Single Nucleotide Modification of VP2 Gene of Highly Virulent Infectious Bursal Disease Virus for Effective Expression Strategy in Tobacco

Plant viral-based expression system offers an alternative tool for the expression of various target proteins via stable transformation or transient expression in plants. In transient expression strategy, generation of in vitro viral transcripts through complete linearization of the recombinant vector containing the cloned gene is crucial to ensure the success of gene expression in plants. Hence, the present study was aimed to generate a modified VP2 gene of highly virulent Infectious bursal disease virus (hvIBDV) for effective cloning and expression of VP2 protein using an engineered Potato Virus X (PVX) vector as a gene delivery system in tobacco. Single nucleotide modification was employed at the SpeI site of the VP2 gene through the substitution of adenine (A) to thymine (T) without changing the amino acid leucine. The modification had facilitated the generation of viral transcripts whereby the recombinant vector of PVX-VP2Mt was completely linearized through a single digestion at the SpeI site of the PVX vector. The modified VP2 gene was delivered and expressed in Nicotiana benthamiana via viral transcripts infection approach. Western blot analysis of plant crude extracts indicated detection of expected bands which confirmed the successful expression of a modified VP2 protein. The VP2 protein expression profile was determined by conducting a kinetic analysis analyzed by dot blot assay. The present study demonstrates the successful development of a modified VP2 gene of hvIBDV through a single nucleotide modification strategy which facilitate the expression of the VP2 protein using a Potato virus X expression system.