cDNA cloning and sequencing
Total RNA from uninfected or APM infected A. polyphaga was extracted using RNeasy extraction kit (Qiagen) as previously described . The Capsid protein 1 cDNA was synthsesized from APM infected A. polyphaga RNA by using the Superscript One-Step RT-PCR with Platinum Taq kit (Invitrogen) with the following primers:
Q5UQL7NcoIF: 5'-GAAGGAGATATACCATGGCAGGTGGTTTACTCCAATTA-3' and Q5UQL7SmaIR: 5'-GATGAGAACCCCCCCCGGGATTACTGTACGCTAATCCG-3'. Underlined are the APM L425 gene specific sequences: nt 560 926 – 560 449 for the forward primer; nt 557 533 – 557 515 for the reverse primer. The resulting 1782 bp cDNA fragment was then cloned into the pIVEX 2.3 expression vector (Roche) at the NcoI and SmaI sites. Recombinant plasmids were selected, purified and then incorporated into the d-Rhodamine Terminator Cycle Sequencing Ready Reaction buffer kit with Amplitaq Polymerase FS (Applied Biosystems). Reaction products were resolved by using an ABI 3100 automated sequencer and sequence analysis was performed using the software package ABI Prism DNA Sequencing Analysis Software version 3.0 (Applied Biosystems). T7 promoter and T7 terminator primers were used as well as two primer pairs targeting internal regions of the cDNA: QUQL7-SF1: 5'-GCTGGCAGTAGTAATTCTGC-3'; QUQL7-SR1: 5'-GCAGAATTACTACTGCCAGC-3'; and QU5QL7-SF2: 5'-GAAGGTAATGATGGTAGAAG-3'; QU5QL7-SR2: 5'-CTTCTACCATCATTACCTTC-3'.
RNA was extracted from uninfected or APM-infected amoebae at 0, 2, 4, 8 and 16 hours post infection. 100 ng of each RNA was submitted to RT-PCR amplification using the SuperScript One-Step RT-PCR kit (Invitrogen) with the Q5UQL7NcoIF/Q5UQL7SmaIR primer pair. cDNA synthesis was performed in one cycle of 30 minutes at 50°C, 3 minutes at 94°C and subsequent PCR reaction with 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, 30 seconds at 72°C, and one cycle of 10 minutes at 72°C. Amplified products were analysed by electrophoresis on 1% agarose gel. GeneRuler 1 kb DNA ladder (MBI-Fermentas) was used as a DNA size marker.
Expression of the recombinant Capsid protein 1
Expression of the Capsid protein 1 was performed using a cell-free translation system , the High Yield RTS 500 Escherichia coli Circular Template kit (Rapid Translation System, Roche). Reactions were performed at 30°C for 24 h, with a stirrer speed of 120 rpm, with 15 μg of recombinant plasmid used as DNA template. The RTS sample was then centrifuged for 10 min at 10 000 rpm and the pellet was resuspended in Laemmli buffer, heated for 5 min at 95°C, resolved by 7.5% SDS-PAGE, followed by Coomassie blue staining or transfer onto nitrocellulose membrane for immunoblot analysis using a anti-Histidine monoclonal antibody. The recombinant Capsid protein 1 was extracted from polyacrylamide gels by the electroelution method using the ElutaTube™ Protein Extraction Kit (Fermentas Life Sciences) according to the manufacturer's protocol and used to immunise mice for the production of monoclonal antibodies.
Monoclonal antibodies production
Three six week-old female BALB/c mice were inoculated three times intraperitoneally at 14-days interval with 2 μg electroeluted protein mixed with 400 μg aluminium hydroxide and 10 μg CpG as previously described . Four days after the last immunisation, spleen cells fusion was performed with X63.Ag 8.653 myeloma cells (2:1) using 50% 1500 polyethylene glycol (Roche). Cells were grown in RPMI medium (Invitrogen) with 15% heat inactivated fetal calf serum (Invitrogen) and hypoxanthine-aminopterin-thymidine selective medium (Invitrogen) at 37°C with 5% CO2. Hybridoma supernatants were screened 10 days after by ELISA using plates coated overnight with 10 μg/ml of APM extract in sodium carbonate buffer 100 mM, pH 9.6. Positive hybridomas were subcloned by limiting dilution and submitted to isotyping using a mouse monoclonal isotyping kit (Sigma-Aldrich). Hybridomas producing the highest mAb titers were then subcloned, expanded, and tested by Western blot analysis using APM extract subjected to 2D gel electrophoresis and transferred onto nitrocellulose.
APM particles were purified through a 25% sucrose gradient and APM extract was prepared for 2-D gel electrophoresis as previously reported . Immobiline™ DryStrips (7 cm, pH 3–10 GE Healthcare) were rehydrated overnight using 125 μl rehydration buffer [8 M urea, 2% (w/v) CHAPS, 60 mM DTT, 0,5% (v/v) IPG buffer (GE Healthcare)] containing 20 μg of solubilized APM proteins and IEF was carried out according to the manufacturer's protocol (IPGphor II, GE Healthcare). Before the second dimension electrophoresis was performed, strips were equilibrated twice in 5 ml equilibration buffer [30% (v/v) glycerol, 3% (w/v) SDS, 6 M urea, 50 mM Tris-HCl, bromophenol blue, pH 8.8] for 15 min. This buffer was supplemented with 65 mM DTT for the first equilibration and with 100 mM iodoacetamide for the second one. The strips were then embedded in 0.5% agarose and the proteins resolved by 10% SDS-PAGE (Mini-Protean III, Bio-Rad). Gels were stained either with silver or transferred onto nitrocellulose for Western blot analysis using Capsid protein 1 specific mAb; anti-Phosphothreonine, anti-Phosphotyrosine or anti-Phosphoserine (10-4 dilution, Sigma) mAbs; or rabbit polyclonal antiserum anti-methylated Lysine (dilution 4.10-2, Biomol GmbH). The detection of glycosylated proteins in 2D gels was performed according to the Pro-Q Emeral 300 glycoprotein stain kit's procedure (Invitrogen) and visualized using a 300 nm UV illuminator.