中文摘要：

目的构建携带绿色荧光蛋白的Rac1-GTPase显性负效突变体（Rac1N17）和组成型活性突变体（Rac1L61）的慢病毒。方法构建Rac1N17和Rac1L61慢病毒表达质粒,并以酶切和测序等方法鉴定。利用ViraPowerTM慢病毒表达系统包装制备Rac1-GTPase突变体慢病毒上清,用其感染大鼠前皮质神经元,分别进行Rac1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Rac1N17和Rac1L61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1×106TU/ml。用制备的慢病毒上清感染原代培养的前皮质神经元,Rac1生物学活性检测结果显示Rac1N17慢病毒显著抑制表皮生长因子诱导的Rac1活性的升高,而Rac1L61慢病毒感染神经元Rac1活性显著升高。感染效率鉴定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Rac1N17和Rac1L61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一个重要工具。

英文摘要：

Objective To construct lentiviruses carrying dominant negative mutant of Rac1-GTPase（Rac1N17） or the constitutive active mutant of Rac1-GTPase（Rac1L61） and expressing enhanced green fluorescent protein（EGFP） bicistronically.Methods The lentiviral expression plasmid Plenti6/v5-Rac1N17 and Plenti6/v5-Rac1L61 were constructed and identified by restriction enzyme digestion and DNA sequence analysis.The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses Rac1L61 and Rac1N17,which were used to infect the prefrontal cortex neurons（PFCs） from neonatal SD rats.The transfection efficiency and biological activity of different Rac1 mutants were evaluated and the morphology of the transfected PFCs was observed.Results The results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction.The packaged lentiviral titer was 1×106 TU/ml.Analysis of Rac1 biological activity showed that Rac1N17 lentivirus particles infection significantly inhibited epidermal growth factor-stimulated Rac1 activity in the PFCs,while Rac1L61 lentivirus particles enhanced the Rac1 activity.The transfection efficiency of these Rac1 mutant lentivirus particles exceeded 80% in the PFCs.Morphologically,the PFCs exhibited distinct dendritic branches after infection by these lentiviruses.Conclusion The lentiviruses carrying Rac1 dominant negative mutant and constitutive active mutant have been successfully constructed.The lentiviral particles can efficiently infect neonatal rat PFCs and lend important support for the study of Rac1-GTPase.