Production optimization of porcine reproductive and respiratory syndrome (prrs) virus nucleocapsid protein in nicotiana benthamiana

Abstract

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is a considerable pathogen to occur disease in pigs and effect to economic on the swine industry around the world including Thailand. Routine observation, rapid diagnosis of PRRSV infection is essential for effective monitoring and disease control. The serological assays using highly conserved viral nucleocapsid (N) protein are widely used for the detection of antibodies to the PRRSV. Accordingly, this study was aimed to produce the N-protein of the PRRSV in the plant system by using Nicotiana benthamiana. The N-protein gene was designed into four constructs that contained with and without the plant signal peptide including the different locations of poly His-tag and cloned in a geminiviral vector, pBYR2e-K2Md, for transient expression in N. benthamiana. The conditions of expression such as effective gene construct, leaf harvesting time (dpi), and Agrobacterium cell density (OD600) were optimized for maximal protein production. Further, by using a western blot assay, the results of protein expression of all constructs indicated N-protein size with an approximate molecular weight of 38 kDa. However, the construct that consists of the plant signal peptide with poly his-tagged in the N-terminus shows a higher-level expression compared to the other constructs. Subsequently, this construct was optimized for the high-level expression at 4 dpi and 0.6 of the Agrobacterium cell density. Therefore, this proof-of-concept study indicated that plant-produced N-protein can be used as an alternative production platform to produce recombinant PRRSV antigens for the diagnosis of PRRSV infection.