Expression of Newcastle Disease Virus Epitope in Nicotiana Benthamiana

Viral diseases have greatly affected human lives and civilizations from time immemorial. The Newcastle disease virus (NDV) is causing lethal and highly contagious diseases in birds especially chickens and having a negative impact worldwide on the poultry industry. Although this virus has been effectively vaccinated, it is still prevalent in Pakistan and disease-related outbreaks have led to serious losses regarding mortality and loss in meat and egg production for the poultry industry in the country. Different genes of this negative single-stranded RNA virus have been sequenced, cloned, and expressed in plasmid-vectors in various organisms. There are different strategies to produce vaccines of NDV and plant-based vaccine (PBV) is one of them. Fusion (F) gene has the most virulence impact among the all-other genes of NDV. In this study, the epitope gene of NDV was cloned into the plant expression vector. Agrobacterium-mediated transformation of F gene in Nicotiana benthamiana was performed through the leaf disc method. The epitope was checked by amplification using specific primers through polymerase chain reaction (PCR). Real-time polymerase chain reaction (RT-PCR) was carried out to analyze the expression of the transgene in transgenic plants. Through this research, we might be able to fight any pandemic disease within few months instead of producing a vaccine in several years in the future.


Introduction
Viruses are dominating on the earth as a competitor of humanity as a parasite and replicating their genetic material in their hosts [1].
Healthy food of a high quality at reasonable commercial prices is the priority of the consumers. There are different sources of meat proteins in the world, but chicken meat is the cheapest among them.  [2]. F glycoprotein is the primary virulence factor of the NDV [3]. The precursor F0 is composed of 553 amino acids and has a 55 kDa molecular mass [4,5]. The F glycoprotein has five or six prospective sites. There are probably 20 residues of fusion peptide conserved in the hydrophobic zone of F1 fragment amino terminus. Viral and host cell membrane connection is regulated through this region [6].
Natural pathogens such as Agrobacterium tumefaciens are utilized to integrate antigenic DNA stably in the plant's nucleus.
The inherent capacity of pathogens to infect and transfer their virulent genes to the host cell nucleus benefits transformation [7].
The transgene can be integrated into the nuclear or plastid genome either through transient or stable transformation [8]. Stable transformation is achieved through the permanent integration of the transgene. Many plants are restricted to agrobacteriummediated transformation [9]. Many plants have been used for the expression of transgenes but Nicotiana benthamiana has significant importance for the expression of therapeutic recombinant proteins [10]. N. benthamiana is an Australian native with many features, including the rapid growth and natural capacity for biopharmaceuticals in heterologous gene sequences [11]. The viral or binary vector system is often involved in the vacuum infiltration of leaf tissue with agrobacterium containing transgene for a protein of interest [12,13]. The availability of recombinant protein in the plant is based on an extensive amount of protein in one gram of leaf tissue. Plant leaf biomass is counted before agro-infiltration and after it [14,15]. Recombinant proteins in N. benthamiana and original plant proteins have maximum structural resemblance and stability in plant cells environment [16,17].
In 1992, the concept of the production of vaccines in transgenic plants was introduced. In 1995, a biopharmaceutical company Pros gene from Texas (USA) carried out clinical studies on pigs, which were supplied with an edible maize vaccine, and they were found to be protected against TGEV. Patents were also obtained for this and other edible transgenic crop vaccines. Such vaccines provide technology, particularly in the developing world, for the production and supply of cheap vaccines, wherever they succeed. Plant-based platforms are one of the preferred expression systems active for recombinant vaccine assembly [18].

Vector Designing
The binary vector pHNF was designed by the ligating pBR 322 ori with pVS1 oriV, pVS1 RepA, pVS1 StaA, and kanamycin sequence  SWISS-MODEL tool (https://swissmodel.expasy.org/) was used to predict the 3D structure of the optimized codons' protein.

Vector Cloning and Confirmation
The vector pHNF was cloned into E. coli through heat shocking of 90 seconds in the water bath at 42ᵒC. The plasmid was isolated through Thermo Scientific Gene JET Plasmid Miniprep kit (www. thermoscientific.com/onebio) from 5ml culture of a transformed E.
coli single-cell colony. The vector was confirmed by XhoI restriction digestion. The reaction was incubated for 3 hours at 37ᵒC and run on 1% agarose gel along with Fastlane 1Kb DNA ladder.

