Sample collection and screening of SPFMV through serological assays
Leaf samples of sweet potato plants showing characteristic feathering and ring spot symptoms were collected from the field of ICAR- Central Tuber Crops Research Institute (ICAR-CTCRI) (Fig 1).
Preliminary screening was done in order to find out the presence of Sweet potato feathery mottle virus in these samples using both the serological methods like DAC-ELISA and DIBA using antibodies obtained from DSMZ, Germany. Samples which were highly positive for DAC-ELISA and DIBA were taken for subsequent molecular analysis.
RNA isolation
For molecular analysis, total RNA was isolated from the DAC-ELISA and DIBA positive plant leaves by Trizol method (TRI reagent protocol, Sigma-aldrich). Fresh Leaf samples (100 mg) were chilled and pulverized to a fine powder in liquid nitrogen using chilled mortar and pestle and mixed well by adding 1ml TRI reagent (cat#T9424, Sigma-aldrich). The homogenate was transferred to a sterile 2 ml centrifuge tube. To this, 200µl of chloroform was added, mixed well by gentle inversion for 10-30 sec and incubate for 15 min on ice. Then it was centrifuged at 20,000g for 15 min at 4°C. The supernatant was transferred to a fresh tube and extracted twice with an equal volume of 25: 24: 1 (v/v) phenol/chloroform/isoamyl alcohol and mixed well by inversion for 2-3 min. Then it was centrifuged at 20,000g for 10 min at 4°C. To the aqueous phase, 1 ml chilled isopropanol was added and mixed by inversion. The mixture was then incubated at -20oC for 2 hr to precipitate the RNA. The precipitated RNA was pelletized by centrifugation at 20,000g for 15 min at 4°C. The supernatant was decanted and the pellet was washed twice in 0.5 ml ethanol (70 %) and centrifuged at 20,000g for 5 min at 4°C. Supernatant was discarded and the pellet was air dried for 30-40 min and dissolved in 50 µl deionised water (Incubation at 55oC for 10 min) followed by storage in a deep freezer (-80°C) for later use. Isolated RNA was used as a template for cDNA synthesis using a first strand cDNA synthesis kit according to the manufacturer’s instructions (Bioline, Memphis).
PCR amplification and cloning
The cDNA was used as a template to amplify the full-length coat protein gene of SPFMV using the forward primer 5’-GCGGGATCCTCTAGTGAACGTACTGAA -3’(BamHI enzyme site was underlined) and reverse primer 5’-AAAGAGCTCTTGCACACCCCTCATTCC -3’ (SacI enzyme site was underlined) to produce an amplicon of 963 bp in length. Reaction mixture per tube were prepared each containing 2.5μl of 10x Taq polymerase buffer (containing 100 mM Tris HCl, 500 mM KCl and 15 mM MgCl2), 0.5μl of dNTPs (10 mM), 0.5μl each of forward and reverse primers, 0.25 μl (2.5 units) of Taq DNA polymerase and 2 μl of template DNA (100 ng) and 13.75 μl of sterile distilled water to make a final volume of 20 μl/tube. The negative controls were the buffer used for RNA extraction and RNA from healthy leaves. The thermal cycling profile was 5 min of initial denaturation at 94°C followed by 35 cycles of: 1 min at 94°C, 45 sec at 57.7oC, 1 min at 72oC and 7 min of final extension at 72oC. Reaction was carried out in DNA BioRad C1000 Touch Thermocycler (Germany). The PCR products (963 bp) were separated by electrophoresis in 1% agarose gel having ethidium bromide as stain (0.5µg/ml) at 70V for about 1hour in 1X- Tris-Acetate–EDTA (TAE) buffer of pH 8.0. The PCR product (963 bp) was excised from the agarose gel using a sharp, disposable blade, put into an Eppendorf tube, and continued with purification using Qiagen gel extraction kit (QIAGEN, United States). The purified amplicon of full-length SPFMV CP was then directly cloned (ligated) into pTZ57R/T (TA cloning vector) using a InsTA clone TA cloning kit (Thermo Fisher Scientific, United States). The