ScFvs targeting the PEDV N protein were previously generated and described (36 (link)). Briefly, a porcine recombinant scFv library was constructed and four scFvs targeting the PEDV N protein were generated. ScFvs were amplified using upstream primers containing the restriction enzyme SalI (Thermo Fisher Scientific, Waltham, MA, USA) and downstream primers with HindIII (Table 1). PCR thermal cycling conditions were: 95°C for 5 min; 35 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 55 s; and 72°C for 5 min. PCR products were purified and cloned into the shuttle vector pAdTrack-CMV (Addgene, Watertown, MA, USA). The linearized recombinant vector pAdTrack-CMV-scFv, digested with the restriction enzyme PmeI, was transformed into BJ5183 competent cells which contained the adenoviral backbone vector pAdEasy-1. Recombinant adenovirus plasmids in BJ5183 cells were selected using streptomycin and kanamycin, and termed pAdEasy-scFv (Addgene). After this, pAdEasy-scFvs were linearized with PacI and transfected into HEK293 cells using TurboFect Transfection Reagent (Thermo Fisher Scientific). Until fluorescence and cytopathic effects appeared, cells were freeze-thawed three times to generate the recombinant adenovirus rAdV-scFv. A recombinant rAdV-wild-type adenovirus was used as a control.
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