Babesia bigemina Kayseri/Türkiye Suşlarının Apical Membrane Antigen-1 (AMA-1) Proteininin Moleküler Genotiplendirilmesi, Ekspresyonu ve İmmunolojik Karakterizasyonu

Arif Çiloğlu

Babesia bigemina Kayseri/Türkiye Suşlarının Apical Membrane Antigen-1 (AMA-1) Proteininin Moleküler Genotiplendirilmesi, Ekspresyonu ve İmmunolojik Karakterizasyonu

Thesis Type: Doctorate

Institution Of The Thesis: Erciyes Üniversitesi, Sağlık Bilimleri Enstitüsü, Turkey

Approval Date: 2016

Thesis Language: Turkish

Student: Arif Çiloğlu

Principal Supervisor (For Co-Supervisor Theses): Abdullah İNCİ

Open Archive Collection: AVESIS Open Access Collection

Abstract:

This study was performed to genotyping, expression and immunogenic characterization of AMA-1 protein molecules of in vivo and in vitro cultivated and passaged (in vivo: 1st passage; in vitro: 1st passage) "Babesia bigemina Kayseri/Türkiye" strains, and also to detect the polymorphisms of these molecules depend on the attenuation of the strains. For this aim, total genomic DNAs were extracted from the B. bigemina in vivo and in vitro cultured strains which have 4.1% and 11% parasitemia, respectively in order to determinate the nucleotide sequences of AMA-1, research the intronless genes in the obtained sequences and analyzed the fragments in open reading frame (ORF). The PCR was carried out with specific AMA-1 primers which amplified full-length B. bigemina AMA-1 (BbigAMA-1) gene. The PCR products were purified and cloned into pJET1.2/blunt vector. The cloned isolates were sequenced and the obtained sequences were deposited in GenBank with accession numbers KP000032-33. Pairwise analyses of the sequences and multiple alignments with some other BbigAMA1 isolates available in GenBank were performed and phylogenies were investigated. The truncated open reading frames (ORF) of the BbigAMA-1 fragments were obtained and new primers were designed for expressions. Total RNAs were extracted from the same strains and the cDNAs were prepared from the obtained RNA samples. The obtained cDNAs were amplified by PCR using predesigned expression specific primers and cloned into the expression plasmid vectors. The plasmids were subsequently transformed into E. coli BL21 (DE3) cells and expressed with IPTG concentrations. The expressed samples were analyzed by using SDS-PAGE and Western Blot techniques and target protein weights were calculated. Pairwise alignment of the sequences from Kayseri/Turkey IV1 strain from in vivo passage 1 and Kayseri/Turkey IT2 strain from in vitro passage 1 of B. bigemina showed 99.77% identity to each other. According to the phylogenetic comparisons, Kayseri/Turkey IV1 strain and Kayseri/Turkey IT2 strains showed most similarity to "Turkey" strain (GenBank accession no: JN572801). Moreover, the amino acid sequences of these two strains showed 99.83% identity to each other because of a mutation in 103rd nucleotides. BbigAMA-1 strains were expressed with a molecular mass of 72 kDa in different expression periods. Depend on the data obtained from this study, BbigAMA-1 was evaluated some controversial as target protein for vaccine candidate to developing immunization against B. bigemina infections in cattle. Because of, this protein should be tested with both of in vitro and in vivo experimental immunoreactions using homolog local B. bigemina strains in all regions of Turkey. In conclusion, molecular characterization and expression level of target AMA-1 protein found in the two different passages of B. bigemina were comparatively and firstly determined with this study and polymorphic regions were revealed due to the attenuation which showed mutational differences between these two strains.