Li Ruofei, Gao Dalong, Dang Xiaoqian, Wang Kunzheng, Yang Leigang, Tu Zhongmin
No. 1 Department of Orthopedics, Xianyang Center Hospital, Xianyang Shaanxi, 712000, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):172-6.
To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nerve growth factor beta (NGF-beta) gene.
By PCR amplification, the structural element of pAAV-multiple cloning site (MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA ligase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell line was cultured, and total RNA was extracted, then human NGF-beta gene was obtained by RT-PCR. T-easy-NGF-beta vector was constructed by cloning human NGF-beta gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-beta gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-beta gene was obtained and identified by RT-PCR, digestion, and DNA sequencing.
The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMV-EGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-beta was digested; DNA sequencing result of T-easy-NGF-beta was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-beta was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-beta showed that sequences were completely correct.
The AAV shuttle vector pAAV-U6/CMV-NGF-beta is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
构建表达神经生长因子β（NGF-β）基因的重组腺相关病毒（AAV）穿梭载体。
通过PCR扩增分别获得pAAV-多克隆位点（MCS）的结构元件和pGenesil-1.1的功能元件，并分别克隆至T-easy载体；对重组T-easy载体进行酶切，回收目的片段后用DNA连接酶连接，从而获得含U6启动子和CMV启动子的重组AAV穿梭载体pAAV-U6/CMV-增强型绿色荧光蛋白（EGFP）。将该载体转染至293细胞。培养人Miapaca-2细胞系并提取总RNA，然后通过RT-PCR获得人NGF-β基因。将人NGF-β基因克隆至T-easy载体构建T-easy-NGF-β载体，并通过RT-PCR、酶切及DNA测序进行鉴定。将NGF-β基因克隆至pAAV-U6/CMV-EGFP载体，获得表达NGF-β基因的重组AAV穿梭载体，并通过RT-PCR、酶切及DNA测序进行鉴定。
pAAV-U6/CMV-EGFP酶切时检测到800 bp和4250 bp的条带。pAAV-U6/CMV-EGFP转染至293细胞时检测到GFP。T-easy-NGF-β酶切时检测到736 bp和3015 bp的条带；T-easy-NGF-β的DNA测序结果完全一致。pAAV-U6/CMV-NGF-β酶切时检测到736 bp和4250 bp的条带。pAAV-U6/CMV-NGF-β的DNA测序结果显示序列完全正确。
成功构建了AAV穿梭载体pAAV-U6/CMV-NGF-β，为脊髓损伤修复研究提供了实验依据。
