构建与制备RhoA-GTPase 突变体慢病毒

中国临床解剖学杂志 ›› 2011, Vol. 29 ›› Issue (5): 541-545.

构建与制备RhoA-GTPase 突变体慢病毒

李良平¹，　顾晶晶²，　王　斌²，　李　娟²，　张　磊³，　张　琳³，　张　璐²

1. 广州市红十字会医院神经外科，广州 510220；　2.南方医科大学病理生理学教研室广东省功能蛋白质组学重点实验室；
3. 南方医科大学组织学与胚胎学教研室，广州 510515

收稿日期:2011-03-05 出版日期:2011-09-25 发布日期:2011-09-29
通讯作者: 张璐，教授，博士生导师，Tel：（020）61648726，E-mail: zhanglu@fimmu.com E-mail:yuyu66006@163.com
作者简介:李良平（1962-），男，博士研究生，研究方向：神经系统疾病信号转导机制
基金资助:
国家自然科学基金(30770821,81071120,30973129 )；教育部新世纪优秀人才支持计划（NCET-09-0088）；广东省自然科学基金重点项目（92510515000008）；广东省科技计划项目(2009A030200015)；教育部高等学校博士学科点专项基金(20094433120012); 教育部科学技术研究重点项目(20103388008)

Construction and identification of RhoA-GTPase lentivirus

LI Liang-ping¹, GU Jing-jing², WANG Bin², LI Juan², ZHANG Lei³, ZHANG Lin³, ZHANG Lu²

1. Department of Neurosurgery, Guangzhou Red Cross Hospital; 2. Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province, Southern Medical University; 3. Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China

Received:2011-03-05 Online:2011-09-25 Published:2011-09-29

摘要/Abstract

摘要：

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。

关键词: RhoA-GTPase, 慢病毒, 绿色荧光蛋白, 生物学活性

Abstract:

Objective To construct lentiviruses carrying dominant negative mutant of RhoA-GTPase (RhoAN19) or the constitutive active mutant of RhoA-GTPase (RhoAL63) and expressing enhanced green fluorescent protein (EGFP) bicistronically. Methods The lentiviral expression plasmid Plenti6/v5- RhoAN19 and Plenti6/v5- RhoAL63 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 RhoAL63 and RhoAN19, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different RhoA 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 RhoA biological activity showed that RhoAN19 lentivirus particles infection significantly inhibited lysophospatidic acid stimulated RhoA activity in the PFCs, while RhoAL63 lentivirus particles enhanced the RhoA activity. The transfection efficiency of these RhoA mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses. Conclusions The lentiviruses carrying RhoA dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs. Thus providing important support for the study of RhoA signaling.

Key words: RhoA-GTPase, Lentivirus, Green fluorescent protein, Biological activity

中图分类号:

李良平, 顾晶晶, 王斌, 李娟, 张磊, 张琳, 张璐. 构建与制备RhoA-GTPase 突变体慢病毒[J]. 中国临床解剖学杂志, 2011, 29(5): 541-545.

LI Liang-Beng, GU Jing-Jing, WANG Bin, LI Juan, ZHANG Lei, ZHANG Lin, ZHANG Lu. Construction and identification of RhoA-GTPase lentivirus[J]. Chinese Journal Of Clinical Anatomy, 2011, 29(5): 541-545.

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