PROKARYOTIC EXPRESSION, PREPARATION AND APPLICATION OF ANTI-PRDM14 POLYCLONAL ANTIBODY OF MEDAKA (ORYZIAS LATIPES) PRDM14

PRDM14, a member of PRDM family, plays an important role in germ cells and the maintenance of pluripotency in embryonic stem (ES) cells in mice and humans, but its functions in other species are largely unknown. Medaka (Oryzias latipes) is an excellent fish model to study the developmental biology and stem cell pluripotency. In order to study the potential function of medaka prdm14, the recombinant medaka Prdm14 protein was expressed and used to generate the rabbit anti-Prdm14 polyclonal antibody. First, the recombinant expression vector pET32a-prdm14^∆600 was constructed by inserting a part of prdm14 CDS into pET32a vector. Second, the vector was transformed into Escherichia coli Rosetta (DE3) and the protein (60 kD) was induced by isopropyl-β-d-thiogalactoside (IPTG). Third, the protein was purified and used as the antigen to immunize rabbit (Oryctolagus cuniculus). Last, six weeks after injection, the antiserum was collected, and the antibody titer and specificity were detected by ELISA and Western blot. The re-sults showed the recombinant Prdm14 could be highly induced at 37℃ with 0.6 mmol/L IPTG for 3h. The polyclonal anti-Prdm14 antibody reacted specifically with Prdm14 in medaka adult tissues or Prdm14: EGFP fusion protein ectopi-cally expressed in HepG2. In conclusion, we generated a polyclonal antibody for medaka Prdm14, which provides a powerful tool to analyze the underlying function of prdm14 in fish stem cells.