Development of novel detection system for sweet potato leaf curl virus using recombinant scFv.

Sweet potato leaf curl virus (SPLCV) causes yield losses in sweet potato cultivation. Diagnostic techniques such as serological detection have been developed because these plant viruses are difficult to treat. Serological assays have been used extensively with recombinant antibodies such as whole immunoglobulin or single-chain variable fragments (scFv). An scFv consists of variable heavy (VH) and variable light (VL) chains joined with a short, flexible peptide linker. An scFv can serve as a diagnostic application using various combinations of variable chains. Two SPLCV-specific scFv clones, F7 and G7, were screened by bio-panning process with a yeast cell which expressed coat protein (CP) of SPLCV. The scFv genes were subcloned and expressed in Escherichia coli. The binding affinity and characteristics of the expressed proteins were confirmed by enzyme-linked immunosorbent assay using SPLCV-infected plant leaves. Virus-specific scFv selection by a combination of yeast-surface display and scFv-phage display can be applied to detection of any virus.


Results
Virus antigen detection and selection of yeast cells expressing viral antigen. SPLCV is one of the monopartite begomoviruses in geminivirus family, and SPLCV has 6 ORFs for encoding different proteins for systemic movement (V1), cell-to-cell movement (V2), virus replication (C1), transcription activator (C2) or replication enhancer (C3), and symptom determinant (C4). V1 of SPLCV encodes coat protein which is the only structural protein of geminivirus particles. As a target antigen, V1 protein of SPLCV Haenam 1 strain was amplified by PCR and the products were visualized in the form of a slight single band product on 1% agarose gel containing ethidium bromide ( Fig. 2A). The V1 sequence of SPLCV Haenam 1 strain consisted of 774 bp nucleotides. The amplified DNA fragments from the V1 of SPLCV was cloned into a pCTCON plasmid vector for yeast-surface display.
Yeast display has recently emerged as an alternative strategy, with one important advantage over phage display: the ability to precisely control selective parameters by FACS analysis. This technique can reliably quantify differences in antigen expression levels; antigens are fluorescently labeled with an antibody recognizing the C-terminal c-Myc tag encoded by the plasmid vector ( Fig. 2B,C).
The surface display of SPLCV V1 by Saccharomyces cerevisiae also allows the detection of appropriately labeled antigen-antibody interactions by flow cytometry. The 6 colonies were evaluated by shifting degrees from wild-type yeast. The yeast expression cells grown in SDCAA media were used as controls. Representative flow cytometry histograms for selected clones are shown in Fig. 2D. The vertical axis indicates the cell number, and the horizontal axis indicates fluorescence. Similar Gaussian distribution patterns of all selected clones were observed. The mean value of the x axis (x-mean) was used to measure the degree of expression of SPLCV V1 as a statistic. Only one colony of SPLCV showed the shifting of the x-mean value from the control. The x-mean value of the control cell was 7.30 while the highest value of one colony was approximate three times higher (22.91). Two high-expression cell lines (CP 5 and CP 6) were analyzed by western blot to verify antigen expression (Fig. 2E). The anti-c-Myc antibody detected a C-terminal tag of target protein. The data showed a 44 kDa band corresponding to the expected size of SPLCV V1. No band was observed in EBY 100, which was used as the negative control.
