Bovine Ephemeral Fever Virüsün Rekombinant Glikoprotein ve Nükleoproteinlerinin Üretilmesi*

Yıl 2017, Cilt: 14 Sayı: 3, 149 - 156, 15.12.2017

Hasan Abaylı , Şükrü Tonbak , Ahmet Kürsat Azkur , Hakan Bulut

Öz

Bu çalışmada, bovine ephemeral fever virüsün (Üç gün hastalığı virüsü; BEFV) glikoprotein (G) ve nükleoprotein-lerinin (N) rekombinant DNA tekniği ile üretilmesi amaçlanmıştır. Bu amaçla, Türkiye izolatlarından çoğaltılan BEFV G ve N genlerini taşıyan pcDNA4/HisMax-TOPO ökaryotik ekspresyon vektörleri oluşturulmuş ve elde edilen rekombinant vektörlerle transfekte edilen hücrelerde bu proteinlerin in vitro üretilmesi gerçekleştirilmiştir. Geçici ve stabil transfekte hücrelerde RT-PCR ile mRNA düzeyinde genlerin transkripsiyonu doğrulanmıştır. Ayrıca, transfekte hücrelerden pürifi-ye edilen rekombinant proteinler Western blotlama deneyi ile gösterilmiştir. Bu çalışma sonucunda, BEFV teşhis ve korunmasında iki önemli proteinin üretimi ve pürifikasyonu gerçekleştirilmiştir. Elde edilen bu proteinlerin teşhis kitleri-nin hazırlanması ve sub-ünit aşı olarak kullanım potansiyeli bulunmaktadır.

Anahtar Kelimeler

BEFV glikoprotein , BEFV nükleoprotein , bovine ephemeral fever , rekombinant DNA teknolojisi

Production of Recombinant Glycoprotein and Nucleoprotein of Bovine Ephemeral Fever Virus

Öz

This study is aimed to create a recombinant glycoprotein (G) and nucleoprotein (N) of bovine ephemeral fever virus (BEFV) by using DNA technology. For this purpose, G and N gene of BEFV Turkish isolate were cloned in to pcDNA4/HisMax-TOPO eukaryotic expression vector. Obtained recombinant vectors were transfected in Vero E6 cell line to produce recombinant protein. Presence of recombinant N and G gene of BEFV in Vero E6 was confirmed by using RT-PCR following transient transfection. Additionally purified recombinant N and G proteins of BEFV from Vero E6 were shown through Western blotting after stable transfection. With the help this study, we have achieved purifica-tion and in production of two recombinant proteins which play important role protection and diagnosis of BEFV. The obtained recombinant proteins will be used to create a sub-unit vaccine and to produce diagnosis kit for BEFV in future.

Anahtar Kelimeler

BEFV glycoprotein , BEFV nucleoprotein , bovine ephemeral fever , recombinant DNA technology

Kaynakça

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