人NT3、BDNF基因Tet-on可调控重组腺病毒载体的构建和鉴定  

Construction and identification of recombinant adenovirus vector containing Tet-on adjustable NT3 and BDNF genes

作　　者：李志全[1] 朱庆生[1] 朱锦宇[1] 郝伟[1] 许彦鸣[2] 王成济[2] 杨安钢[2] 

机构地区：[1]第四军医大学西京医院骨科研究所,陕西西安710032 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2004年第3期344-347,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目 (No .30 2 71 31 5)

摘　　要：目的 :分别构建含人神经营养素 3(NT3)基因和脑源性神经营养因子 (BDNF)基因的重组四环素可诱导的腺病毒载体 ,并进行PCR和酶切鉴定。方法 :将NT3和BDNF基因依次分别亚克隆到pIND载体和pTRE Shuttle2载体中 ,构建重组载体pTRE Shuttle2 NT3和pTRE Shuttle2 BDNF。用PI SceI和I CeuI双酶切后将所获NT3及BDNF基因片段再与线性化的腺病毒载体pAdeno X连接 ,构建成pAdeno NT3及pAdeno BDNF的重组腺病毒载体。以电穿孔转化E .coli后挑取克隆进行PCR及酶切鉴定。结果 :重组腺病毒载体的PCR鉴定表明 ,在 312bp处出现特定的条带 ;酶切鉴定证实 ,得到约 2 3kb的预期片段 ,说明人NT3、BDNF基因与腺病毒载体pAdeno X已正确连接。结论 :成功地构建了含人NT3和BDNF基因的四环素可诱导重组腺病毒载体 。AIM: To construct Tetracycline(tet) inducible recombinant adenovirus vectors containing human neurotrophin 3(NT3) and brain derived neurotrophic factor(BDNF) genes, respectively and perform PCR and restrictive enzyme digestion analysis. METHODS: Full length NT3 and BDNF cDNAs were subcloned into pIND vector, followed by being cloned into pTRE shuttle2 vector. The NT3 and BDNF gene fragments resulted from the pTRE shuttle2 NT3 and pTRE shuttle2 BDNF digested with I Ceu I and PI Sce I were linked to the linear adeno X virus DNA. The recombinant adenovirus vectors were confirmed by PCR and restriction enzyme digestion analysis. RESULTS: The PCR identification showed that a given band with 312 bp, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human NT3 and BDNF genes had been connected with pAdeno X vectors correctly. CONCLUSION: Tet inducible recombinant adenovirus vector of Human NT3 and BDNF genes have been constructed successfully, which upon packaged in HEK293 cells, will be used to introduce the target genes into Schwann’s cells in vitro or in vivo .

关 键 词：神经营养素3 脑源性神经营养因子 四环素 重组腺病毒载体 

分 类 号：R338[医药卫生—人体生理学]