ligation product was then transformed into E. coli DH5α competent cells using the heat shock method [38]. The transformed white colonies were patched on to a fresh petri plate with Ampicillin (50 µg/ ml final concentration) and LB agar medium containing 40 μl of X- gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) (20 mg/ ml in Dimethyl formamide) and 20 μl of IPTG (Isopropyl-β-D-1-thiogalactopyranoside) (0.5 M in sterile water). Selected colonies of bacteria were picked and colony PCR was conducted to determine if the colony contains the DNA fragment or plasmid of interest. The PCR program for the colony PCR was the same as the previous program for the cloning procedure. Putative positive transformants, were selected and plasmid isolation was done by alkali lysis method [37]. Presence of insert was confirmed by restriction with enzymes BamH1 and HindIII. Restriction digestion was performed according to the manufacturer’s instructions (New England Biolabs, United States). Positive clones (plasmid) were sent for sequence analysis (Agrigenome labs, Cochin) and confirmed that intact SPFMV coat protein gene is present (PCR amplicon), through NCBI BLASTn program.
Subcloning into expression vector
The Insert (whole coat protein gene CP) was subcloned (ligated) into pET28A(+) expression vector and transformed into DH5α cells [38]. The transformed white colonies (Kanamycin selection) were patched on fresh petri plates with Kanamycin (50 ug/ ml final concentration) and LB agar medium. Confirmation of the insert was done using colony PCR (coat protein gene) and restriction digestion using BamHI. Transformation into pET28A(+) was successful from which putative positive clones were selected through kanamycin selection and plasmid DNA isolated using alkaline lysis method [37]. The positive plasmid (SPFMV-CP in pET28A(+) constructs) was transformed into BL21DE3 cells (NiCo21-DE3 cells cat#C2529H New England Biolabs, United States). Positive clones were identified using restriction digestion with BamH1 and one positive colony from pET28A(+) clones were selected for protein induction and expression studies.
Protein expression and solubility check
Bacterial culture (SPFMV CP positive clone in BL21DE3 cells) was grown in LB medium with Kanamycin (final concentration 50 µg/ml) at 37°C and 200 rpm. After reaching the optical density of 0.8, 1mM concentration of IPTG was added to the bacterial culture and incubated for 3 hrs at 37°C for the IPTG induced production of the recombinant protein (SPFMV CP). Similarly, another set of bacterial culture was grown seperately in LB medium with Kanamycin at 37°C and 200 rpm for 3hrs without IPTG induction and this was used as uninduced control. Cells from both IPTG induced culture and uninduced control were harvested by centrifugation at 20,000g for 10 min at 4°C and the total protein was extracted using lysis buffer (containing 20 mM Tris pH 8, 100mM NaCl, 0.1% Triton-X-100, 1mM EDTA pH 8, 2M Urea and 10X Protease Inhibitory Cocktail (PIC) (cat#G6521, Promega, United States)). Total cell lysate was loaded on 15% acrylamide-bisacrylamide gel and run the SDS PAGE to confirm the protein induction. Western Blotting was conducted to confirm the expression of protein (SPFMV CP) using crude extracts of the sample and the results were analysed through staining with BCIP/NBT solution.
Solubility of the recombinant protein was checked according to the procedures in ‘The QIAexpressionist handbook for high level expression and purification of 6xHis-tagged proteins’ (QIAGEN, United States). Both insoluble and soluble fractions were collected from IPTG induced bacterial culture and SDS PAGE analysis was done to confirm the presence of protein of interest is in which fraction. The same was confirmed through Dot Immunobinding Assay (DIBA).
