap-1R as primers; Cloning the amplified fragment (1440 bp) into TOPO 10 vector and transforming competent E. coli cells; Selection of clones and purification of recombinant plasmids; Sequencing and analysis of rap-1α1 from the B. bigemina isolates, obtained from at least 3 recombinant clones per isolate, sequencing the inserts in both directions; and sequence alignments and assembling with the CLC application genomic workbench 4.8. The identity of the rap-1α1 sequences was determined by a homology search with BLAST tools. The results showed amplification of rap-1α1 in all the B. bigemina isolates. The comparative analysis of nucleotide sequences revealed a high degree of conservation (98-99% identity) between the rap-1α1 genes of the different Mexican isolates, compared to the reference rap-1α1 sequence. BLASTX analysis revealed identities up to 99% in the deduced amino acid sequences of RAP-1 compared to the sequence of the reference isolate. The high degree of conservation in rap-1α1 sequences among geographically distant Mexican isolates suggests that there is no strong bovine immune pressure that translates into genetic variation of this particular gene. The RAP-1 antigen is a viable candidate for inclusion in a diagnostic test for bovine babesiosis caused by B. bigemina in Mexico.