Folia Microbiol. 53 (5), 438–442 (2008)
http://www.biomed.cas.cz/mbu/folia/
Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus Y J. FOLWARCZNAa,c, H. PLCHOVÁa, T. MORAVECa, H. HOFFMEISTEROVÁa, P. DĚDIČb, N. ČEŘOVSKÁa aIntitute of Experimental Botany, Academy of Sciences of the Czech Republic, 160 00 Prague, Czech Republic
e-mail
[email protected] bPotato Research Institute, 580 01 Havlíčkův Brod, Czech Republic cDepartment of Biochemistry, Faculty of Science, Charles University, 128 40 Prague, Czech Republic
Received 18 January 2008 Revised version 6 May 2008
ABSTRACT. The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.
Abbreviations CP DAS IC IPTA PAGE PBS
coat protein double antigen sandwich (ELISA) immunocapture indirect plate trapped antigen (ELISA) polyacrylamide gel electrophoresis phosphate-buffered saline
PCR PVY RT SDS WBA
polymerase chain reaction potato virus Y reverse transcription sodium dodecyl sulfate Western blot analysis
PVY is one of the most common viral pathogens found in potato, and is the type member of the genus Potyvirus (family Potyviridae), being distributed world-wide and causing losses in the form of reduced yield or quality of potato crop. PVY has long flexuous rod-shaped particles; its genome consists of a single-stranded, positive-sense RNA molecule of ≈9.7 kb, with a VPg protein attached covalently to its 5´ end and a polyA tail at its 3´ end (Shukla et al. 1994). RNA encodes a single large polypeptide that is cleaved by three virus-encoded proteinases into nine products (from the 5´ to the 3´ end of the RNA: P1, Hc-Pro, P3, 6K1, CI, 6K2, NIa, NIb and CP (Dougherty and Carrington 1988). Potato virus strains are commonly subdivided into three main groups, viz. PVYN, PVYO and PVYC (de Bokx and Huttinga 1981). PVYN isolates induce systematic venial necrosis symptoms on Nicotiana tabacum cv. Xanthi leaves and very mild mottling with occasional necrotic leaves on potato. PVYO isolates induce mottling and mosaic symptoms on tobacco plants and mild to severe mosaic and leaf drop on potato. PVYC isolates induce stripple streak symptoms on some potato cultivars. The main objective of this paper is to evaluate the possibility of using a recombinant PVY CP for antiserum production and diagnostics. MATERIALS AND METHODS Virus source and IC-RT-PCR. We used the PVYN Wilga (PVYN-W) isolate as a virus source for recombinant antigen design. All PVY isolates used were kindly provided by the Institute for Potato Research (Havlíčkův Brod, Czechia). cDNA of PVY RNA was obtained by IC-RT-PCR as follows. The tubes were coated with 100 μL anti-PVY IgG (1 μg/mL) (PRI Wageningen, The Netherlands) in coating buffer (in mmol/L: NaHCO3 35, Na2CO3 15, NaN3 3) for 3 h at 37° C. The wells were then washed (4 × 150 μL PBS + T) and 100 μL of the homogenate of PVY infected leaves in conjugate buffer (1 : 10, 1 L PBS + T, pH 7.4, 0.2 % ovalbumin, 0.5 mmol/L polyvinylpyrrolidon) was added. The samples were incubated overnight at 4 °C and washed again 4× with PBS + T. Then the reverse transcription and amplification with Superscript II (Promega) and
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Taq polymerase (Fermentas) were done according to Pečenková et al. (2005). The RT and subsequent PCR were done using PVY specific primers CAT AAA GCT TGC AAA TGA CAC AAT CG CAT AAG ATC TTC ACA TGT TCT TGA CT
as a forward primer and as a reverse primer,
based on the sequence of PVYN-W coat protein available in GenBank database under accession no. Z70238 (Chachulska et al., unpublished results). Primers contained HindIII (forward) and BglII (reverse) restriction sites, which were relevant for further experiments. The PCR was carried out in iCycler (BioRad) for 30 cycles: denaturation (30 s 94 °C), annealing (30 s 56 °C) and elongation (1 min 72 °C). Sequencing. The 824-bp fragment was cloned into pMPM-A4Ω (ampicillin selection; Čeřovská et al. 2002) using Hind III and BglII sites and sequenced using ALFexpressII Sequencer with the AutoRead Sequencing Kit (AP Life