Ssap-NtrB prokaryotik geninin ökaryotik ekspresyon vektörü pcDNA3.1 / V5 / His B içine klonlanması

Abstract

İntihar gen terapisi son zamanlarda kanser tedavilerinde kullanılan bir yöntem olarak karşımıza çıkmaktadır Bu terapilerde hücrede ifade olan enzimlerden faydalanılmaktadır. Bu çalışma kapsamında, Staphylococcus saprophyticus supsp. saprophyticus’tan elde edilen Ssap-NtrB geninin ökaryotik ekspresyon vektörü olan pCDNA3.1 / V5 / His B’ye klonlanması gerçekleştirilmiştir. Bu amaçla, ilk olarak genin içinde bulunduğu pET14B vektöründen restriksiyon kesimleri ile gen bölgesi alınarak, pGEM-T-Easy vektörüne aktarılmıştır. Bu adımdan sonra seçilen uygun enzimler olan KpnI/ApaI ile kesim yapılarak hazırlanmış olan pCDNA3.1 / V5 / His B’ye ligasyonu sağlanmıştır. Kontrol kesimleri ile gen klonlamanın doğrulanması sağlanmıştır. Sonuç olarak, intihar gen tedavisinde kullanıma hazır hale getirilen Ssap-NtrB geninin klonlanması ve kontrolü gerçekleştirilmiştir. Bu sayede ökaryotik kanser hücrelerinde kullanıma hazır hale getirilmiştir.

Keywords

• Ssap-NtrB,nitroredüktaz,ekspresyon vektörü,pcDNA3.1/HisB

Cloning of the Ssap-NtrB prokaryotic gene into the eukaryotic expression vector pcDNA3.1 / V5 / His B vector

Abstract

Suicide gene therapy has recently emerged as a method used in cancer treatments. These therapies utilized enzymes that are expressed in the cell. In this study, Staphylococcus saprophyticus supsp. saprophyticus Nitroreductase gene (Ssap-NtrB) was subcloned into the eukaryotic expression vector namely pcDNA3.1 / V5 / His B. For this purpose, Nitroreductase gene region was firstly amplified from the pET14B vector using PCR strategy and cloned into the pGEM-T-Easy vector. After this step, the Ssap-NtrB gene was restricted with KpnI/ApaI and was ligated into pcDNA3.1 / V5 / His B vector. Recombinant colonies were verified using KpnI/ApaI restriction enzymes. As a result, the Ssap-NtrB gene was cloned into pcDNA3.1/V5/His B vector and was readyfor use in suicide gene therapy in eukaryotic human cancer cells.

Keywords

• Ssap-NtrB,Cloning,Nitroreductase,Expression Vector,pcDNA3.1/HisB

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