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Fırat Üniversitesi Sağlık Bilimleri Dergisi (Veteriner)
2000, Cilt 14, Sayı 1, Sayfa(lar) 189-195
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Mehmet Ziya DOYMAZ1, Ali Osman KILIÇ2, Hakan BULUT3, Aykut ÖZDARENDELİ3, Yusuf BOLAT3
1Faculty of Medicine, Fırat University, Elazıg-TURKEY
2Faculty of Medicine, Karadeniz Technical University, Trabzon-TURKEY
3Faculty of Veterinary Medicine, Fırat University, Elazıg-TURKEY
Keywords: Rinderpest virus, hemagglutinin, cloning, bacterial expression

SUMMARY: In this study, cloning and prokaryotic expression of rinderpest virus (RPV) hemagglutinin gene was reported. For this purpose, Vero cells were infected with Bovine O Kabete (RBOK) strain of RPV and total RNA was isolated at the height of the infection. Reverse transcription was carried out with random primers and viral cDNA was obtained. Hemagglutinin specific primers were used in polymerase chain reaction (PCR). Later, PCR amplified H gene segment was electrophoresed on 1,5% agarose gel and was found to be approximately 1830 base pair long. Hemagglutinin gene segment was cloned into the prokaryotic expression vector pEZZ18 and the resulting plasmid pRPVH4 was used in transforming Escherichia coli. Cloned H gene segment was subjected to restriction endonuclease analysis in order to confirm the identity of H gene and it was noticed that the product contained the unique restriction endonuclease sites present in the published H sequences of RPV. Recombinant H protein synthesized and secreted by E.coli was purified over affinity chromatography and electrophoresed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant H protein was observed as a 65 kDa protein in Coomassie Brillant blue staining. Furthermore, in Western immunoblot experiments, recombinant H protein was identified with RPV hyperimmune rabbit serum.

* This work was supported by a grant from the Turkish State Planning Organisation (Devlet Planlama Teşkilatı, Proje No: 95K120370)

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