Science, Sweden). The sequence analyses were done using programs available on ExPASY server (Gasteiger et al. 2003). The deduced amino acid sequence was aligned with the PVY sequence available in GenBank under accession no. Z70238. Bacterial expression of CP gene and preparation of CP fractions. Expression of CP gene was performed in E. coli strain MC1061 which was evaluated by our previous research as the most suitable. To produce the recombinant PVY CP, 5 mL of an overnight culture was added to 500 mL of fresh LB with ampicillin (0.1 mg/mL), the culture was grown to the absorbance A600 of 0.6 and then induced with arabinose (SigmaAldrich) (final concentration of 0.2 %) overnight at 37 °C. Cells were then harvested by centrifugation (3450 g, 15 min, 4 °C) and stored at –80 °C until used. The pellet obtained from the culture was resuspended in 100 mL of 20 mmol/L Tris-HCl (pH 7.5) with 0.2 mg/mL lysozyme (Sigma) and 100 μg/mL DNAse–RNAse (Roche) and incubated (20 min, 37 °C). After disintegration (glass homogenizer, on ice) the lysate was centrifuged (14 926 g, 15 min, 4 °C). The supernatant containing main part of CP was purified by high-speed centrifugation through 30 % sucrose cushion (90 000 g, 2 h, 4 °C; 27 000 RPM Beckman Ti 50.2 rotor, USA) (Čeřovská et al. 1997). Protein concentration (2 mg/mL) was determined by Bradford’s (1976) method. SDS-PAGE and WBA. A 1 mL aliquot of cultured bacterial cells was collected, centrifuged (14 000 g, 1 min), resuspended in 100 μL of Laemmli buffer, boiled for 2 min and 10 μL aliquots were loaded on 12 % polyacrylamide gel containing SDS (Laemmli 1970). Proteins separated by SDS-PAGE were electroblotted to a nitrocellulose membrane (0.45 μm; Schleicher & Schuell Protran) in semidry system (Omni-Trans apparatus; Omnibio, Czechia) according to Hirano and Watanabe (1990). The membrane was stained nonspecifically with Ponceau S (Sigma-Aldrich, USA), incubated for 1 h in 4 % bovine serum albumin in PBS + T and then washed 4× in PBS + T. Recombinant proteins were detected with rabbit anti-PVY (PRI Wageningen) and the polyclonal anti-rabbit conjugate with alkaline phosphatase was used as a second antibody (SWAR–AP, Sigma-Aldrich; diluted 1 : 30 000 in conjugate buffer). The bands of interest were visualized by reaction with a substrate, 5-bromo-4-chloro-3-indolylphosphate–nitro blue tetrazolium tablets (Sigma) according to Sambrook et al. (1989). ELISA. Individual PVY CP fractions from different steps of the purification process were subjected to IPTA ELISA according to Čeřovská et al. (2003). We tested 9 isolates of PVY in the natural host Solanum tuberosum (4 of PVYN-W, 5 of PVYN) and 11 isolates in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 (6 of PVYNTN, 2 of PVYN, 3 of PVYO) (Table I). Plates were coated with antigens diluted in standard carbonate coating buffer (pH 9.6) and incubated overnight at 4 °C. The ELISA plates were washed with PBS + T 4×. Commercial (PRI Wageningen) anti-PVY IgG (5 μg/mL) in conjugate buffer was added and incubated (2 h, 37 °C). Anti-rabbit IgG conjugated with alkaline phosphatase (SWAR–AP; Sigma-Aldrich) diluted 1 : 30 000 in conjugate buffer) was added and incubated (3 h, 37 °C). The plates were then washed and substrate, 1 mg/mL 4-nitrophenyl phosphate in 0.1 mol/L diethanolamine buffer (pH 9.8), was added. Antisera production. The antisera against the bacterially expressed PVY CP were prepared in New Zealand rabbits by three subcutaneous and one intramuscular injections of the purified coat protein in 3-week intervals. The purified protein was emulsified with an equal volume of Freund’s complete adjuvant for the first injection and with Freund’s incomplete adjuvant for subsequent two injections; for the last injection the antigen was diluted in PBS. The rabbits were bled two weeks after the last injection. The serum fractions were collected and stored at –20 °C. The immunoglobulin fraction was obtained from the antisera using caprylic acid fractionation (Steinbuch and Audran 1969).
