Development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus

Lyoo, Kwang-Soo; Yeom, Minjoo; Kim, Jungho; Kim, Donghyuk; Ha, Gunwoo; Na, Woonsung; Le, Van Phan; Song, Daesub

To express the PEDV-N protein, cDNA was generated by RT-PCR using RNA extracted from the PEDV-DR13 strain (accession no. JQ023161) provided by Green Cross Veterinary Product (Suwon, Korea). The N protein gene was amplified using primers 5-GGA TCC ATG GCT TCT GTC AGC TTT-3 and 5-GTC GAC TTA ATT TCC TGT GTC AAA-3. The PCR products were cloned into the BamH I/Sal I restriction site (underlined) of the pFastBac vector (Invitrogen, Carlsbad, CA, USA), and this recombinant plasmid was transformed into Escherichia coli DH10Bac containing a baculovirus shuttle vector (Bacmid, Invitrogen). The recombinant Bacmid DNA carrying the PEDV-N protein gene was transfected using Cellfectin Reagent (Invitrogen) into Spodoptera frugiperda (Sf-9) cells. Expression of the N protein gene of PEDV was confirmed by immunoblot analysis using SDS-PAGE. For immunoblotting, Sf-9 cells expressing N protein of PEDV were lysed in radioimmunoprecipitation assay buffer (RIPA) lysis buffer (1 per cent Triton X-100, 1 per cent deoxycholate and 0.1 per cent SDS). The proteins were then separated on 10 per cent SDS polyacrylamide gels, and then transferred onto a nitrocellulose membrane. The membrane was blocked in 5 per cent skimmed milk buffer and incubated with polyclonal anti-PEDV from mice immunised with PEDV at 4°C overnight. For protein detection, the membrane was incubated with antimouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody and visualised using the ATTO Ez-Capture II system (ATTO, Tokyo, Japan). Recombinant baculoviruses expressing PEDV-N protein were propagated in Sf-9 cells. The recombinant N protein was purified by affinity chromatography using Ni-NTA resin (Qiagen, Hilden, Germany) according to the manufacturer's protocol.