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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.

Citation

Ruofei Li, Dalong Gao, Xiaoqian Dang, Kunzheng Wang, Leigang Yang, Zhongmin Tu. Construction and identification of recombinant adeno-associated virus shuttle vector expressing nerve growth factor beta gene]. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery. 2012 Feb;26(2):172-6

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PMID: 22403879

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