Transformation of NDV Epitope Gene into N. benthamiana
The sterilized leaf discs of N. benthamiana were dipped in

Gene Expression Analysis
Genomic DNA was extracted from young leaves of transgenic seedlings and wild-type N. benthamiana by the CTAB method to determine the presence of foreign genes in the genome of the regenerated kanamycin-resistant seedlings. PCR was carried out by using specific primers (5′ACTATTACAATTACATGCATCA3′ and 5′GAGTTCATCTTTCAAAGTG3′). The 35 cycles for the PCR were 94ᵒC, 60 s for 55ᵒC, and 60 s for 72ᵒC, followed by a final extension of 72ᵒC, which was completed in 10 minutes. As a control, genomic DNA of N. benthamiana was used. 1.5% agarose gel was used to separate the PCR product along with the omega BIO-TEK 100bp DNA ladder.
Total RNA was extracted using a commercial kit of Triazole. Following DNase treatment, cDNA was synthesized by using the cDNA synthesis kit. cDNA was synthesized by using oligo(dT) as reverse

Vector Cloning and Confirmation
The isolated plasmid was digested with restriction enzyme XhoI and run on 1% gel ( Figure 3). This restriction sequence was placed in pHNF at two sites during vector designing. Comparison of digested fragments (8491bp and 891bp) with Fastlane 1Kb DNA ladder confirmed that the isolated plasmid was pHNF and it was successfully cloned in E. coli.

Leaves of N. benthamiana plants were transformed using
A. tumefacien strain LBA4404 with pHNF through the leaf disc method. Almost two hundred pre-cultured leaf discs (Figure 4a) of N. benthamiana were co-cultured (Figure 4b). A thin whitish film of the bacterial growth was seen on the periphery of infected discs which became more visible after 72 hours and also covered the surface of discs after one day of co-culture. It was observed that due to kanamycin selection pressure only the transformed cells were developed into shoots ( Figure 5) after 28 days. The shoots were shifted on selection rooting media to develop the shoots into the whole plant (Figure 6a-6d).

Discussion
Tobacco is the most preferred model in bioreactor plant studies for the expression, due to its easy transformation and its rapid regeneration, of foreign protein [20,21]. Therefore, in the present study, N. benthamiana plants were used as a model for genetic transformation and the evaluation of NDV epitope production. A synthetic gene with 4 tandem repeats of HN epitopes and 3F NDV epitopes has been prepared in the current research. Based on studies, the optimization of a codon can increase protein quality and quantity. The gene construction has been optimized based on the preferred codon. 5′ omega (TMV), which is considered to be a translation enhancement element, has been added to 5′UTR. In terms of the effect of SEKDEL on gene expression improvement.
SEKDEL had been attached to 3′ before the stop codon. To identify and isolate target protein by anti-His sequence, a Histidine tag was added to amino gene terminals [22].
Confirmation of the vector is necessary before using it for the transformation. Vector pHNF was cloned in E. coli to increase the quantity of the vector for the experiments. The plasmid was isolated from single colony culture to avoid contamination. The same size of the bands showed that extracted plasmids were the same and there was no contamination during culture growth and plasmid isolation. The extracted plasmid samples were digested with restriction enzyme XhoI. The specific sequence for the XhoI enzyme was placed at two sites in the vector pHNF. The band size of the pHNF fragments was confirmed through Snapgene viewer software (http://www.snapgene.com/) by finding XhoI double cutter sites in pHNF. Approximately, 8491bp and 891bp fragments were obtained which confirmed that cloned vector was pHNF.
Confirmation of the vector through restriction digestion has been followed by many scientists, Chaudhury et al. [23] is one of them.

Plants of N. benthamiana were transformed through LBA4404
A. tumefacien strain with plasmid pHNF. Genetic transformation by the Agrobacterium strain has various advantages, including the large piece of DNA transformation, increased transformation efficiencies, low copy number addition, and minimum reorganization of transferred DNA [24,25]. Genomic DNA and RNA were isolated at 3-4 leaf stage and subjected to PCR and RT-PCR amplification, respectively. Total DNA was extracted to screen for the foreign gene using PCR after germination of transgenic seedlings. PCR primers have been designed to match the ORF of pHNF. Approximately, 307bp NDV epitope gene fragment was obtained from 9 putative N. benthamiana plants, PCR confirmed the presence of transgene (Figure 7) whereas no band was observed for non-transgenic control plants. Band intensity was almost the same for all the tested plants which were indicated that the integration was the same and successful. The analysis of gene integration in transgene through PCR is a general practice that has been followed by several scientists [26][27][28]. RNA expression in four plants using RT-PCR was semi-quantitatively measured. RT-PCR gave the same results according to the PCR, but the band intensity was different for putative transgenic plants. Different intensity levels indicated the amount of mRNA transcription in cells of each transgenic plant [29][30][31].

Conclusion
The plant-based vaccine of NDV was first time produced in