Abstract：The transcription factor Nanog plays a core role to maintain the self-renewal and pluripotency of stem cells. In order to study the regulatory function of pig (Sus scrofa) Nanog in pluripotent networks, the recombinant Nanog protein was used to generate the mouse anti-Nanog polyclonal antibody. Firstly, the CDS of Nanog cloned from porcine kidney was connected into pET32a vector to construct the recombinant expression vector pET32a-Nanog. Next, the pET32a-Nanog was transformed into Escherichia coli Rosseta (DE3) and Nanog expression was induced by isopropyl-β-d-thiogalactoside (IPTG). After optimizing the induction time, temperature and IPTG concentrations, the recombinant Nanog protein (57 kD) was obtained. The Nanog protein was purified and then used as the antigen to immunize C57BL/6 mice (Mus musculus). After 6 weeks, the positive antiserum was collected, and the antibody titer and specificity were detected by Western blot and immunofluorescence assay. The results showed that the recombinant Nanog could be highly expressed under the induction conditions of 28 ℃, 8 mmol/L IPTG and 2.5 h; the generated polyclonal anti-Nanog antibody could react specifically with Nanog generated from porcine induced pluripotent stem cells, suggesting that the polyclonal antibody can provide a powerful tool for porcine pluripotent stem cell research.
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