Ahmet Kürşat AZKUR, Yusuf BOLAT, Mehmet Ziya DOYMAZ

Sığır vebası virüsü RBOK aşı suşu matriks geninin polimeraz zincir reaksiyonu ile çoğaltılması ve $TOPO^R$ XL klonlama vektörünü yerleştirilmesi

Bu çalışmada sığır vebası virüsü (RPV) RBOK aşı susu matriks (M) geni polimeraz zincir reaksiyonu (PZR) ile çoğaltıldı ve TOPO® XL klonlama vektöründe klonlandı. Bu amaçla Vero hücreleri RPV aşı susu ile infekte edilerek toplam RNA'lar elde edildi. Toplam RNA'lardan tersine transkripsiyonla M geni kopya DNA'ları (cDNA) çoğaltıldı. Matriks genine özgü primerler kullanılarak cDNA'lardan çoğaltılan M geni TOPO® XL klonlama vektörüne direkt yerleştirildi ve bu rekombinant DNA kompetan Escherichia coli (E. coli) JM109 hücrelerine transforme edildi. Rekombinant plazmid pTOPO XL'de M geninin bulunup bulunmadığını anlamak için, hem PZR tarama hem de enzim kesim deneyleri uygulandı.

Amplification of the matrix gene of RBOK vaccine strain of rinderpest virus (RPV) by polymerase chanin reaction and cloning into $TOPO^ R$ XL cloning vector

In this study, the matrix (M) gene of the RBOK vaccine strain of rinderpest (RPV) was amplified by polymerase chain reaction (PCR) and cloned into $TOPO^R$ XL cloning vector. For this purpose, Vero cells were infected with the RBOK vaccine strain of RPV and total RNA was obtained from the infected cells. cDNA of the matrix gene was obtained by reverse transcription from the total RNA. Amplification of the cDNA with PCR was achieved by using the M gene specific primer and PCR products of the M gene were directly ligated into the $TOPO^R$ XL cloning vector and this recombinant plasmid DNA transformed into JM109 competent E. coli cells. To determine the presence of the M gene in the recombinant plasmid pTOPO XL, both PCR and restriction enzyme digestion assays were performed.

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