Prokaryotic expression of nucleoprotein gene of Transmissible gastroenteritis virus and development of ELISA based on the expressed protein

Abstract

AbstractThe recombinant PET-N plasmid, which includes the N gene of the Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21 (DE3), and induced at 37°C with 1.0 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside). An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the TGEV nucleocapsid protein antibody was developed after the reactogenicity of the recombinant protein was demonstrated by Western blot. The operating conditions for the ELISA, an antigen concentration of 15 μg/ml, serum dilution of 1:40, blocking solution of 0.5% fetal bovine serum (FBS), serum sample incubation for 90 min, a concentration of horseradish peroxidase (HRP)-spa of 1:5000, incubated for 60 min, incubation of the substrate at room temperature for 10 min, and a cutoff value for the ELISA OD450⩾0.35, were carried out using a checkerboard titration method. The sensitivity and specificity of this method relative to the Svanova TGEV/PRCV antibody diagnosis kit were 93.5% and 93.8%, respectively.