@article{Vu Anh Tuan Vo_Leksakchai_Kedkovid_Tawatsin_Nuntaprasert_2020, title={Production of recombinant chimeric swine PKR-APAF-1 protein and its apoptotic induction on MARC-145 cells}, volume={50}, url={https://he01.tci-thaijo.org/index.php/tjvm/article/view/243724}, abstractNote={<p><span class="fontstyle0">Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine that has<br>adverse effects on the pig industry as a result of direct and indirect loss (Li </span><span class="fontstyle2">et al</span><span class="fontstyle0">. 2007). Many methods have been applied<br>to control this important viral disease in swine farms, however outbreaks are still taken place. The use of recombinant<br>chimeric swine PKR-Apaf-1 protein (rcPAP) generated from human Double-stranded RNA (dsRNA) Activated<br>Caspase Oligomerizer (DRACO) concept is an alternative way to control swine viruses. This rcPAP consisted of the<br>Human Immunodeficiency Virus Trans-activator transcription (HIV-TAT) domain, the dsRNA-binding domain of<br>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<br>investigate its biological activity. Recombinant vectors pET-P-A and pQE32-P-A were constructed. The His-tag fusion<br>proteins were expressed in </span><span class="fontstyle2">E. coli </span><span class="fontstyle0">and purified under native condition with the HiTrap chelating affinity column. The<br>soluble rcPAP produced from the pET-P-A plasmid with a yield of 12.32 mg per liter of bacterial culture media at 25<br>°C for 18 h were 2.4-fold higher than the pQE32-P-A plasmid and were selected for </span><span class="fontstyle2">in vitro </span><span class="fontstyle0">study. The purified protein<br>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 ±<br>0.008 vs 0.510 ± 0.003 ng/mg protein) when compared to uninfected control. The bioactivity </span><span class="fontstyle2">in vitro </span><span class="fontstyle0">indicates this<br>established protein as a prospective molecule for research to control of PRRS virus infection.</span> </p>}, number={2}, journal={The Thai Journal of Veterinary Medicine}, author={Vu Anh Tuan Vo, Phong and Leksakchai, Thanaporn and Kedkovid, Roongtham and Tawatsin, Achara and Nuntaprasert, Athipoo}, year={2020}, month={Jun.}, pages={137–148} }