Construction of Recombinant Bacmid DNA Encoding Newcastle Disease Virus (NDV) Fusion Protein Gene

Background and Aims: Newcastle disease virus (NDV) is one of the major pathogen in poultry. Vaccination is intended to control the disease as an effective solution; nevertheless this virus is a growing threat to the poultry industry. F gene open reading frame (ORF) from NDV is 1650 bp, encoding a protein of 553 amino acids that can induce protective immunity alone. The F glycoprotein on the surface of NDV is important for virus infectivity and pathogenicity. Towards protection goal, the full-length of F gene was isolated using specific primers and cloned into the baculovirus derived bacmid shuttle vector to produce recombinant F-protein in insect cells. Materials and Methods: F gene ORF from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned firstly into the T/A cloning vector and then subcloned into the pFastBac Dual donor plasmid through NcoI/KpnI sites. After the verification of cloning process by PCR and enzymatic digestion analysis, the accuracy of F gene ORF in the T/A cloning vector was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated and confirmed by PCR using F specific primers and M13 universal primers. Results: Results showed that a recombinant baculovirus containing a correct and in framework sequence of Newcastle F gene under the control of p10 promoter was constructed. Conclusion: The above mentioned F-containing recombinant baculovirus, in addition to its independent application, can be used with other individual recombinant baculoviruses expressing NH and NP genes to produce Newcastle VLPs in insect cell line.