Screening of scfvs by bio-panning. The antigen-binding affinity of randomly selected colonies was measured in each round to determine whether the affinity was higher by bio-panning rounds (Fig. 3A). OD 450 was measured for screening of phage scFvs from randomly selected colonies. As the number of panning rounds increased, the binding affinity of positive libraries for positive antigens tended to increase. SPLCV-infected sweet potato leaves were used for the selection of antigen-specific scFv after bio-panning. SPLCV-infected sweet potato samples of leaves and phloem tissue 39 were identified as shown in Fig. 3B. Both the sensitivity and the specificity used in the diagnosis were critical values. The negative samples (Healthy, TYLCV samples) had OD 450 values of less than 0.20, and 15 phage scFv clones showed binding affinity with SPLCV-infected plants (Fig. 3C). The scFv DNA of selected clones was amplified by PCR and the nucleotide sequences were analyzed. The   The bio-panning result with sweet potato leaf curl virus (SPLCV) displayed on the yeast cells as an antigen. Sixty randomly selected colonies were measured after each panning round. The collected phages were bound to the target antigen and quantitatively confirmed using HRP-conjugated anti-M13 antibodies at an OD of 450 nm. Data are presented as means ± SEMs. (B) PCR detection of SPLCV in different tissues (phloem tissues and leaves) of sweet potatoes. Lane N is a no-template control. Lane H is virus-free sweet potato samples. Lane P1 and P2 are amplified with phloem tissue of SPLCV-infected samples, and 1, 2 are amplified viral DNA from sweet potato leaf samples. (C) Binding of scFv antibodies, determined by ELISA against SPLCV-infected plant leaves. SPLCV-infected sweet potato leaves were coated onto 96-well microtiter plates. Each scFv was detected using HRP-conjugated anti-M13 antibodies and TMB substrate solution. ELISA readings (OD 450 ) were collected after 30 min of incubation in a TMB substrate at 25 °C. Clone numbers 10 and 12 are the selected scFv used for expression. www.nature.com/scientificreports www.nature.com/scientificreports/ complementarity-determining regions (CDRs) were identified as shown in Table 1. The sequence was compared with the IgBLAST KABAT antibody sequence database 40 . Most of the scFv clones consisted of nonsense mutations or junk codons, and some clones could not be analyzed. A comparative analysis of VH and VL sequences showed significant differences in the CDRs, which are associated mainly with different biological activities 41 . Only two scFv clones (scFv "10" was named "G7", and "12" was renamed "F7") had complete sequences, including VH and VL chains.
Bacterial expression and characterization of anti-SPLCV scFv. The two scFv genes, F7 and G7, were subcloned into pET26b (+) (Fig. 4A) and pDEST-periHisMBP plasmids, respectively (Fig. 4B), and expressed in BL21 (DE3) pLysE. The expression test was performed under various IPTG concentrations (0, 0.1, 1, and 2 mM) and confirmed by western blot analysis (Fig. 4C). In the pET26b (+) plasmids, no soluble scFvs were expressed, and the plasmids were soluble in MBP fusion proteins. When the scFv protein was purified using Ni-NTA, the scFv did not bind well to the column. We therefore performed functional analysis using a filtrated medium in which scFv was expressed as soluble. To compare the binding affinity of different soluble scFv fragments, each protein was measured quantitatively by ELISA (Fig. 4D). Analysis of individual P values revealed a significant difference between negative and healthy samples (P < 0.0001, both) for F7 scFv. On the other hand, G7 scFv showed a slightly lower difference in the negative (P = 0.0049) and healthy (P = 0.0013) samples than did F7. Both scFv showed specific binding affinity to SPLCV sample.
Different scFv formats containing only F7 scFv domains including monovalent and bivalent scFv were compared in ELISA (Fig. 5C). The monovalent F7 protein bound only at relatively high concentrations of more than 2 μg/mL. The bivalent F7 scFv bound 25 times as efficiently to SPLCV samples compared with the monovalent scFv.

Discussion
Generally, many of the conventional serological diagnosis tests by using polyclonal-or monoclonal antibodies are less specific and less sensitive than molecular diagnosis approaches. Therefore conventional serological diagnosis tests is not suitable for precise and accurate diagnosis of field samples but it is useful in serologically confirmatory testing and epidemiological studies 42 . To overcome the disadvantages associated with conventional serological diagnosis techniques, molecular diagnosis tests such as PCR, real-time PCR, recombinase polymerase amplification (RPA) and loop mediated isothermal amplification (LAMP) assays have been used for specific and sensitive detection of samples [43][44][45] . But still serological detection tests have many advantages to detect viruses easily and rapidly from many samples if suitable antibodies are provided. Recombinant scFv has been developed to overcome the limitations and drawbacks of conventional polyclonal-and/or monoclonal antibodies. In many review papers, recombinant scFv can provide us many advantages such as no animal immunization required, shorter periods for scFv production, much cheaper than conventional antibody for mass-production and easy maintaining the scFv as a gene in expression vectors 46 . Nowadays, scFv has a well-established protocol to produce a completely functional antigen-binding fragment in bacterial systems. In addition, the advances in scFv applications give us more efficient and generally applicable method to produce better scFv by an antibody engineering techniques.
