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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