Önder DÜZLÜ, Arif ÇİLOĞLU, Alparslan YILDIRIM, Abdullah İNCİ, Zuhal ÖNDER

Babesia bigemina Kayseri/Türkiye suşunun in vivo ve in vitro izolatlarında apikal membran antijen-1 proteininin moleküler karakterizasyonu ve ekspresyonu

Bu çalışma, “Babesia bigemina Kayseri/Türkiye” suşundan in vivo ve in vitro kültivasyon yapılarak ve pasajlanarak elde edilmiş olan izolatların AMA-1 proteinlerinin moleküler karakterizasyonu ve ekspresyonu ile söz konusu proteinin attenüasyona bağlı polimorfizm gösterip göstermediğini ortaya koymak amacıyla yapılmıştır. Bu amaçla, Babesia bigemina Kayseri/Türkiye suşuna ait, in vivo ve in vitro izolatlardan genomik DNA ve total RNA izolasyonu sonrasında hedef gen, PCR ile bütün olarak amplifiye edilmiş ve pürifiye amplikonlar uygun vektörlere klonlanmıştır. Klonlama sonrasında rekombinant plazmidler, sekanslanarak izolatlara ait nükleotid dizilimleri GenBank’a kaydedilmiştir (KP000032-33). Nükleotid ve amino asit sekansların çoklu ve ikili hizalama analizleri yapılmış ve izolatların filogenileri araştırılmıştır. Elde edilen rekombinant plazmidler, E. coli BL21 (DE3) hücrelerine transforme edilerek IPTG ile indüklenmiş ve ekspresyonları sağlanmıştır. Ekspresyon ürünlerinde hedef proteinler SDS-PAGE ve Western Blot teknikleri ile analiz edilmiştir. Kayseri/Turkey IV1 ve Kayseri/Turkey IT2 izolatları nükleotid dizilimlerinin kendi aralarında %99.7 identiklik gösterdiği ve 103. nükleotiddeki mutasyona bağlı olarak amino asit diziliminde farklılık (%0.2) oluştuğu belirlenmiştir. SDSPAGE ve Western Blot analizleri sonucu söz konusu iki izolata ait AMA-1 proteinlerinin 72 kDa moleküler ağırlıklarla farklı IPTG konsantrasyon düzeyleri ve zaman aralıklarında eksprese oldukları belirlenmiştir. Sonuç olarak bu çalışma ile Babesia bigemina Kayseri/Türkiye suşunun in vivo ve in vitro pasajlarından elde edilmiş iki izolata ait AMA-1 nükleotid ve amino asit dizilimleri arasında mutasyonel farklılıkları gösteren attenüasyona bağlı polimorfik alanlar olduğu ortaya konulmuştur.

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

Summary: 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 Kayseri/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 amino acid sequences of these two isolates due to a mutation in 103rd nucleotide. According to results of SDS-PAGE and Western Blot analyses, the BbigAMA-1 isolates were expressed with a molecular mass of 72 kDa in different IPTG concentration levels and time periods. In conclusion, attenuation-related polymorphic sites that indicate the mutational differences between AMA-1 nucleotide and amino acid sequences from in vivo and in vitro isolates of Babesia bigemina Kayseri/Turkey strain were revealed with this study.

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