摘要
为深入研究鱼类干细胞多能性转录因子Nanog的功能,本实验进行了团头鲂Nanog基因的原核表达和多克隆抗体的制备。首先,从团头鲂卵巢克隆出Nanog基因的编码区,将其连接到p ET32a载体上构建原核表达载体。接着将重组载体转化至大肠杆菌BL21(DE3)pLysS,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得预期大小的Nanog重组蛋白,并获得大量蛋白以制备抗体。最后通过ELISA检测抗体的效价和采用Western blot技术检测Nanog抗体的特异性。结果显示,在37°C,0.5 mmol/L IPTG诱导4 h可获得Nanog重组蛋白的高效表达;制备的多克隆抗体能够有效识别原核表达的Nanog蛋白及团头鲂肝脏和精巢的内源Nanog蛋白;该抗体也可应用于检测HepG2细胞中过表达的团头鲂Nanog蛋白。本研究为蛋白的原核表达和特异性抗体的制备及验证提供了研究思路,也为研究鱼类Nanog基因功能提供了特异性抗体。
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, Ma Nanog coding sequence was obtained from the ovary organ, and the fulllength open reading frame or the partial fragment containing the homeodomain was inserted into the p ET32 a vector, respectively. Then the vectors were transformed into Escherichia coli BL21（DE3）p Lys S, and the recombinant Nanog proteins were induced by IPTG. After optimization of expression conditions, the Ma Nanog-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 Ma Nanog protein in adult organs, and the ectopic expressed Ma Nanog proteins in Hep G2. 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.
作者
于淼
方健
李玲玉
薛亭
陈天圣
YU Miao;FANG Jian;LI Lingyu;XUE Ting;CHEN Tiansheng(Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, College of Fishery, Henan Normal University, Xinxiang 453007, China)
出处
《水产学报》
CAS
CSCD
北大核心
2017年第11期1649-1659,共11页
Journal of Fisheries of China
基金
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)
河南省水产养殖重点学科资助
关键词
团头鲂
NANOG
原核表达
多克隆抗体
干细胞
Megalobrama amblycephala
Nanog
prokaryotic expression
polyclonal antibody
stem cell