To select an antigen-specific scFv with bio-panning, a large amount of antigen is required. We used yeast-surface display for this study because the system offers eukaryotic post-translational machinery such as disulfide isomerization and glycosylation 37 . In this study, we prepared a genetically engineered yeast cell that displays the SPLCV V1 as an antigen. Phage display has been used to develop target-specific recombinant antibodies 30,47 . Some advantages of phage display over conventional hybridoma techniques include shorter time, lower cost, and greater application 23 .
Based on a combination of yeast-surface display and scFv-phage display, we screened SPLCV-specific scFv clones by bio-panning as described in 48,49 . Two scFv clones were selected for SPLCV and these genes could be expressed in E. coli for mass production. The scFv was not expressed as soluble in E.coli with short fusion peptides such as a His tag, but was expressed in relatively large protein such as MBP. In addition, a small tag such as a His tag could be detected as an antibody in SDS-PAGE under denaturation conditions. However, the non-denatured www.nature.com/scientificreports www.nature.com/scientificreports/ protein was not purified through a Ni-NTA column. The His tag was likely not exposed to the outside due to the steric structure of the scFv proteins. The binding affinity for antigens and the properties of the expressed scFv clones were clearly identified by ELISA using SPLCV-infected plant leaves. The results showed that the expression of scFv in E. coli can induce reactivity and specificity of a recombinant antibody. Therefore SPLCV-specific scFv can be mass-produced easily and inexpensively in E. coli.
The design of a bivalent scFv-expressing vector enhanced the binding affinity of monovalent scFv protein 50,51 . The avidity effect of SPLCV-specific bivalent scFv-Fc proteins resulted in a 25-fold improvement in antigen binding compared with the monovalent scFv fragment. This recombinant scFv detection method with bivalent scFv will contribute to more efficient virus detection system development by enhancing scFv-antigen binding affinity. Then this method can detect virus from small piece of sweet potato samples compared the conventional serological virus detection method with monovalent scFv.
With these advantages, scFv could play an important role in SPLCV diagnosis. The main purpose of this study was screening specific antibodies for sweet potato viruses as a diagnostic method. Recombinant antibody fragments for specific antigens can be modified with genetic engineering to more specific and stable antibodies for serological methods of detection. In addition, a genetically engineered, recombinant scFv with higher efficiency can be produced, based on different scFv binding actions to the antigen.

Methods
Virus antigen DnA preparation. SPLCV Haenam 1 strain (GenBank No. HM754641)-infected sweet potato leaves (Fig. 1B) were homogenized in liquid nitrogen after sampling. The total genomic DNA of the sweet potato was extracted following a method described in 52 . A small amount of sweet potato (less than 1 g) was collected in a 1.5 mL microcentrifuge tube and 500 μL of Dellaporta extraction buffer (100 mM Tris, pH 8.0), 50 mM of ethylenediamine-tetraacetate EDTA, 500 mM of NaCl, ad 10 mM of β-mercaptoethanol (BME) were added. The microfuge tube was mixed vigorously and incubated for 10 min at 65 °C with 33 μL of 20% sodium dodecyl sulfate (SDS; w/v). Next, 160 μL of 5 M potassium acetate was added, mixed, and spun for 10 min at 15,000 g in a centrifuge. The 450 μL of supernatant was transferred to a new tube and the process was repeated until the supernatant was free of