A large number of plant viruses can successfully infect N. benthamiana, as one of the most widely used experimental hosts in plant virology. In addition, N. benthamiana is gaining popularity in plant viral purification and large quantities of protein both rapidly and economically (Goodin et al. 2008). PVX is also able to establish infection in N. benthamiana (Aguilar et al. 2015). Thus, to investigate the large-scale purified virus, PVX used for inoculation of N. benthamiana in phosphate buffer (pH 7.2). Then, the leaves were harvested and purified within 2–3 weeks after inoculation of N. benthamiana (Senanayake and Mandal 2014). Accordingly, 100 g of the leaves were homogenized with 0.1-M phosphate buffer. Furthermore, the leaves were centrifuged at 7800g for 20 min after filtration with cheesecloth. 0.02-M NaCl and 4% PEG were added to the supernatant and centrifuged at 7800g for 30 min. The pellet was suspended in 1% Triton X-100 and 0.05-M phosphate buffer and centrifuged at 7800g for 10 min. In the next procedure, the supernatant was centrifuged in 30% sucrose gradient at 72,500g for 150 min using ultracentrifuged Backman Ti 70 rotor (Salazar 1993). Finally, the pellet was suspended in phosphate buffer (0.05 M, pH 7.2). Furthermore, the purity of virus was confirmed by SDS-PAGE and UV/Vis spectroscopy.
To obtain PVX-Herceptin nanoparticle (PVX-HER), EDC/sulfo-NHS (1:2.5) was used to conjugate Herceptin (IV infusion) to PVX (Esfandiari et al. 2016). In addition, EDC/sulfo-NHS cross linker was used to conjugate PVX–PVX during a two-step process. First, PVX (5 µg/µl) was incubated with EDC/sulfo-NHS (2 mM/10 mM) for 4 h, and accordingly, PVX-linker and another PVX particle (5 µg/µl) were conjugated for 2 h. Furthermore, free-PVX was purified virus without any conjugation.
Mechanical inoculation
To study the pathogenicity of PVX-HER on plants, N. benthamiana plants as a PVX indicator were set up in three rows and each row contained 12 plants. Next, plants inoculated with PVX-HER nanoparticle and PVX (as a positive control) in a buffered solution containing 0.05-M phosphate buffer pH 7.2, 2% PVP (Esfandiari et al. 2006). The development of symptoms on the plants was observed after 3 weeks (Senanayake and Mandal 2014; Zhao et al. 2001).
Elisa
The virus accumulation increased at 21-day post-inoculation with PVX (Senanayake and Mandal 2014). Accordingly, leaves of N. benthamiana 3 weeks after inoculation with PVX-HER, free-PVX, and healthy plant as a negative control were tested by sandwich ELISA assay in 96-well plates (Nunk Denmark). Each well was coated with PVX-specific antibodies (DSMZ, PV-0027) diluted in coating buffer. Then, the samples were prepared in PBST buffer, pH 7.4 (containing 2% PVP) added to the well and incubated at 37 °C overnight. In addition, the secondary antibody (Goat–anti-rabbit IgG-HRP, ab6721) was diluted and added to each well. After washing the plate three times with PBST buffer pH 7.4, 100 µl of TMB solution (3, 3′, 5, 5′-tetramethylbenzidine, T0565 Sigma) was added to each well and incubated for 15 min at the room temperature. The reaction was stopped by 100 µl/well of stopping solution. Then, the absorbance was read at 450 nm using ELISA reader (ELX 800, Biotek). PVX-HER, free-PVX, and negative control should be performed in triplicate.
SDS-PAGE and Western blot
The leaves of N. benthamiana were separately inoculated with PVX-HER and free-PVX. After 3 weeks, the leaves were extracted in 0.05-M phosphate buffer pH: 7.2 and boiled for 5 min. The boiled samples were loaded into 4–12% acrylamide gels electrophoresis.
PVX-HER, free-PVX, positive control (purified PVX), negative control (without inoculating healthy N. benthamiana leaves), and a protein marker (prestained protein ladder, Thermo; 2616) were run at 100 V for 45 min. Then, proteins were transferred into nitrocellulose membrane at 350 mA for 90 min. After blocking in 4% nonfat dry milk in 37 °C for 2 h, the PVDF membrane was blotted by PVX antibody (DSMZ, PV-0027) at 4 °C overnight. After washing the membrane with PBST buffer, it was incubated with goat polyclonal secondary antibody to rabbit IgG-HRP (Abcam ab6721) for 2 h. In the next procedure, ECL chemiluminescent substrate kit (GE Healthcare, Advance Western Blotting Detection Kit, RPN 2235) was used to detect the visualized protein bands. Finally, PVX-HER, free-PVX, positive control, and negative control were compared to each other.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from 100-mg infected leaves at N. benthamiana 14-day post-inoculation with PVX-HER, free-PVX, and healthy plant (non-inoculated as a negative control) by RNeasy plant mini kit (Qiagen RNeasy Plant Mini Kit (cat#74904)). The cDNA of RNA virus was synthesized using M-MuLV reverse transcriptase (200 U/µl), total RNAs, and the reverse specific primer. This reaction was incubated at 42 °C for 1 h.
In addition, the cDNA synthesized was applied in the PCR reaction containing 0.1-M cDNA, 2.5-µl 10× PCR buffer, 1-µl dNTP (10 mM), 0.8-µl MgCl2 (50 mM), 0.3 µl of each specific primer (forward and reverse 20 pM), 0.3-µl Taq DNA polymerase (Promega), as well as 19.7 µl of DEPC-treated water. The PCR reaction condition occurred at 94 °C for 1 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, with a final extension step of 72 °C for 1 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis including 0.5% µl/ml ethidium bromide, and visualized by ultraviolet transilluminator (UVP). The forward primer: 5′-AAGATGTCAGCACCAGCTAG-3′ and reverse primer 5′-GTAGGCGTCGGTTATGTAGA-3′.
Flow cytometry
Tumor cell lines and MCF-12A non-tumorigenic epithelial breast cell line were implemented to study the toxicity of free-PVX, PVX–PVX, and free-HER. Accordingly, SKBR3 (2 × 105), SKOV3 (1 × 105), and MCF-12A (1 × 105) cells were seeded on a 12-well plate at 37 °C in a humidified atmosphere containing 5% CO2 incubator. SKBR3 and SKOV3 cells were grown in RPMI-1640, and MCF-12A was cultured in DMEM. The growth media were supplemented with 10% FBS (fetal bovine serum), penicillin, and streptomycin 10 μg of free-HER, free-PVX, and PVX–PVX were added on a 12-well plate for each cell, separately. At 24 h after the treatment, the cells were collected by trypsinization into their culture media, washed with PBS, and stained with Annexin V/propidium iodide (PI) Kit (BMS500 FI/100, eBioscience).The Annexin V/PI assay was conducted to determine cell apoptosis. In the next stage, the cells were analyzed after 10-min incubation by FACScan flow cytometer. Finally, Annexin V-binding and PI-binding were recognized to be apoptotic and necrotic, respectively. It is worth noting that all measurements were performed in triplicate.
Statistical analysis
To analyze the data, Mean ± SEM along with analysis of variance (ANOVA) with LSD post hoc test were used by SPSS 16.0 software. Before performing the parametric tests, normality distribution of the data sets was tested by Kolmogorov–Smimov and Shapiro–Wilk tests. The difference in values was considered as significant if the P value was less than 0.001. All experiments were performed in a triplicate and repeated at least three times.