团头鲂转录因子Oct4的原核表达和多克隆抗体的制备  

Prokaryotic expression and polyclonal antibody preparation of Oct4 protein in Megalobrama amblycephala

作　　者：于淼 方健 李玲玉 潘启华 薛亭 邓羽 陈凯 陈天圣 

机构地区：[1]华中农业大学水产学院农业部淡水生物繁育重点实验室,湖北武汉430070 [2]河南师范大学水产学院河南省水产动物养殖工程技术研究中心,河南新乡453007 [3]水产高效健康生产湖南省协同创新中心,湖南常德415000

出　　处：《上海海洋大学学报》2018年第1期8-15,共8页Journal of Shanghai Ocean University

基　　金：国家自然科学基金(31672653;31771648);国家重点基础研究计划(2013CB967700);华中农业大学科技自主创新基金(2013RC014;2662015PY049);河南省自然科学基金(102300410104);河南师范大学青年科学基金(2013qk15)

摘　　要：为进一步分析鱼类干细胞多能性转录因子Oct4的功能,将团头鲂(Megalobrama amblycephala)Oct4进行原核表达并制备了兔抗Oct4多克隆抗体。采用RT-PCR方法从团头鲂卵巢中扩增Oct4基因的部分编码片段,插入载体pET32a中构建重组原核表达载体pET32a-MaOct4;将表达载体转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导、Ni^(2+)亲和柱层析纯化获得分子量约49 ku的可溶性重组蛋白;以纯化的重组蛋白为抗原免疫新西兰兔制备抗体,通过ELISA法测定其效价,Western blot鉴定其特异性。实验结果表明:该重组载体在37℃,0.5 mmol/L IPTG条件下诱导4 h可获得Ma-Oct4重组蛋白的高效表达;制备的团头鲂Oct4多克隆抗体能够分别与纯化的Oct4蛋白、原核表达的Oct4蛋白、团头鲂胚胎中的内源Oct4蛋白以及HepG2细胞中过表达的Ma-Oct4:DsRed融合蛋白特异结合。为后续深入研究团头鲂Oct4在干细胞多能性调控中的作用提供了有效的分子工具。To understand the potential function of pluripotency-associated gene Oct4 in blunt-snout bream（Megalobrama amblycephala）,the present study investigated the prokaryotic expression of the recombinant Oct4 protein and generated the rabbit anti-Oct4 polyclonal antibody.Firstly,the expression vector pET32 aMaOct4 was constructed by inserting the C-terminal coding region of Ma-Oct4 into p ET32a.Then the expression vector was transformed into Escherichia coli BL21（DE3） p Lys S,and the recombinant Oct4 protein was induced by IPTG.After optimization of expression conditions,the protein was largely induced and purified to immunize the New Zealand rabbits.Subsequently,the titer and specificity of the generated antibody were assayed by ELISA and Western blot.The recombinant Oct4 protein was highly induced by 0.5 mmol/L IPTG for 4 h at 37 ℃.The polyclonal anti-Oct4 antibody effectively recognized the purified recombinant Ma-Oct4 antigen,the induced Ma-Oct4 protein in E.coli,the endogenous Ma-Oct4 protein from the fish embryos,and the overexpressed Ma-Oct4： DsRed fusion protein in Hep G2 cells.In conclusion,this research provided an effective antibody to study the potential function of Oct4 in blunt-snout bream.

关 键 词：团头鲂 OCT4 原核表达 多克隆抗体 转录因子 

分 类 号：S917.4[农业科学—水产科学]