debris. Isopropanol (0.5 volumes) was added with a vortex and spun for 10 min at 15,000 g. When the supernatant was removed, all nucleic acids were in the bottom of the tube. The pellet was washed with 70% ethanol and spun repeatedly for 5 min at 9,000 g. After removal of the supernatant, the pellets were dried at room temperature for 30 min. Finally, the pellet was resuspended in 200 μL of RNase-treated TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The extracted genomic DNA was purified and then stored at −86 °C. SPLCV coat protein gene amplification. SPLCV coat protein (V1) was amplified by PCR with genomic DNA as a template. The forward primer 5′-CCTAGGATGACAGGGCGAATTCCC-3′ and reverse primer 5′-AGATCTATTATTGTGCGAATCATAGAAA-3′ were designed to amplify the coat protein. The amplification reaction was performed in PCR tubes containing 1 μL of the template, 10 pmol of each primer and 2 × PCR premix (Takara, Tokyo, Japan), for a total volume of 20 μL. PCR amplification was performed with a thermal cycler machine (T100; BioRad, California, USA) under the following conditions: 10 min at 96 °C for pre-denaturation, followed by thermal cycling for 35 cycles (30 s at 96 °C, 30 s at 55 °C, and 1 min at 72 °C), 10 min at 72 °C for the final extension and storage at 12 °C. The amplified product of the V1 gene (774 bp) was identified on 1.5% agarose gel containing ethidium bromide. The DNA was purified with a gel extraction kit (Macrogen, Seoul, Republic of Korea). Purified DNA was cloned into the TA-cloning vector pGEM ® -T easy (Promega, Madison, WI, USA) and introduced into E. coli DH5α according to the manufacturer's instruction. After transformation, a single colony was placed onto a Luria-Bertani (LB) agar (1.5% w/v) plate containing 50 μg/mL of ampicillin, 100 μg/mL of X-gal, and 1 mM of isopropylthio-β-D-galactoside (IPTG). The selected colony was cultured in 3 mL of LB broth with 50 μg/mL of ampicillin, and the plasmid was extracted with a plasmid mini-prep kit (Bioneer, Daejeon, Republic of Korea) after cell incubation. The plasmid sequence was analyzed by the Macrogen-sequencing service (Seoul, Republic of Korea) with T7 and SP6 primers and a basic local alignment search tool (BLAST) from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Subcloning for yeast surface display. The SPLCV V1 fragment was inserted into the pCTCON plasmid.
The V1 gene in T-vector was digested with AvrII and BglII (Takara, Tokyo, Japan), and inserted between the NheI to BamHI sites of pCTCON according to the manufacturer's instructions. The ligate was inserted into a DH5α-competent cell, and colony selection onto LB agar plate with ampicillin, colony culture selection in broth, mini-scale plasmid extraction, and sequencing analysis were performed.
Yeast transformation and yeast surface display. Yeast (Saccharomyces cerevisiae strain EBY100)-competent cell preparation was performed following a Clontech manual (Tokyo, Japan) 53 . Pre-culture was carried out by streaking cells onto a yeast-peptone-glucose (YPD) plate containing 2% dextrose (w/v) and letting them grow for 3 days at 30 °C. A single colony was inoculated with fresh YPD and cultured to an optical density at 600 nm (OD 600 ) of 1.5 and diluted 10-fold in YPD and grown at 30 °C to an OD 600 of 0.4-0.6. The cells were harvested and washed with distilled water. The yeast was resuspended in sterilized TE/LiAc buffer (100 mM lithium acetate, 100 mM Tris-HCl, 10 mM EDTA, pH 7.5). The plasmid DNA containing V1 and cell were mixed with carrier DNA (sheared salmon sperm DNA) and sterilized PEG/LiAc (40% of PEG 4000, 100 mM lithium acetate, 100 mM Tris-HCl, 10

Bio-panning with phage-displayed scfv libraries. A higher initial antibody library size significantly
