Padtrack cmv

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.

Mouse TRPC7 cDNA [16 (link)] was kindly provided by Professor Yasuo Mori (Kyoto University). For constructing TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TRPC7, restriction endonuclease sites KpnI and NotI were added to 5′- and 3′- end of mTRPC7 cDNA, respectively, by PCR and the cDNA was ligated into the adenoviral shuttle plasmid Blue-pAdTrack-CMV which was modified from pAdTrack-CMV plasmid (Addgene plasmid # 16405). For constructing Tag-TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TagTRPC7 KpnI site plus 3× Flag was added to the 5′- end, 3× hemagglutinin (HA) plus NotI site was added to the 3′- end of mTRPC7 cDNA, the cDNA was then ligated into Blue-pAdTrack-CMV. For constructing TRPC7 knockdown plasmids pAdTrack-U6-shRNA458 and pAdTrack-U6-shRNA459, two sequences of shRNA, shRNA-458 (targeting mouse TRPC7) and shRNA-459 (targeting rat TRPC7), adopted from Genetic Perturbation Platform were synthesized and ligated into the adenoviral shuttle plasmid pAdTrack-U6 at AgeI and XhoI restriction sites. pAdTrack-U6 was modified from pAdTrack plasmid (Addgene plasmid # 16404). The plasmid pAdTrack-U6-shRNAluc harboring shRNA targeting luciferase (shRNA-luc) was constructed as a negative control [31 (link)]. Targeting sequences of these shRNAs were shRNA-458: 5′-GCCGAATCAAACTCGCCATTA-3′, shRNA-459: 5′-GCCAACATTGAGACTGAATTT-3′, shRNA-luc: 5′-CCTAAGGTTAAGTCGCCCTCG-3′.