Immunodiagnostic Applications of Polyclonal Antibodies that Recognize the Southern Bean Mosaic Virus Movement Protein

Plant viruses invade and spread through their hosts either via the short-distance movement which involves the spreading of the virus from the infected cell to the adjacent cell, or, via the long-distance movement where the virus spreads through the vascular system and infects cells from different regions of the plant. In the first case, viruses penetrate the cell wall through the plasmodesmata, which are channels unique in the plant kingdom, typically with diameters of about 25-30 nm, that interconnect cells [1-3]. Different species of plant viruses encode specialized proteins referred to as Movement Proteins (MPs) that dilate the plasmodesmata permitting the passage of complexes formed by viral nucleic acids and proteins or even of whole virions [4].


Introduction
Plant viruses invade and spread through their hosts either via the short-distance movement which involves the spreading of the virus from the infected cell to the adjacent cell, or, via the long-distance movement where the virus spreads through the vascular system and infects cells from different regions of the plant. In the first case, viruses penetrate the cell wall through the plasmodesmata, which are channels unique in the plant kingdom, typically with diameters of about 25-30 nm, that interconnect cells [1][2][3]. Different species of plant viruses encode specialized proteins referred to as Movement Proteins (MPs) that dilate the plasmodesmata permitting the passage of complexes formed by viral nucleic acids and proteins or even of whole virions [4].
There is no common movement mechanism that is shared by all viruses and viruses, independent of their taxonomy, may resort to different strategies depending on the MP sequence, participation of virus encoded proteins, type of infected host and interaction with host factors [5][6][7]. MPs, as well as replicases are non-structural proteins that are generally produced in very low concentration in plants thus making it difficult to identify, localize and purify them [8].
The aim of this study is the production of antibodies for the efficient detection of low concentrations of MPs from the Southern Bean Mosaic Virus (SBMV) which belongs to the Sobemovirus genus. This virus has a strict host range and some of them infect common bean and soy, which are of economic relevance. SBMV is the type species from this genus, has isometric particles (28-30 nm) and contains genomic RNA of 4.0-4.5 Kb with positive polarity and a capsidal protein with a molecular mass of 29-30 kDa.
The SBMV genome consists of four Open Reading Frames (ORFs) with sequence overlaps among them. ORF 1 encodes a movement protein with 17 kDa [9].

Virus Purification and RNA Extraction
SBMV was purified from infected leaves of Phaseolus vulgaris following the method of [10]. Viral RNA was extracted using the initial denaturation at 95 °C for 10 min followed by 30 consecutive cycles of denaturation at 95°C for 30 s, annealing for 1min at 55°C, extension at 72°C for 1 min 30s, then final extension at 72°C for 10 min. Electrophoresis on a 1% agarose was used to confirm the specific PCR product obtained and it was purified using PCR gel purification kit (Qiagen).

Cloning in pGEM-T Easy Vector
The resulting PCR product and pGEM-T Easy vector were ligated at 4°C overnight using T4 DNA ligase to yield the pGEM-T-MP construct that was transformed into Escherichia coli DH5αcells.
To confirm successful cloning, blue white screening colony and PCR was performed. One clone was used for plasmid extraction and the recombinant vector was sequenced by the automatic ABI 377 DNA Sequencer.

Subcloning in pET28a Vector and pMALc2x
Recombinant pGEM-T-MP vector was digested with BamHI and HindIII restriction enzymes and a 500bp insert (ORF) was purified from agarose 1% gel by using gel extraction and purification kit

Expression of Recombinant Protein in E. coli using the pMALc2x Vector
For the expression of the MP protein tagged with MBP, BL21 E.coli cells were transformed with the pMAL-MP construct and incubated in LB broth (with 0.2% glucose and 100 µg/ml ampicillin for selection). The medium was inoculated with an overnight culture (1:100 dilution) and the culture was incubated under constant agitation at 25°C until an OD6000.5 was reached. Subsequently, 0.3 mM IPTG was added and the culture was further incubated either at 37°C or at 25°C for 6 hours.

Purification of the MP-MBP Recombinant Protein
Cells were harvested by centrifugation at 6,000 x g, at 4°C for 20 min and lysed in a buffer containing 20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA (including protease inhibitors: 100 µg/ ml PMSF, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin and 1 µg/ml pepstatin) by sonication for 2 min on ice. The crude extract was isolated by centrifugation of the samples at 15,000 x g at 4°C for 1 hour and was applied on to a column containing an amylose resin pre-equilibrated with the lysis buffer. The recombinant protein was eluted after the addition of the lysis buffer with 10 mM maltose and a final step of gel filtration using a Superdex G75 10/300 GL column, was performed. The presence of the MP through all the steps of expression and purification steps was monitored by SDS-PAGE gels.

Preparation of Polyclonal Antibodies against the Recombinant MP
Antibodies against the recombinant MP were raised in a male New Zealand white rabbit which was injected subcutaneously with 1 mg of the purified recombinant MP-MBP protein in 5 mM Tris-HCl pH 7.5; 100 mM NaCl. Four booster injections were administered with 1 mg of recombinant protein each at an interval of one week.
One week after the final injection, 50 ml of blood was collected and maintained overnight at room temperature. The crude antiserum was collected by centrifugation (4200 x g for 5 min) and the globulin fraction was isolated by three rounds of selective precipitation with ammonium sulfate (40% saturation). After the final round of precipitation, the proteins were dissolved in 20 ml of 5 mM Tris-HCl

Expression of Recombinant Protein in E. coli using the pET28a Vector
For expression of the His-tagged MP, BL21 E.coli cells were transformed with the pET28a-MP construct and incubated in LB broth (100 µg/ml kanamicin for selection). The medium was inoculated with an overnight culture (1:100 dilution) and the culture was incubated under constant agitation at 30°C until an OD 600 0.5 was reached. Subsequently, 0.3 mM IPTG was added and the culture was further incubated either at 30°C or at 18°C for 8 hours.