increases the selectivity of high-affinity antigen-specific antibodies [54][55][56] . The human scFv library pRGA-scFv was provided by Prof. Myung-Hee Kwon (Department of Microbiology, Ajou University School of Medicine) 57 . The scFv gene fragments were inserted between the Sfi I and Not I restriction sites of a modified pCANTAB 5E phagemid vector. Titers of the propagated phage library were measured as kanamycin-resistant colony forming units (CFUs). Selection of anti-SPLCV phage scFvs by bio-panning was performed as described in 48,49 . Briefly, wells of 96-well plate (SPL Life Sciences, Pocheon, Korea) were coated with the yeast cells [blank (PBS), negative samples (TYLCV V1-displaying yeast cells) and positive samples (SPLCV V1-displaying yeast cells)] diluted to OD 600 value of 0.6 with carbonate coating buffer (pH 9.6). After that, cover the plate with wet paper towel and incubate at 4 °C overnight. After a brief washing with TBS-T buffer (0.1% Tween 20 in TBS), all wells were blocked with blocking buffer (TBS, 0.1% Tween 20, 3% skim milk) for 2 h at 37 °C. For selection of phage scFv proteins, 10 8 mL/CFU recombinant phages in blocking buffer were added in blank wells, and incubated for 2 h at room temperature. Non-binding phages were transferred to negative-sample wells and incubated overnight at 4 °C. Collected non-binding phages were transferred to positive-sample wells and incubated for 1 h at room temperature. The positive-sample wells were washed vigorously with TBS-T 6 times. SPLCV-binding recombinant phages were eluted with 100 mM triethylamine and neutralized with 1 M Tris-HCl (pH 7.0). The eluted phages was titrated with XL1-blue (Agilent, Santa Clara, CA, USA) CFUs on an LB agar plate containing tetracycline (50 μg/mL) and carbenicillin (50 μg/mL). Three rounds of bio-panning were carried out, and phage titration was performed at every panning round.
Isolation and production of SPLCV-specific scFv. Randomly selected XL1-blue colonies containing individual clones from each bio-panning round were grown in 96-well mini tubes for 6 h at 37 °C and shaken with 2TY growth media containing 25 μg/mL tetracycline, 50 μg/mL carbenicillin, and 1% (w/v) glucose. After incubation, the cells were inoculated into new tubes with fresh 2TY growth media and incubated for 1 h at 37 °C with shaking. The M13KO7 helper phage was inoculated to cells at a multiplicity of infection of 50 and incubated 1 h at 37 °C without shaking. The soup was removed after centrifuging at 2,500 g, and resuspended in 2TY broth containing 25 μg/mL tetracycline, 50 μg/mL carbenicillin and 50 μg/mL kanamycin overnight at 30 °C. phage eLiSA. To select putative antigen-binding phage scFv clones, a phage enzyme-linked immunosorbent assay (ELISA) assay was performed in 96-well microplates coated with sweet potato leaves (and healthy leaves as controls) in a general-extraction buffer (10 mM sodium sulfite, 2% (w/v) polyvinylpyrrolidone (MW 40,000), 0.2% (w/v) sodium azide, 2% (w/v) BSA, 2% (v/v) tween-20 with PBS, pH 7.4; GEB) 58 overnight at 4 °C. Following rinsing with TBS-T 6 times, each phage clone culture's supernatants were added to each antigen and control well and incubated for 2 h at room temperature. The wells were washed 3 times with TBS-T, and the plates were incubated with HRP-conjugated anti-M13 (1:1000; 11793, Sino Biological Inc., Beijing, China) antibodies at room temperature for 1 h. After washing 6 times with TBS-T, TMB substrate solution (Agdia) was added for 30 min. The enzymatic reaction was stopped by the addition of 1 M sulfuric acid. Absorbance was read at OD 450 using a microplate spectrophotometer (BioTeK, Winooski, VT, USA).
