水生生物学报2017，Vol.41Issue(4)：748-754,7.DOI:10.7541/2017.93
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 ANTI-PRDM14 POLYCLONAL ANTIBODY OF MEDAKA (ORYZIAS LATIPES) PRDM14
摘要
Abstract
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 to study the potential function of medakaprdm14, 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 ofprdm14 CDS into pET32a vector. Second, the vector was transformed intoEscheri-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 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 ofprdm14 in fish stem cells.关键词
青鳉/Prdm14/原核表达/多克隆抗体/干细胞Key words
Medaka (Oryzias latipes)/Prdm14/Prokaryotic expression/Polyclonal antibody/Stem cells分类
生物科学引用本文复制引用
李玲玉,方健,于淼,陈天圣..青鳉prdm14的原核表达、多克隆抗体制备及其应用[J].水生生物学报,2017,41(4):748-754,7.基金项目
国家重点基础研究计划(2013CB967700) （2013CB967700）
华中农业大学自主科技创新基金(2013RC014和2662015PY049)资助[Supported by the State Key Development Program for Basic Research of China (2013CB967700) （2013RC014和2662015PY049）
the Fundamental Research Funds for the Central Universities (2013RC014, 2662015PY049)] （2013RC014, 2662015PY049）
