Sığır vebası virusu RBOK aşı suşu nükleoprotein geninin bakteri hücresinde klonlanması ve pürifikasyonu

Purification and prokaryotic cloning of nucleoprotein (NP) gene of rinderpest virus (RPV) RBOK vaccine strain

Abstract

In this study, it was aimed to expres the NP protein of rinderpest virus (RPV) RBOK vaccine strain in a prokaryotic system. Vero cells were infected with RBOK vaccine strain. Total RNA was isolated from infected cells and reverse transcription was carried out and cDNA of NP gene was obtained. The NP gene was amplified by polymerase chain reaction (PCR) with the primers specific to the NP gene. Nucleoprotein gene of RPV-RBOK vaccine strain was cloned into vector PinPointTM Xa-3 by using the Hindlll and Kpnl restriction enzyme sites. Recombinant NP protein synthesized by E.coli was purified. To determine the recombinant NP protein which is approximately 80kDa, western blotting was performed.

Özet

Bu çalışmada sığır vebası virusu'RBOK aşı susu (SV-RBOK) nükleoprotein (NP) geninin prokaryotik hücrede klonlanması amaçlanmıştır. Vero hücreleri RBOK aşı susu ile infekte edildi. İnfekte hücrelerden toplam RNA'lar elde edilerek tersine transkripsiyonla NP geni kopya DNA'ları elde edildi. NP geni özgül primerler kullanılarak polimeraz zincir reaksiyonu ile çoğaltıldı. SV-RBOK NP geni Hindlll ve Kpnl enzimleri kullanılarak PinPointTM Xa-3 plazmidine klonlandı. E.coli hücresinde sentezlenen rekombinant NP protein pürifiye edildi. Yaklaşık 80kDa moleküler ağırlığındaki NP proteinini belirlemek için western blotlama yapıldı.