Streptococcus pyogenes virulans enziminin rekombinant üretimi ve spesifik DNA aptamerlerinin geliştirilmesi
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Dosyalar
Tarih
2020
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Grup A streptococcus (GAS) olarakta bilinen Streptococcus pyogenes, dünya çapında en tehlikeli ilk on patojenden biridir. Nekrotizan fasiit, toksik şok sendromu, farenjit ve impetigo enfeksiyonlarına neden olur. Gelişmiş ülkelerde ölüm oranı düşük olmakla beraber, gelişmemiş ve gelişmekte olan ülkelerde ölüm oranları hayli yüksektir. GAS enfeksiyonlarının neden olduğu komplikasyonlar genelde diğer hastalıklarla karıştırılır. Bu nedenle enfeksiyonun tanısında geç kalınmaktadır. GAS enfeksiyonlarının tanısında hızlı, hassas ve ucuz yöntemlere ihtiyaç vardır. Streptopain (SpeB), S.pyogenes tarafından salgılanan bir virulans enzimdir. SpeB insanda immunoglobulinler ve kompleman aktivasyon sisteminde görev alan proteinleri parçalayarak patojeniteye katkı sağlar. Aptamerler hedeflerine özgü üç boyutlu yapıda katlanarak hedeflerini yüksek spesifiklik ve afinite ile tanıyan tek zincir DNA veya RNA moleküleridir. Aptamerler, SELEX (Systematic Evolution of Ligands by EXponential Enrichment) adı verilen bir yöntem kullanılarak geliştirilir. Bu tez çalışmasında SpeB virülans enzimini yüksek spesifiklikle tanıyacak aptamerler geliştirildi. Aptamer adaylarının SpeB'ye afinitesi ELONA (Enzyme Linked Oligonucleotide Assay) testi ile belirlendi. Apt_22'nin SpeB'ye afinitesinin düşük nM düzeyde olduğu belirlendi.
Streptococcus pyogenes, also known as Group A streptococcus (GAS) is one of the top ten most dangerous pathogens woldwide. S.pyogenes causes necrotizing fasciitis, toxic shock syndrome, pharyngitis and impetigo. Although, the mortality rate low in developed contries, it is high in underdevelop and developing countries. Complications caused by GAS infections are generally confused with different diseases. Therefore, there is usually a delay in the diagnosis of the infection. For this reason, fast, sensitive and cheap methods are needed for the diagnosis if GAS infections. Streptopain (SpeB) is a virulence enzyme produced by S.pyogenes. SpeB contributes to pathogenicity by breaking down the immunoglobulins and proteins involved in the complement activation system. Aptamers are single strand DNA or RNA oligonucleotides that can recognize target molecules with high specifity and affinity thanks to target specific three-dimensional folding. Aptamers are developed by a method, which is called SELEX (Systematic Evolution of Ligands with Exponential Enrichment). In this thesis study, aptamers were developed to recognize SpeB with high specificity and affinity. The affnity of aptamer candidates to SpeB was measured by ELONA (Enzyme Linked Oligonucleotide Assay)assay.It was determined that Apt_22 displayed a low nM affinity for SpeB.
Streptococcus pyogenes, also known as Group A streptococcus (GAS) is one of the top ten most dangerous pathogens woldwide. S.pyogenes causes necrotizing fasciitis, toxic shock syndrome, pharyngitis and impetigo. Although, the mortality rate low in developed contries, it is high in underdevelop and developing countries. Complications caused by GAS infections are generally confused with different diseases. Therefore, there is usually a delay in the diagnosis of the infection. For this reason, fast, sensitive and cheap methods are needed for the diagnosis if GAS infections. Streptopain (SpeB) is a virulence enzyme produced by S.pyogenes. SpeB contributes to pathogenicity by breaking down the immunoglobulins and proteins involved in the complement activation system. Aptamers are single strand DNA or RNA oligonucleotides that can recognize target molecules with high specifity and affinity thanks to target specific three-dimensional folding. Aptamers are developed by a method, which is called SELEX (Systematic Evolution of Ligands with Exponential Enrichment). In this thesis study, aptamers were developed to recognize SpeB with high specificity and affinity. The affnity of aptamer candidates to SpeB was measured by ELONA (Enzyme Linked Oligonucleotide Assay)assay.It was determined that Apt_22 displayed a low nM affinity for SpeB.
Açıklama
Anahtar Kelimeler
S.pyogenes, Streptopain, Aptamer, SELEX, ELONA
