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

Abstract

The recombinant PET-N plasmid, which includes the N gene of Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21(DE3) and expressed by 1.0 mmol/L IPTG at 37 ℃. The indirect ELISA for detecting TGEV nucleocapsid protein antibody was established after the reactionogenicity of the recombinant protein was proved by Western blot. The optional working circumstances for the ELISA are as follows: antigen concentration 15 μg/mL, serum dilution 1∶40, blocking solution 0.5% FBS, serum sample incubated for 90 min, concentration of HRP-spa 1∶5 000 incubated for 60 min, the substrate incubated at room temperature for 10 min, and the threshold value of ELISA OD450≥0.35 tried out with chess titration. Sensitivity , specificity and concordance of this method were 93.5%, 93.8% and 93.5%, respectively compared to Svanova TGEV/PRCV antibody diagnosis kit.