Molecular Cloning Of Porcine Akirin2 Gene And Its Expression In Vitro

Innate immune system is the first line of host defense that prevents host body from being infected by pathogen. Akirin2 is strictly localized to the nucleus, and it plays an essential role in the innate immune system. Although Akirin2 acts in NF-κB signaling pathway, its mechanism is unclear. Knockout of Akirin2 in mice shows that Akirin2 has an essential function downstream of the Toll-like receptor, tumor necrosis factor and IL-1βsignaling pathways. In vitro transfection demonstrated that over expression of Akirin2 can induce some inflammatory factors expressing, including IL-6. We in silico cloned porcine Akirin2 gene according to human Akirin2 sequence, and then conformed it by experiments. Our results are as following.1. We cloned porcine Arikin2 gene, and analyzed the characteristics and evolution progression of porcine Akirin2 by using bioinformatics software. Our results showed that porcine Akirin2 encoded 203 amino acids, and that the N and C-terminal of Arikin2 protein was highly conserved. There is similarity of 98% between porcine and human Akirin2.2. We subcloned Akirin2 into prokaryotic expression vetor pET-32a, and induced and expressed it in E. coli BL21(DE3) using IPTG. SDS-PAGE analysis showed that we got fusion protein of 43ku. We selected the optional conditions for expressing His-Akirin2 fusion protein with final concentration of IPTG at 0.7 mmol/L for 6h. The fusion protein was purified by HiTrap Chelating HP.3. We inserted porcine Akirin2 into eukaryotic expression vetor pEGFP-C1 which contains a green fluorescence reporter gene and transfected this plasmid into SUVECs (swine umbilical vein endothelial cells) by lipofectamine-2000. Fluorescence microscope showed that fusion protein EGFP-Akirin2 was at the best expression level at 24 hours post transfection. According to the fluorescence position of the cells, we concluded that porcine Akirin2 was localized to the nucleus. Western blot analysis confirmed the expression of EGFP-Akirin2 in the cells in vitro.