Sequence analysis of selected SPLCV-specific phage scFv. Selected scFv clones by bio-panning were re-grown in 2TY media containing tetracycline and carbenicillin, and plasmid extraction was performed. Using these plasmids as a template, scFv sequences were amplified by PCR under the following conditions: 5 min at 96 °C for denaturation, followed by thermal cycling for 35  www.nature.com/scientificreports www.nature.com/scientificreports/ using the primer sets pCANTAB5E-S1 5′-CAACGTGAAAAAATTATTATTCGC-3′ and pCANTAB5E-S6 5′-CATTTACTTAAAAGACATACTCC-3′. The amplified DNA was sent to Macrogen (Seoul, Republic of Korea) and analyzed with a DNA-standard sequencing service. Amino acid sequences were deduced from the obtained nucleotide data (ExPASy, https://web.expasy.org/translate). From the results of sequence analysis of screened scFv bound to each antigen, complementarity-determining regions (CDRs) of scFv were found with IgBlast (https:// www.ncbi.nlm.nih.gov/igblast) 40,59 . Bacterial expression of scfv. The selected scFv genes were cloned into a pET26b (+) (Merck) vector and expressed in E. coli. The genes of scFv were amplified by PCR using primers ( Table 2) that added NcoI and EcoRI sites for the pET26b (+) plasmid to the 5′ and 3′ ends of the PCR product, respectively. The His-tag for pET26b (+) at the C-terminus was used for purification. The digested gene products were separated on 1% (w/v) agarose gels and the DNA was excised using a gel extraction kit (Bioneer). The digested PCR products and vectors were ligated overnight at 16 °C and inserted into E. coli strain BL21 (DE3) pLysE cells (Merck). To optimize the expression condition, 3 mL of exponential growth culture (with an OD 600 of approximately 0.6) was induced at 26 °C overnight with 0.1, 1, and 2 mM IPTG in a shaking incubator. Large-scale expression of scFv genes in 200 mL of LB medium was induced by the addition of IPTG during 6 h of growth at 26 °C. The cells were harvested by centrifugation (2,500 g, 4 °C for 15 min) and the proteins were extracted from cell periplasm by cold osmotic shock 60 and filtrated with a vacuum filter (Merck). For purification, supernatants of scFv expressed in pET26b(+) plasmids were applied to an nickel-nitrilotriacetic acid (Ni-NTA) 61 . 10% SDS-PAGE was performed to identify and separate each protein. Western blot analysis was carried out with primary anti-His-tag mouse monoclonal antibodies (1:5,000; R&D Systems, Minneapolis, MN, USA) and HRP-conjugated secondary anti-mouse IgG (Cell Signaling Technology) to detect scFv proteins.

Maltose-binding protein fusion expression.
To improve protein expression, the scFv genes were subcloned into maltose-binding protein (MBP) encoding a plasmid vector, pDEST-periHisMBP (Addgene, 11086, Cambridge, MA, USA) 62 using gateway cloning 63 . The scFv genes were amplified by PCR using primers (Table 3) in which the AttB1 and AttB2 sites were added to the 5′ and 3′ ends of the PCR product, respectively, for recombination site insertion. To produce entry clones, the BP reaction was carried out with BP Clonase ™ II Enzyme mix (Invitrogen, Carlsbad, CA, USA) and pDONR221 (Invitrogen) as a donor plasmid. After addition of proteinase K, recombinant plasmids were transformed with DH5α and selected on the LB media containing kanamycin (50 μg/ mL). Subcloning of an entry clone into a destination vector, pDEST-periHisMBP, was performed by LR Clonase ™ II Enzyme mix (Invitrogen). After addition of proteinase K, the expression clones were transformed with DH5α and selected in an ampicillin (50 μg/mL) selection medium. The expression clone sequences were analyzed using a DNA sequencing service (Macrogen, Seoul, Republic of Korea). MBP-fusion scFvs were expressed in BL21 (DE3) pLysE cells (Merck). An expression test was then carried out as with the pET26b (+) clone. functional analysis of scfv proteins. To determine the binding activity of scFvs purified from E.coli, indirect ELISA was carried out. Briefly, sweet potato samples were coated on a 96-well microplate directly with a general extract buffer overnight at 4 °C. Following washing with TBS-T 6 times, the microplate was blocked