Production of recombinant chimeric swine PKR-APAF-1 protein and its apoptotic induction on MARC-145 cells

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine that has
adverse effects on the pig industry as a result of direct and indirect loss (Li et al. 2007). Many methods have been applied
to control this important viral disease in swine farms, however outbreaks are still taken place. The use of recombinant
chimeric swine PKR-Apaf-1 protein (rcPAP) generated from human Double-stranded RNA (dsRNA) Activated
Caspase Oligomerizer (DRACO) concept is an alternative way to control swine viruses. This rcPAP consisted of the
Human Immunodeficiency Virus Trans-activator transcription (HIV-TAT) domain, the dsRNA-binding domain of
porcine Protein kinase R (PKR) gene and the caspase recruitment domain (CARD) of the porcine Apoptotic ProteaseActivating Factor-1 (Apaf-1) gene. This study aimed to produce rcPAP using a bacterial expression system and
investigate its biological activity. Recombinant vectors pET-P-A and pQE32-P-A were constructed. The His-tag fusion
proteins were expressed in E. coli and purified under native condition with the HiTrap chelating affinity column. The
soluble rcPAP produced from the pET-P-A plasmid with a yield of 12.32 mg per liter of bacterial culture media at 25
°C for 18 h were 2.4-fold higher than the pQE32-P-A plasmid and were selected for in vitro study. The purified protein
reacted with the mouse anti-rcPAP polyclonal antibodies. The rcPAP (80 μg/mL) induced apoptosis in PRRS virusinfected MARC-145 cells at 48 hpi by increasing the monkey active caspase-3 value by 17.9 fold was higher (9.150 ±
0.008 vs 0.510 ± 0.003 ng/mg protein) when compared to uninfected control. The bioactivity in vitro indicates this
established protein as a prospective molecule for research to control of PRRS virus infection.