Anti-TAT monoclonal antibody, clone FQS7232

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 Protease-Activating 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 degrees 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 mu g/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.

Maladaptive plasticity involving increased expression of AMPA-type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell-permeable, high-affinity (similar to 2 nM) peptide inhibitor, Tat-P-4-(C5)(2), of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat-P-4-(C5)(2) disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA-receptor surface expression in vivo. Moreover, Tat-P-4-(C5)(2) administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat-P-4-(C5)(2) as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non-tandem protein-protein interaction domains.