转基因大豆毛状根hbFGF的检测

目的通过检测转基因大豆毛状根的hbFGF,为利用毛状根生产药物实现工业化提供实验基础。方法采用PCR、RT-PCR、SDS-PAGE电泳、Westem blot印迹、质谱检测等方法,利用植物基因组DNA提取试剂盒,兔抗人bFGF单克隆抗体进行对转基因大豆毛状根hbFGF检测。结果用PCR检测转基因大豆毛状根,并在60条Hyg抗性大豆毛状根中鉴定出19条大豆毛状根整合了hbFGF,阳性率为32%。半定量RT-PCR检测总RNA,表明RNA完整性好,降解较少。SDS-PAGE大致确定重组蛋白的分子量和浓度,分子量约18KDa,hbFGF含量可达110mg/L。兔抗人bFGF单克隆抗体检测表明,hbFGF在转基因大豆毛状根中稳定表达。结论表明转基因大豆毛状根能够稳定表达hbFGF基因,在工业生产中具有广阔的前景。

Detection of hbFGF in the hairy roots of transgenic soybeans

Object The paper provides experimental bases for the mass production of medicines making use of hairy roots through testing hbFGF in the hairy roots of transgenic soybeans. Methods Using plant genome DNA extraction kit and rabbit anti-human bFGF monoclonal antibody, hbFGF of transgenic soybeans’ hairy roots is tested through methods like PCR, RT-PCR, SDS-PAGE electrophoresis, Westem blot engram and mass spectrometric detection, etc. Result Tested through PCR, 19 soybeans’ hairy roots contain hbFGF out of sixty Hyg resistance soybean’s hairy roots, 32% positive. Semi-quantitative RTPCR is used to test RNA which shows integrity and low degradation. SDS-PAGE is used to roughly decide the molecular weight and molecular concentration of recombinant proteins. The molecular weight is about18KDa and the content of hbFGF is 110mg / L. the test with rabbit anti-human bFGF monoclonal antibody shows hbFGF is stable in transgenic soybeans’ hairy roots. Conclusion hbFGFis stable in in transgenic soybeans’ hairy roots which have a great potential in industrial production.