Magic™ Anti-His tag antibody, clone JKU.J0

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.

Background Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). Methods The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed inE. colias an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. Results ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. Conclusion A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.