大鼠Somatostatin基因重组慢病毒载体的构建与检测  

Construction and detection of a recombinant lentiviral vector containing rat somatostatin gene

作　　者：魏云艳 赵莉娟[1] 孙蒙蒙[1] 王瑾[1] 于佳田 王德广[1] 

机构地区：[1]徐州医学院人体解剖学教研室,江苏徐州221004

出　　处：《徐州医学院学报》2015年第11期764-768,共5页Acta Academiae Medicinae Xuzhou

基　　金：江苏省教育厅课题（14KJB310021）

摘　　要：目的克隆大鼠生长抑素somatostatin（SST）基因和神经元突触素Synapsin（Synl）的启动子序列，构建神经元特异性表达SST基因的重组慢病毒载体。方法采用RT—PCR法和PCR法分别扩增SST基因和Synl的启动子序列，并将其克隆至重组慢病毒载体系统（Lenti—EGFP），构建出以Synl启动子序列为启动子、携带SST基因的表达质粒Lenti—pSyn—SST-2A—EGFP；再将该表达质粒与pMDL、pRey和pVSVG用磷酸钙共沉淀法共转染HEK293T细胞，获得重组的慢病毒载体，将病毒注射人大鼠海马，检测EGFP和SST的表达效果。结果经测序克隆的SST和Synl启动子与GenBank上的序列相比完全一致，并测得SST重组慢病毒纯化后病毒滴度为1．5×10^9TU／ml，该病毒注射人海马可以转染神经元并表达SST。结论成功构建出以Synl启动子序列为启动子，携带SST基因的重组慢病毒载体。Objective To clone rat somatostatin （SST） gene and a promoter sequence of synapsin （ Synl ） into a lentiviral vector to express neuronal - specific SST gene. Methods SST gene and the promoter sequence were amplified by RT- PCR and PCR, respectively and then inserted into a Lenti -EGFP vector, resulting in a recombinant plasmid Lenti -pSyn -SST -2A -EGFP. The resultant plasmid and three additional plasmids pMDL, prey and pVSVG were co - transfected into HEK 293T cells using calcium phosphate - mediated transfection to obtain recombinant lentivirus parti- cles. The packaged lentivirus （Lenti- pSyn- SST- 2A- EGFP and Lenti- pSyn- EGFP） were injected into the rat hippocampus. The levels of EGFP and SST were detected by Nissl staining and immunostaining. Results DNA sequen- cing analysis showed the sequences of SST and Synl promoter were consistent with those in GenBank, and the resultant lentivirus vector produced a viral titer of 1.5 ~ 109 TU/ml. The lentivirus vector could induce the expression of EGFP and SST gene after transfeetion into hippocampus neurons. Conclusion The recombinant lentiviral vector containing rat SST gene and Synl promoter sequence is successfully constructed.

关 键 词：生长抑素 神经元突触素启动子 慢病毒载体 构建 

分 类 号：Q784[生物学—分子生物学]