Purification of His-Tagged MP
Expression of MP using pET28a in E. coli was fraught with difficulties, including poor expression, degradation, and sequestration as insoluble protein in inclusion bodies; therefore it was purified under denaturing conditions and subsequently Spectropolarimeter (Jasco, Tokyo, Japan). For each spectrum, 10 accumulations were performed. Secondary structure percentages for each tested condition were calculated with CONTINLL software of CDPro package, using the reference set of proteins SMP56 [11].

Western Blot Evaluation
To determine the specificity of polyclonal antiserum, MP Histagged that has no MBP (10 µg) was used in immunoassays to make sure that the polyclonal antibody has affinity for the sequence

Western Blot Analysis of Proteins Extracted from Infected Bean Leaves
Following inoculation of bean leaves with the purified virus for different time intervals, the bean leaves were harvested, and 1 gram was homogenized by grinding in 1 ml of 50 mM Tris-HCl, pH 7.5, 9 M urea, 10% SDS, 5% 2-mercaptoethanol. The sample was boiled for 5 min, centrifuged at 10,000 x g for 10 min and 20 µL of the supernatant was applied in 12% SDS-PAGE gels. The proteins were electro-blotted onto a nitrocellulose membrane and submitted for Western blot assays as described above.

Dot Blot
The purified recombinant movement protein was also analyzed

Prediction of Linear Epitopes
Putative linear epitopes of MP of SBMV were predicted using the program BepiPred [12]. The prediction score is based on hydrophilicity and secondary structure prediction from a single sequence.

Virus Purification, RNA extraction and Cloning of ORF1 from SBMV
From infected bean leaves it was possible to purify viral particles with high purity (Figure 1), serving as a source of RNA for cDNA synthesis. In this study, ORF1 of SBMV was cloned into the expression vectors pMALc2x and pET28a.

Expression and Purification of Recombinant MP-MBP
Using pMALc2x it was possible to purify a recombinant movement protein of approximately 60 kDa (Figure 2). This expression vector was chosen not only based on solubility and stability of MBP-MP, but also because MBP might enhance immune responses to vaccine fusion proteins [13,14] facilitating the production of antibodies as it has been demonstrated in mice and rabbits eliciting a strong IgG antibody response against both the dengue virus [15,16] and Plasmodium falciparum [17].  The protein was concentrated to 1mg/mL, the purity was checked by SDS-PAGE gels ( Figure 2) and was used for the production of polyclonal antibodies in rabbits.

Expression and Purification of Recombinant Protein in E. coli using pET28a Vector
This system was successful in expressing the N-terminal Histagged MP that was subsequently purified under denaturing conditions.

Circular Dichroism Spectroscopy of Refolded MP
The refolding of MP was analyzed based on the percentages of secondary structure calculated by the CONTINLL software of the CDPro package, using the reference set of proteins SMP56 [11] and indicate a structural content of 25 % α-helix, 39 % β-sheet, 18 % turns and 18 % random coil (Figure 3). Using the purified

Western Blot
Western blot showed the specificity of the antibodies reacting positively with MP His-tagged (20 kDa), but not with BSA and recombinant C-terminal portion of SBMV polymerase, even 5 times more concentrated than the MP (Figure 4).

Dot Blot
Twenty ng of the refolded His-tagged MP spotted onto strips of nitrocellulose membrane was used to determine the efficiency of the obtained antibody and we found that the antiserum at different dilutions (1:1.000; 1:2.000; 1:4.000; 1:8.000; 1:16.000; 1:32.000) reacted with the recombinant protein. The same assay was performed using denatured and refolded MP and no difference was found between them ( Figure 5). The minimal dilution of antiserum able to react with 20 ng of MP was 1:16000. Analyzing serial dilutions of recombinant MP His-tagged, it was possible to conclude that at least 1.8 ng of protein is detectable using the antiserum diluted 1:2000 ( Figure 6).

Prediction of Linear Epitopes
The Bepipred Linear Epitope Prediction tool was used in order to explain the antibodies recognition of denaturing and native form of MP guided by algorithms that predict immunogenic epitopes.
Four main linear epitopes were predicted (Figure 7). Antibodies may recognize epitopes made up of contiguous (conformational epitope) or non-contiguous (linear epitope) amino acids and according to these results, it is suggested that the MP has surfaceexposed linear epitopes in native state. The advantage of using an antiserum that recognizes both native and denatured proteins is the possibility to use additives that can change the folding of the target protein without disturbing its recognition.

Conclusion
The movement protein was cloned and over expressed in E. coli and was subsequently used as an antigen to produce antibodies against the recombinant protein with high specificity. Since the prepared polyclonal antibody is able to react specifically with MP protein of SBMV under both denaturing and native conditions, it may serve as a tool to identify its sub-cellular localization and can help to understand its role in virus replication. Based on immunoblotting assays of infected bean leaves we conclude that the MP concentration per gram of sample of infected leaves is less than 90 ng or that the MP is degraded during the viral infection cycle.