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Table I. Comparison of specificity of anti-PVY CP antibodies and commercial polyclonal antiPVY antibody by IPTA ELISA (absorbance A405)a
Isolate of PVY
Originb
Antibodies anti-PVY CPc
anti-PVYd
Host plante
PVYO 19 33 1427 Ecosse Irlande Ke 49 Le 88 LW
CZ CZ CZ CZ FR CZ CZ PL
0.00 0.13 0.22 0.63 0.79 0.16 0.14 0.69
0.74 0.77 0.15 0.30 0.24 1.19 1.75 0.77
N.t. N.t. N.t. S.t. S.t. N.t. N.t. S.t.
1.64 0.38 0.63 1.03 1.72 0.27 0.17 0.99 0.80 0.48 0.29 0.46
N.t. S.t. S.t. N.t. S.t. N.t. S.t. N.t. S.t. S.t. S.t. S.t.
0.85 0.73 0.67 0.14 0.59 0.28 0.38
N.t. N.t. N.t. N.t. N.t. N.t. N.t.
PVYN 8 B 203 Carrera Gu Irlande LH 58 Lukava Nord 242 Ramos Saturna Saturna 48 Velox
CZ FR CZ CZ FR CZ CZ CZ CZ CZ CZ CZ
0.12 0.59 0.62 0.00 1.10 0.00 0.00 0.21 0.37 0.47 0.36 0.43 PVYNTN
H Igor LB-Ranka Lukava Nicole Orleans Vital
FR FR CZ CZ CZ FR FR
0.87 0.47 1.30 0.13 1.00 0.54 1.03 PVYN-W
Angela Ditta Ramos Velox
CZ CZ CZ CZ
0.49 0.41 0.32 0.57
0.59 0.37 0.68 0.66
S.t. S.t. S.t. S.t.
Healthy Healthy
– –
0.10 0.10
0.11 0.09
N.t. S.t.
aVarious cultivars of S. tuberosum, infected with various strains of PVY were tested. bCZ – Czechia cSD = 0.298 dSD = 0.239 FR – France PL – Poland eN.t. – Nicotiana tabacum S.t. – Solanum tuberosum
RESULTS Cloning of PVY CP gene, expression and purification of PVY CP. Primers for CP amplification were designed with restriction sites HindIII and BglII. The 824-bp PCR product containing CP coding region was cloned into pMPM-A4Ω, which allows tightly regulated expression from arabinose-induced pBAD promoter. The above described construct was used to transform E. coli. After optimization of cultivation (time, temperature, and inductor concentration) several clones containing the recombinant vector were isolated and tested for PVY CP expression. The noninduced clones produced much lower but visible amounts of the protein while in nontransformed E. coli no such protein was detected.
