## 3 Results  
### Gene Cloning, Production, and Purification of N and HEV/RBD Recombinant Proteins  
The plant codone-optimized N gene was cloned into a pEAQ-_HT_ vector using _Age_ I and _Xlo_ I restriction sites (Figure 1a). The plant codon-optimized HEV/RBD gene was cloned into the pEff vector (Figure 1b) [30]. HEV/RBD expression was targeted to the endoplasmic reticulum (ER). We achieved this by adding an ER-targeting signal peptide at the N-terminus and an ER retention signal HDEL at the C-terminus of the fusion protein [30]. To facilitate purification of the N and HEV/RBD recombinant proteins, a 6-histidine tag and an 8-histidine tag were added to the C-terminus of the N and HEV/RBD recombinant proteins, respectively (Figure 1). The sequences of the inserts were confirmed by sequencing,and the recombinant vectors were transformed into _A. tunefacients_ and used to infiltrate _N. benthamiana_ leaves. _N. benthamiana_ leaves were agroinfiltrrated with the recombinant vectors pEAQ-_HT_-N and pEff-HEV/RBD. Morphological changes were observed in the plant leaves agroinfiltrated with _Agrobacteriumrium_ carrying pEff-HEV/RBD. The leaves started to show serious necrosis at 5 dpi (Figure 2a). In plants agroinfiltrated with pEAQ-HT-N, chlorosis began on the base leaves at 5 dpi (Figure 2b). Leaf tissues for isolation of protein samples were harvested four days after agroinfiltration based on the morphological characteristics of infiltrated leaves and expression analyses. The N and HEV/RBD protein purifications were performed using metal-affinity chromatography under native conditions. After purification, the protein samples were dialyzed against PBS and subjected to SDS-PAGE gel analysis stained with Coomassie Blue. The purified N protein was analyzed by SDS-PAGE (Figure 3a) and Western blot with anti-his Ab (Figure 3b). The purified N protein was observed as a monomer (49.5 kDa),  
Figure 1: Scheme of the recombinant vectors pEAQ-_HT_-N (**a**) and pEff-HEV/RBD (**b**). 35S, promoter of the cauliflower mosaic virus RNA; NosT, terminator of the napaline synthase gene from _A. tumefaciens_; P24, P19, gene of silencing suppressor from grapevine leafroll-associated virus-2 and from the Tomato bushy stunt (Tombus) group virus, respectively; LB and RB are the left and right borders of T-DNA; 5′-UTR from cowpea mosaic virus RNA-2; 3′-UTR from cowpea mosaic virus RNA-2; RDRP, RNA-dependent RNA polymerase of PVX; Sgp1, the first promoter of the subgenomic RNA of PXV; AMV, the leader sequence of RNA 4 of alfalfa mosaic virus. Figure 2: Morphological changes of _N. benthamiana_ leaves after agroinfiltration with _Agrobacteriumrium__tunefaciens_ carrying pEff-HEV/RBD construct at different days post infiltration (dpi) started at day 1 till day 5 (**a**); _N. benthamiana_ leaves changes after agroinfiltration with _Agrobacteriumrium__tumefaciens_ carrying pEAQ-HT-N construct from 1 days after infiltration (dpi) till 5 dpi (**b**). dimer, and tetramer (Figure 3a). The final yield of the N protein after purification was about 30 mg/kg of WF. The _N. benthamiana_ leaves infiltrated with the pEff-HEV/RBD construct were harvested on 4 dpi, and the recombinant protein was purified by IMAC. The SDS-PAGE revealed that HEV/RBD fusion protein is expressed very well in plants (Figure 4a). We observed a clearer band with a molecular mass of 81 kDa; the Western blot analysis with an anti-RBD mAb further confirms that the 81 kDa protein is HEV/RBD (Figure 4b). The IMAC method applied here obtained the \(\sim\)100 mg HEV/RBD of kg fresh tissue weight (FW). Figure 4: **(a)** SDS-PAGE Coomassie brilliant blue-stained gel of HEV/RBD fusion protein. **(b)** Western blot with a monoclonal anti-RBD mAb. M-prestained protein molecular marker (kDa). The position of HEV/RBD protein (calculated molecular weight 81 kD) is shown by a black arrow.