كلونينگ ژن F ويروس بيماري نيوكاسل در باكتري Escherichia.coli

Cloning of fusion (F) gene of newcastle disease virus in E.coli

Newcastle disease virus (NDV) might be the most important viral disease of avian species, including poultry. The emergence of genetic engineering technology provided the industry with new methods of manufacturing vaccines. The fusion protein, along with haemagglutinin-neuraminidase, serves as the target for the immune response of the host. After extraction of RNA from the B1 vaccinal strain of NDV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplificated by the polymerase chain reaction (PCR) and analyzed by agarose gel electrophoresis. To confirm the nature of the PCR product, the amplicon was digested with SacI and EcoRI restriction enzymes. The intact PCR product of fusion gene was cloned in pUC18 vector which had been digested with XbaI and BamHI enzymes. After of successful ligation, the constructed plasmid was transformed into DH5aα E.coli. Plasmid DNA from transformed bacteria was extracted in white clony and potential positive clones were confirmed with restriction digestion. Construction of a recombinant pUC18 plasmid containing the fusion gene was achieved as a first step towards the expression in transgenic plant.