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Among several methods of isolation of a specific fraction from protein bacterial lysate we chose the simple fractionation for its easy performance. The highest content of recombinant PVY CP was found in soluble cytoplasmic fraction; it was proved by comparison of all fractions by ELISA testing. Production of polyclonal antisera to a recombinant PVY. The anti-PVY CP sera had titres of 1 : 2000 when tested in an indirect ELISA with purified protein. The antisera gave strong reaction with homologous recombinant antigen in IPTA ELISA, while no reaction was obtained using DAS ELISA (data not shown); here we were not able to detect the reaction with plant materials (natural host S. tuberosum, laboratory host N. tabacum), in IPTA ELISA we obtained a significant difference in absorbance A405 between the healthy and infective materials after overnight incubation with the substrate. Our polyclonal antibodies were able to detect the whole strain spectrum of PVY. WBA. The polyclonal antibodies reacted with the homologous recombinant PVY CP in WBA. Western blot revealed a strong band at a position corresponding to M ≈30 kDa, which is the expected molar mass of PVY CP. We did not detect specific reaction with the cells from the culture transformed with the empty pMPM-A4Ω (Fig. 1).
Fig. 1. Immunochemical detection of antigens with anti-PVY CP antibodies by WBA. Both the homologous and heterologous antigens were detected by our polyclonal antibodies; M – Protein Elfo Page RulerTM prestained protein marker, A – bacterial expression of PVY CP, B – pMPM-A4Ω, 1 – S. tuberosum Ramos, healthy, 2 – N. tabacum cv. Samsun, healthy, 3 – PVY infected S. tuberosum Ditta, 4 – PVY infected N. tabacum cv. Samsun.
No reaction was seen with the extracts from leaves of healthy S. tuberosum cv. Ramos. A slight reaction was apparent with the infected leaves of S. tuberosum cv. Ramos and cv. Marena. Stronger reactions were detected with the infected leaves of S. tuberosum cv. Ditta and cv. Satina. The stronger reaction which was obtained with these two cultivars relative to that obtained with cv. Ramos and cv. Marena could be explained by their higher virus titer. DISCUSSION The expression of viral CP in E. coli, followed by purification and polyclonal antiserum production, has been reported for a number of different plant viruses. These antisera have mostly been used successfully for plant virus detection by WBA and IPTA ELISA but they failed in DAS ELISA (Nikolaeva et al. 1995; Jelkman and Keim-Konrad 1997). According to us the failure to get positive results, when the obtained antibodies were used for coating ELISA plates (DAS ELISA), suggests that our antibodies may not recognize native epitopes but only epitopes, which are affected by some denaturation steps (e.g., binding to the surface of ELISA plates in binding buffer with high pH or SDS-PAGE in case of WBA), as this binding may affect their conformation (Korimbocus et al. 2002). However, there are some reports, in which antibodies produced against recombinant viral proteins of tomato spotted wilt virus (Vaira et al. 1996) and grapevine leafroll-associated closterovirus-3 (Ling et al. 2000) were found to be effective in detecting the viruses by DAS ELISA. Petrzik et al. (2001) have demonstrated the successful use of an antiserum against Prunus necrotic ringspot virus recombinant CP for the detection of the virus by DAS ELISA. Our results with CP are in agreement with the results obtained for antibodies raised against recombinant potato virus A CP, potato mop-top virus CP (Čeřovská et al. 2002, 2003; cf. Čeřovská et al. 2007), and potato mop-top virus CP described by Helias et al. (2003) who obtained only one serum reacting in DAS ELISA (which gave in comparison with commercial polyclonal antiserum 10× lower absorbance values). In contrast to the conventional method of antigen preparation (which can take two or more months), the purification procedure described by us can be completed in 3 d. Five-hundred mL of bacterial culture fluid yielded ≈2 mg of PVY CP, i.e. an amount sufficient for a repeated immunization of laboratory animals for antisera preparation. We believe that the recombinant viral CPs expressed in bacterial cells have great