Optimisation of protein expression
Three different temperatures namely 25oC, 31oC and 37oC, four different concentrations of IPTG namely 0.5 mM, 1mM, 1.5mM and 2mM and five different time points namely 30 min, 1 hr, 2 hr, 3 hr and 4hr were selected for SPFMV CP protein standardization experiments. Using these conditions, bacterial culture (SPFMV CP positive clone in BL21DE3 cells) were grown, induced with IPTG and total protein was extracted. Insoluble fractions were collected from the total protein and SDS PAGE analysis was done in 15% acrylamide-bisacrylamide gel to check the optimum temperature, concentration of IPTG and the desired time point after IPTG induction in which maximum yield of protein is to be obtained.The results show that an IPTG concentration of 1 mM is giving maximum induction and a time period of 3 hrs after IPTG induction at 37oC gives the maximum yield.
Protein purification and polyclonal antibody production
Using the above stated conditions, cells were grown and induced with IPTG and purified the protein (under denaturing conditions) using Ni-NTA resin affinity chromatography (QIAExpress Type IV kit). SDS PAGE analysis and western blotting were conducted to confirm the expression of purified protein (SPFMV CP). Large scale production and purification of the recombinant protein (SPFMV CP) was carried out using the optimized conditions and the concentration of the purified protein was determined according to Bradford (1976). Purified protein was given to two New Zealand white rabbits for producing polyclonal antibody.
Immunization of rabbits, absorption and purification of antibodies
Immunization of rabbits for polyclonal antibody production and purification were given for outsourcing (Abgenix India Private limited) where the purified recombinant SPFMV CP was used as the antigen to raise antibodies in the two New Zealand White rabbits. First and third bleed serum were collected one and two months respectively after immunization from both rabbits. Indirect ELISA was done using both the bleeds with 1:5000 dilution against custom antigen (purified recombinant SPFMV CP protein) to evaluate the strength of reactivity and the optimal concentration of the antiserum raised.
After completing the sensitivity assay, antisera raised was purified and confirmed the reactivity of the purified antibody through ELISA and western blot analysis.
Optimization and calibration of developed polyclonal antibody
Specificity analysis of polyclonal antibody using DAC-ELISA and DIBA
DAC-ELISA was done using polyclonal antibody (IgG) developed against the recombinant SPFMV coat protein using the standard protocol (Hegde et al. 2010). Crude leaf extracts from SPFMV positive samples and leaf samples showing feathering symptoms were tested along with healthy leaves from aseptically raised sweet potato plants and non-host plants (tissue culture raised cassava plants) (negative control). The result was evaluated after adding universal alkaline phosphatase–conjugated anti-rabbit IgG (Sigma Aldrich, United States) by measuring light absorbance at 405 nm wavelength. The samples were positively identified if the mean DAC-ELISA (Absorbance at 405 nm) value of samples exceeded at least twice the mean of the healthy control.
Crude leaf extracts from SPFMV positive samples and field samples showing feathering symptoms were spotted on nitrocellulose membrane (NCM) and used for DIBA analysis according to the satnderd protocol (Hegde et al. 2010) for analyzing the specificity of purified antibody. The universal alkaline phosphatase-conjugated anti-rabbit IgG (Sigma Aldrich, United States) was used as the secondary antibody at a dilution of 1: 10,000. The target proteins were finally revealed as purple spotes on NCM by adding to substrate 5-bromo-4-chloro-3- indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)
Sensitivity analysis of polyclonal antibody using DAC-ELISA
To determine the sensitivity of polyclonal antibody (IgG) developed against the recombinant SPFMV coat protein, DAC-ELISA was done using the standard protocol (Hegde et al. 2010) wherein different dilutions were used for optimizing the reactivity of the IgG (1:100, 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 and 1:10000). SPFMV positive samples along with negative control (healthy leaves from aseptically raised sweet potato plants) were tested. The result was evaluated after adding universal alkaline phosphatase–conjugated anti-rabbit IgG (Sigma Aldrich, United States) by measuring light absorbance at 405 nm wavelength.