青鳉prdm14的原核表达、多克隆抗体制备及其应用

PROKARYOTIC EXPRESSION, PREPARATION AND APPLICATION OF ANTIPRDM14 POLYCLONAL ANTIBODY OF MEDAKA(ORYZIAS LATIPES)PRDM14

青鳉(Oryzias latipes)是研究遗传发育和细胞多能性的重要模式鱼类,为探究prdm14同源基因的潜在作用,实验将青鳉prmd14经原核表达后制备了兔抗Prdm14多克隆抗体。首先,将prdm14基因的部分编码区连接到p ET32a质粒中,构建重组表达载体p ET32a-prdm14?600。随后将重组载体转化至大肠杆菌(Escherichia coli)Rosetta(DE3),经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside,IPTG)诱导表达,获得分子量为60 k D的Prdm14重组蛋白。接着大量诱导蛋白表达并切胶纯化,免疫家兔(Oryctolagus cuniculus),6周后获得阳性抗体,最后通过ELISA和Western blot检测抗体效价及其特异性。结果显示,在37℃、0.6 mmol/L IPTG、诱导3h的条件下,可获得Prdm14重组蛋白的高效表达;制备的兔抗青鳉Prdm14多克隆抗体能够特异性识别青鳉组织中表达的Prdm14蛋白以及在Hep G2细胞中过表达的青鳉Prdm14:EGFP融合蛋白。综上所述,研究首次制备了一种能有效识别青鳉Prdm14的多克隆抗体,该抗体的获得为后续研究prdm14基因在鱼类多能性干细胞中的作用提供了有力工具。

PRDM14, a member of PRDM family, plays an important role in germ cells and the maintenance of pluripo-tency 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 tostudy the potential function of medaka prdm14, the recombinant medaka Prdm14 protein was expressed and used togenerate the rabbit anti-Prdm14 polyclonal antibody. First, the recombinant expression vector pET32a-prdm14？600 wasconstructed by inserting a part of prdm14 CDS into pET32a vector. Second, the vector was transformed into Escheri-chia coli Rosetta （DE3） and the protein （60 kD） was induced by isopropyl-β-d-thiogalactoside （IPTG）. Third, the pro-tein 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 polyclonalanti-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 apowerful tool to analyze the underlying function of prdm14 in fish stem cells.