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potential as an alternative source of antigens for raising specific antibodies to plant viruses. They can be produced in large quantity and can be manipulated or modified as needed for specific use. This research was supported by grant no. 1M06030 of the Ministry of Education, Youth and Sports of the Czech Republic. The authors wish to thank Ms. R. Hadámková, D. Cibochová, J. Dědičová and I. Zavadilová for their excellent technical assistance. REFERENCES BRADFORD M.M.: A rapid and sensitive method for the quantification of micrograme quantities of protein utilizing the principle of protein dye binding. Anal.Biochem. 72, 248–254 (1976). DE BOKX J.A., HUTTINGA H.: Potato virus Y. Descriptions of Plant Viruses No. 242. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew (UK) 1981. ČEŘOVSKÁ N., FILIGAROVÁ M., ŠUBR Z.: Optimization of purification procedure for potato virus Y strain NN. Acta Virol. 41, 47–49 (1997). ČEŘOVSKÁ N., MORAVEC T., VELEMÍNSKÝ J.: Polyclonal antibodies to a recombinant coat protein of potato virus A. Acta Virol. 46, 147–151 (2002). ČEŘOVSKÁ N., MORAVEC T., ROSECKÁ P., DĚDIČ P., FILIGAROVÁ M.: Production of polyclonal antibodies to a recombinant coat protein of potato mop-top virus. J.Phytophatol. 151, 1–6 (2003). ČEŘOVSKÁ N., PEČENKOVÁ T., FILIGAROVÁ M., DĚDIČ P.: Sequence analysis of the Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov. Folia Microbiol. 52, 61–64 (2007). DOUGHERTY W.G., CARRINGTON J.C.: Expression and function of potyviral gene products. Ann.Rev.Phytopathol. 26, 123–143 (1988). GASTEIGER E., GATTIKER A., HOOGLAND C., IVANY I., APPEL R.D., BAIROCH A.: ExPASy. The proteomics server for in-depth protein knowledge and analysis. Nucl.Acids Res. 31, 3784–3788 (2003). HELIAS V., JACQUOT E., GUILLET M., LEHINGRAT Y., GIBLOT-DUCRAY D.: Production of recombinant potato mop-top virus coat protein in Escherichia coli and generation of antisera recognizing native virus protein. J.Virol.Meth. 110, 91–97 (2003). HIRANO H., WATANABE T.: Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application on an insoluble protein. Electrophoresis 11, 573–580 (1990). JELKMANN W., KEIMKONRAD R.: Immuno-capture polymerase chain reaction and plate-trapped ELISA for the detection of apple stem pitting virus. J.Phytopathol. 145, 499–503 (1997). KORIMBOCUS J., PRESTON S., DANKS C., BARKER I., COATES D., BOONHAM N.: Production of monoclonal antibodies to sugar yellow leaf virus using recombinant read-through protein. J.Phytophatol. 150, 488–494 (2002). LAEMMLI U.K.: Cleavage structural protein during the assembly of the head of bacteriophage T4. Nature 227, 748–753 (1970). LING K.S., ZHU H.Y., JIANG Z.Y., GONSALVES D.: Effective application of DAS-ELISA for detection of grapevine leafroll associated clostervirus-3 using a polyclonal antiserum developed from recombinant coat protein. Eur.J.Plant Pathol. 106, 301–309 (2000). NIKOLAEVA O.V., KARASEV A.V., GUMPF D.J., LEE R.F., GARNSEY S.M.: Production of polyclonal antisera to the coat protein of citrus tristeza virus expressed in Escherichia coli – application for immunodiagnosis. Phytophatology 85, 691–694 (1995). PEČENKOVÁ T., FILIGAROVÁ M., ČEŘOVSKÁ N.: Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus. Protein Expr.Purif. 41, 128–135 (2005). PETRZIK K., MRÁZ I., KUBELKOVÁ D.: Preparation of recombinant coat protein of prunus necrotic ringspot virus. Acta Virol. 45, 61–63 (2001). SAMBROOK J., FRITSCH E.F., MANIATIS T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) 1989. SHUKLA D.D., WARD C.W., BURNT A.A.: The Potyviridae. Cambridge University Press, Cambridge (UK) 1994. STEINBUCH M., AUDRAN R.: The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch.Biochem.Biophys. 34, 279– 284 (1969). VAIRA A.M., VECCHIATI M., MASENGA V., ACCOTTO G.P.: A polyclonal antiserum against a recombinant viral protein combines specificity with versatility. J.Virol.Meth. 56, 209–219 (1996).
