Expression of F protein gene in maize for production edible vaccine against Newcastle disease Virus

Abstract

The aim of present study is the cloning of F and HN gene in plant expression vector to develop plant-based edible vaccine against Newcastle disease virus (NDV) of poultry. NDV was collected from Veterinary Research Institute, Lahore, Pakistan on request. ~1662bp F gene and ~1712bp HN gene were PCR amplified from cDNA of NDV that were TA cloned and further evaluated through sequencing. BLAST results determined the specificity of these genes in accordance with previously reported Mukteswar strain (Accession # GU182327). Both F and HN genes were ligated into pET30a expression vector to produce recombinant pET-F and pET-HN. Both recombinant constructs were transformed in E. coli Rosetta cells to study the prokaryotic expression of immunogenic protein. Induction by adding IPTG generated increased yield of protein and SDS polyacrylamide gel electrophoresis confirmed protein on desired size (~67kDa for F and ~69kDa for HN). Further western blot analysis, confirmed specificity of protein through antigen antibody reaction at proper size. Protein purification using IMAC affinity chromatography was performed and the appearance of single band of F protein at ~67kDa and HN protein at ~69kDa confirmed the specificity of our desired immunogenic protein. 2D and 3D structural analysis of F and HN proteins through Immune epitope database (IEDB) analysis resource tool revealed that more than 70% of its sequence is antigenically active and the predicted protein regions behave as epitopes. After prokaryotic expression of both F and HN genes, the next major objective of this study was to construct pCAMBIA 1301 with both F and HN for plant transformation. After confirming F and HN inserts, recombinant pCAMBIA-F+HN plasmid was electroporated into Agrobacterium cells (LBA4404) using a Bio-Rad electroporation device. After confirming the presence of F or HN genes in Agrobacterium, embryos from inbred lines of maize were transformed with recombinant pCAMBIA-F+HN by Agrobacterium mediated nuclear transformation. Further, the presence of F and HN genes in transgenic maize plants were confirmed through different molecular biological tools. The putative plants were confirmed through polymerase chain reaction (PCR). PCR confirmed a short fragment of ~181bp and ~191 for F and HN genes respectively. RT-PCR analysis confirmed the expression of F and HN genes in corn leaves and seeds respectively. The comparitive analysis of Ct values obtained by qRT-PCR revealed that the expression of F gene increased from 2-7.1 fold. Similarly, comparison of Ct value by qRTPCR confirmed that expression of HN gene increased from 0.5 to 4 fold as compared to negative control. Protein expression of F and HN genes were confirmed through ELISA and western blot by using gene specific antibodies. In ELISA, both F and HN proteins expression were observed in putative plants. The maximum obtained concentration of F gene was in the range of 0.15μg/ml to 0.166 μg/ml. Similarly, maximum obtained concentration of HN protein was in the range of 0.195μg/ml 0.24μg/ml. The plants with high mRNA expression of F and HN genes were confirmed through western blot analysis. Transgenic plants produced a fragment of 67kDa for F protein and 69kDA for HN protein which confirmed the expression of transgene. Furthermore, immuniztion of chicks with transgenic maize and immune response generated by ELISA results showed production of anti-NDV antibodies in sera of chicks. On the other side ELISA results from the sera of chicks having non-transgenic diet did not induce any significant immune response. This is a key achievement of this study, which can lead towards development of plantbased edible vaccine against Newcastle disease virus of poultry.