Nano glo luciferase kit

The inactivated ASFV-positive and -negative serum samples were kept and provided by the ASF Regional Laboratory of China (Lanzhou) (Shi et al., 2021 (link)), which including inactivated 138 serum samples collected from pig farms in Henan Province after ASFV was introduced into China and 110 serum samples collected from pig farms in Gansu Province before 2015. The sera against four swine pathogens (classical swine fever virus, CSFV; porcine reproductive and respiratory syndrome virus, PRRSV; type-2 porcine circovirus, PCV2; porcine epidemic diarrhea virus, PEDV) were provided by Lanzhou Veterinary Reseach Institute. A bicinchoninic acid (BCA) protein assay kit was purchased from Solarbio Life Sciences, China. INgezim PPA COMPAC (5 plates kit, Lote/Batch: 050819) was purchased from Ingenasa (INGEZIM 11.PPA.K3; Spain). Nano-Glo luciferase kit was purchased from promega (Promega, N1110). HEK293T cell are cultured in DMEM containing 10% FBS at 37°C/5% CO2.

Luciferase analysis was performed as previously described in Que et al. (22 (link)). Briefly, the nano-luciferase (NL) activities of HBV-NL transfectants were determined using Nano-Glo Luciferase Kit (Promega) and PowerScan HT (BioTek). The NL gene was inserted into the core region of the HBV genome in the HBV-NL chimeric construct, and the NL activity represents pgRNA expression in the transfected hepatocyte cell line (22 (link), 23 (link)). Samples were duplicated or triplicated within each biological replicate.

Serum samples from mice immunized with NiV G-protein (n = 9) and negative mice (n = 9) were prepared in our laboratory. Sera from NiV-, Ebola virus (EBOV)-, Rift Valley fever virus (RVFV)-, Crimean–Congo hemorrhagic fever virus (CCHFV)-, and West Nile virus (WNV)-positive horses were provided by Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Sera from patients infected with Chikungunya virus (CHIKV) and Dengue virus (DENV) were provided by Guangdong Center for Disease Control and Prevention (China), and normal human and hepatitis C virus (HCV) sera were provided by Nan Fang Hospital, Southern Medical University, Guangdong, China. These sera were kept in our laboratory and were validated in previous studies. A bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime (Shanghai, China). The pNLF1-N (Promega, Madison, WI, USA, N1351) plasmid was maintained in our laboratory for subsequent vector construction. A Nano-Glo luciferase kit (Promega, N1120, USA) was purchased from Promega. HEK 239T cells were maintained in our laboratory and cultured in 5% CO₂ at 37 °C using Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum.