LINE1-ORF1抗体的制备及神经祖细胞分化中LINE-1转座水平的检测

Preparation of LINE1-ORF1 antibody and detection of LINE-1 retrotransposition during the differentiation of neural progentitor cells

目的研究转座子LINE-1在神经细胞分化中的转座水平的变化。方法用原核表达系统表达LINE-1 ORF1蛋白,免疫后制备LINE-1 ORF1蛋白抗体,用Western blot法检测胚胎干细胞分化至神经祖细胞过程中LINE-1转座水平。结果制备的抗体具有较好的特异性和灵敏度,不同类型真核细胞中LINE-1转座水平不完全一致。同时,对细胞分化过程中的LINE-1 ORF1蛋白水平直接进行分析,首次在蛋白水平证实了神经细胞分化过程中LINE-1转座水平在分化前期增高,后期降低。结论 LINE-1在神经系统发育中具有较高的转座活性,提示,其在神经系统发育中扮演着重要的作用。

Objective To explore endogenous LINE- 1 expression during Neural progenitor cells （NPCs） differenti- ation. Methods Recombinant LINE-10RF1 protein was expressed in bacteria, followed by purification and the purified protein was used to immunize rabbits to produce anti-ORF1 polyclonal antibody. The antibody was used to detect LINE-1 retrotransposition during neural progenitor cells （NPCs） differentiation by Western blotting. Results The anti-ORF1 antibody possessed high sensitivity and specificity, and observed the variability of LINE-1 retro- transposition in different eukaryotic cell lines. Using anti-ORF1 antibody to detect LINE-10RF1 protein level dur- ing NPC differentiation, it was found that when stem cells commits to NPCs, LINE-1 retrotransiposition increases in the early stage and decline in the late stage. Conclusions LINE-1 retrotranspose increases neuronal commitment, suggested LINE-1 play a pivotal role in neural development.