Molecular characterization and expression of the apical membrane antigen-1 from in vivo and in vitro isolates of Babesia bigemina Kayseri/Turkey strain

Ciloglu A., Inci A., Yildirim A., Onder Z., Duzlu Ö.

ANKARA UNIVERSITESI VETERINER FAKULTESI DERGISI, vol.65, pp.239-246, 2018 (SCI-Expanded) identifier identifier


This study was performed to molecular characterization and expression of AMA-1 protein molecules of in vivo and in vitro cultivated and passaged isolates from "Babesia bigemina Kayseri/Turkey" strain, and also to determine the possible polymorphisms of these molecules due to the attenuation of the isolates. For this aim, target gene was completely amplified by PCR after isolation of genomic DNA and total RNA from the in vivo and in vitro cultivated isolates which were obtained from Babesia bigemina Kaysen/Turkey strain. The isolates were cloned into suitable vectors after PCR analysis. The cloned isolates were sequenced and the obtained sequences were deposited in GenBank with accession numbers KP000032-33. Multiple and pairwise alignments of the sequences were performed and phylogenies were investigated. The obtained recombinant plasmids were transformed into E. coli BL21 (DE3) cells and expressed after induction with IPTG. The target proteins were analyzed by using SDS-PAGE and Western Blot techniques. Nucleotide sequences of Kayseri/Turkey IV1 and Kayseri/Turkey IT2 isolates showed 99.7% identity to each other and a difference (0.2%) was also determined in ammo acid sequences of these two isolates due to a mutation in 103(rd) nucleotide. According to results of SDS-PAGE and Western Blot analyses, the BbigAMA-1 isolates were expressed with a molecular mass of 72 kDa m different IPTG concentration levels and time penods. In conclusion, attenuation-related polymorphic sites that indicate the mutational differences between AMA-1 nucleotide and ammo acid sequences from in vivo and in vitro isolates of Babesia bigemina Kayseri/Turkey strain were revealed with this study.