水产学报2017，Vol.41Issue(11)：1649-1659,11.DOI:10.11964/jfc.20160910544
团头鲂转录因子Nanog的原核表达及其多克隆抗体制备
Prokaryotic expression and polyclonal antibody preparation of Nanog protein in Megalobrama amblycephala
摘要
Abstract
To investigate the function of the pluripotency transcription factor Nanog in fish, this study examined the prokaryotic expression of the recombinant Nanog protein and generation of the rabbit anti-Nanog polyclonal antibody in blunt-snout bream.Firstly,MaNanog coding sequence was obtained from the ovary organ, and the full-length open reading frame or the partial fragment containing the homeodomain was inserted into the pET32a vector, respectively.Then the vectors were transformed into Escherichia coli BL21(DE3)pLysS, and the recombinant Nanog proteins were induced by IPTG.After optimization of expression conditions, the MaNanog-S protein was induced on a large scale and applied to antibody generation.Subsequently, the specificity of the antibody was examined by ELISA and Western blot.Results showed that the recombinant Nanog protein was highly expressed with 0.5 mmol/L IPTG induction for 4 h at 37 °C.The polyclonal antibody could identify effectively the induced Nanog protein in E.coli, the endogenous MaNanog protein in adult organs, and the ectopic expressed MaNanog proteins in HepG2.Taken together, these results provided the research methods for prokaryotic expression of protein and preparation of specific antibodies, and also provided an effective tool to investigate the function of Nanog gene in fish.关键词
团头鲂/Nanog/原核表达/多克隆抗体/干细胞Key words
Megalobrama amblycephala/Nanog/prokaryotic expression/polyclonal antibody/stem cell分类
生物科学引用本文复制引用
于淼,方健,李玲玉,薛亭,陈天圣..团头鲂转录因子Nanog的原核表达及其多克隆抗体制备[J].水产学报,2017,41(11):1649-1659,11.基金项目
国家重点基础研究计划(2013CB967700) （2013CB967700）
华中农业大学科技自主创新基金(2013RC014, 2662015PY049) （2013RC014, 2662015PY049）
河南省自然科学基金(102300410104) （102300410104）
河南师范大学青年科学基金(2013qk15) （2013qk15）
河南省水产养殖重点学科资助 National Key Basic Research Program of China (2013CB967700) （2013CB967700）
Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (2013RC014, 2662015PY049) （2013RC014, 2662015PY049）
the Natural Science Foundation of Henan Province (102300410104) （102300410104）
the Youth Science Fund Project of Henan Normal University (2013qk15) （2013qk15）
the Key Subjects of Fishery in Henan Province （）