with TBS-T containing 3% BSA solution and incubated for 2 h at room temperature. The wells were washed 3 times with TBS-T. The microplates were then coated with purified scFv proteins diluted in a blocking buffer at a concentration of 0.1 μg/μL for 1 h at room temperature. Following rinsing with TBS-T, anti-His tag mouse monoclonal antibodies (R&D Systems) and HRP-conjugated secondary anti-mouse IgG (Cell Signaling Technology) were applied to detect scFv proteins. After washing 6 times with TBS-T, TMB substrate solution (Agdia) was added for 30 min, and the enzymatic action was stopped with the addition of 1 M sulfuric acid. The absorbance was read at OD 405 using a microplate spectrophotometer (Tecan Sunrise, Tecan, Switzerland). cloning and soluble expression of homodimer scfv. The synthesized gene was cloned into maltose-binding protein (MBP), encoding a plasmid vector, pDEST-periHisMBP (Addgene, 11086, Cambridge,

SPLCV_F7_scFv_26b_F
CCA TGG ATC TGG TGC AGT CTG GGG   SPLCV_F7_scFv_26b_R  GAA TTC GGG GTG ACC TTG GTC CCT C   SPLCV_G7_scFv_26b_F  CCA TGG ATG TGC AGT CTG GGG CTG A   SPLCV_G7_scFv_26b_R  GAA TTC GGG GCT CCA GTC TGC TGA T   Table 2. Nucleotide sequence of the primer sets used for cloning of scFvs.  Table 3. Nucleotide sequence of the primer sets used for MBP fusion expression.
MA, USA) 62 , by gateway cloning 63 . The BP reaction was carried out with BP Clonase ™ II Enzyme mix (Invitrogen, Carlsbad, CA, USA) and pDONR221 (Invitrogen) as a donor plasmid. The proteinase K was added and, recombinant plasmids were transformed into DH5α and selected on the LB agar plates containing kanamycin (50 μg/ mL). To produce expression clones, additional recombination was performed by LR Clonase ™ II Enzyme mix (Invitrogen) with entry clones and pDEST-periHisMBP. After addition of proteinase K, the expression clones were transformed into DH5α and selected on LB agar plates containing ampicillin (50 μg/mL) selection medium. The expression clone, named pPHM-F7-di-scFv (Fig. 5A), was analyzed by a DNA sequencing service (Macrogen, Seoul, Republic of Korea). Bivalent F7 scFv protein was expressed in BL21 (DE3) pLysE cells (Merck). An expression test was then carried out as for the protein A fusion clone. For purification, the supernatant products of scFv expression were applied to a nickel-nitrilotriacetic (Ni-NTA) agarose column (Thermo Scientific, Waltham, MA, USA) 64 . SDS-PAGE (10%) was performed to identify and separate proteins. Western blot analysis was conducted as described by 65 using primary anti-His tag mouse monoclonal antibodies (1:5000; R&D systems, Minneapolis, MN, USA) and HRP-conjugated secondary anti-mouse IgG (Cell Signaling Technology) to detect target proteins.
functional analysis of bivalent scfv. To determine the binding activity of bivalent F7 scFv, indirect ELISA was carried out. Sweet potato samples were coated on a 96-well microplate directly with GEB at 4 °C for overnight in a humidified container. After washing six times with TBS-T, the microplate was blocked with TBS-T containing 3% BSA solution and incubated at room temperature for 2 h. The wells were rinsed three times with TBS-T. The microplates were then coated with purified bivalent scFv serially diluted in blocking buffer (five-fold dilution from 300 μg/mL) at room temperature for 1 hour. The serially diluted F7 scFv also used to compare. Each concentration of scFv protein was determined after calculation based on the values measured with a spectrophotometer with the molecular weight of the scFv protein and coefficient factor. We used estimates based on the values measured with a spectrophotometer to determine the protein concentration Following rinsing with TBS-T, anti-His tag mouse monoclonal antibodies (1:1000; R&D systems) and HRP-conjugated anti-mouse IgG antibodies (1:1000; Cell Signaling Technology) were treated for detection. After six more rounds of washing, TMB substrate solution (Agdia, Evry, France) was added for 30 min, and the action was stopped by the addition of 1 M sulfuric acid. Absorbance was read at 405 nm against a reference wavelength of 620 nm (A450-A620) using a microplate spectrophotometer.