Zhang Shuai, Huang Yanhong, Zhu Jingling, Shan Liya, Gao Jianfeng, Zhang Yunhua, Yu Na, Yang Lei, Huang Jin
The Key Laboratory of Xinjiang Endemic & Ethnic Diseases and Department of Biochemistry, Shihezi University School of Medicine, Shihezi, Xinjiang 832002, China.
Hangzhou Normal University, Hangzhou, Zhejiang 310036, China.
Gene. 2018 Mar 20;647:129-135. doi: 10.1016/j.gene.2018.01.026. Epub 2018 Jan 7.
Neuritin plays an important role in the development and regeneration of the nervous system, and shows good prospects in the treatment and protection of the nervous system. To characterize neuritin function, we constructed a baculovirus expression system of neuritin, and identified the biological activity of the neuritin protein. The results and showed that the expression product could promote the neurite growth of dorsal root ganglion in chicken embryos. The neuritin open reading frame was amplified and cloned into the plasmid pFastBac™HTA. The pFastBac™HTA-neuritin was confirmed to be correct by PCR and DNA sequencing, and then transformed into Escherichia coli DH10Bac. The high purity recombinant Bacmid-neuritin (shuttle vectors) was obtained from DH10Bac through screening and identification. Recombinant virus, including the neuritin gene (virus-neuritin), was produced by transfection of SF9 cells using the bacmid-neuritin, and then amplified repeatedly to express the neuritin fusion protein. Finally, we identified the fusion protein with SDS-PAGE and western blotting, and optimized the best expression time of the neuritin fusion protein. We also analyzed the activity of the expressed protein by dorsal root ganglion from chicken embryos.
神经突素在神经系统的发育和再生中发挥着重要作用，并且在神经系统的治疗和保护方面展现出良好前景。为了表征神经突素的功能，我们构建了神经突素的杆状病毒表达系统，并鉴定了神经突素蛋白的生物活性。结果表明，表达产物能够促进鸡胚背根神经节的神经突生长。扩增神经突素开放阅读框并将其克隆到质粒pFastBac™HTA中。通过PCR和DNA测序确认pFastBac™HTA-神经突素正确无误，然后将其转化到大肠杆菌DH10Bac中。通过筛选和鉴定从DH10Bac中获得高纯度重组杆粒-神经突素（穿梭载体）。使用杆粒-神经突素转染SF9细胞产生包含神经突素基因的重组病毒（病毒-神经突素），然后反复扩增以表达神经突素融合蛋白。最后，我们通过SDS-PAGE和蛋白质免疫印迹鉴定融合蛋白，并优化神经突素融合蛋白的最佳表达时间。我们还通过鸡胚背根神经节分析了表达蛋白的活性。
