Cloning and expression of fusion (F) protein gene of Newcastle disease virus (NDV)

Abstract

The present study was undertaken to amplify, clone and express Fusion (F) protein gene of Newcastle disease virus. The freeze-dried virus of pigeon origin was propagated in embryonated chicken eggs. RNA was isolated from the infected allantoic fluid and cDNA was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Primers specific to F gene incorporated with restriction enzyme sites, viz. NdeI and XhoI were used to amplify the F gene using cDNA as a template by polymerase chain reaction (PCR). The 1680 bp F gene amplicons were gel-purified and first cloned into TOPO vector. Positive clone was confirmed by colony PCR and restriction enzyme digestion. TOPO released insert was ligated with NdeI and XhoI digested pET22b expression vector using T4 DNA ligase and transformed into E.coli. Recombinant clones were selected by colony PCR and restriction enzyme digestion and induced with 0.5 mM final concentration of Isopropyl-1-thio-b-D-galactosidase (IPTG) for the expression of the recombinant F protein. The expressed protein of 55 kDa was obtained during the 4 h post-induction. The obtained recombinant protein reacted with rabbit anti serum in Western blot confirming it to be NDV specific.