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目的构建携带绿色荧光蛋白的RhoA显性负效突变体(RhoAN19)和组成型活性突变体(RhoAL63)慢病毒。方法构建RhoAN19和RhoAL63慢病毒表达质粒,并以酶切及序列测定方法进行鉴定。利用ViraPowerTM慢病毒表达系统包装制备RhoA突变体慢病毒上清,用其感染大鼠前皮质神经元,分别进行RhoA生物学活性检测、细胞转染效率鉴定与神经元形态学观察。结果构建的RhoAN19和RhoAL63慢病毒表达质粒经酶切与测序鉴定正确,包装的慢病毒滴度为1×106 TU/ml。用制备的慢病毒上清感染原代培养的前皮质神经元,生物学活性检测结果显示RhoAN19慢病毒显著抑制溶血磷脂酸(LPA)诱导的RhoA活性的升高,而RhoAL63慢病毒感染神经元后RhoA活性显著升高。感染效率鉴定结果显示病毒上清可感染80%以上的前皮质神经元。形态学观察显示经慢病毒感染后的神经元其胞体与树突分支清晰可见。结论成功制备了RhoA突变体慢病毒,并成功实现了慢病毒感染前皮质神经元,为进一步研究Rho蛋白家族信号通路提供了研究工具。
Objective To construct RhoA dominant negative mutant (RhoAN19) and constitutive active mutant (RhoAL63) lentivirus carrying green fluorescent protein. Methods The RhoAN19 and RhoAL63 lentiviral expression plasmids were constructed and identified by restriction enzyme digestion and sequencing. The RhoA mutant lentivirus supernatant was prepared by packaging with ViraPowerTM lentiviral expression system, which was used to infect rat prefrontal cortex neurons. The biological activity of RhoA was detected, and the transfection efficiency and morphology of neurons were observed. Results The constructed RhoAN19 and RhoAL63 lentiviral expression plasmids were identified by restriction enzyme digestion and sequencing. The titer of the packaged lentivirus was 1 × 106 TU / ml. The cultured primary cultured cortical neurons were infected with the lentivirus supernatant. The biological activity of RhoAN19 lentivirus significantly inhibited the increase of RhoA activity induced by lysophosphatidic acid (LPA), while RhoAL63 lentivirus infected neurons RhoA activity was significantly increased. Infection efficiency identification results show that the virus supernatant can infect more than 80% of the anterior cortical neurons. Morphological observations revealed that the cell bodies and dendritic branches of neurons infected with lentivirus were clearly visible. Conclusion The RhoA mutant lentivirus was successfully prepared and the cortical neurons before lentivirus infection were successfully established, which provided a research tool for further study of the Rho protein family signal pathway.