大鼠脑源性神经营养因子基因T载体的构建及鉴定  

Construction and identification of brain derived neurotrophic factor gene cloned into a T-vector in rat

作　　者：吴燕峰[1] 王鹏[2] 唐勇[2] 黄霖[2] 杨睿[2] 沈慧勇[2] 

机构地区：[1]中山大学附属第二医院脐血库中山大学脊髓损伤研究所,广州市沿江西路107号510120 [2]中山大学附属第二医院骨科中山大学脊髓损伤研究所,广州市沿江西路107号510120

出　　处：《中国脊柱脊髓杂志》2006年第4期291-294,共4页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金资助项目(基金编号30271311);广东省自然科学基金资助项目(基金编号36642);广东省科技厅重大项目(基金编号2004A30201002);广州市科委重大项目(2003Z-E-0011)

摘　　要：目的:探讨利用T载体完成大鼠脑源性神经营养因子(BDNF)聚合酶链式反应(polymerasechainreac-tion,PCR)产物克隆的可行性。方法:提取大鼠脑组织总RNA,利用M-MLV逆转录酶及oligo-dT引物逆转录合成cDNA第一条链,再利用exTaq酶及BDNF上、下游引物扩增出BDNFcDNA,最后利用BufferⅠ连接液将BDNF基因克隆入T载体用于测序及下一步克隆。结果:成功构建了BDNF基因T载体,经双脱氧链终止法测序证实为大鼠BDNF基因。结论:利用T载体克隆BDNF基因PCR产物简便、快捷,成功率高。Objective： To construct a T-vector cloned with a BDNF gene of a SD rat. Method： The total RNA of the brain of a SD rat were extracted, the first strand cDNA was synthesized using M-MLV reverse transcriptase and oligo-dT primers, and then BDNF cDNA was amplified from the total cDNA via a PCR using an upstream sense primer and downstream antisense primer, finally the PCR product was subcloned into the pMD18-T simple vector using a metheod of A-T cloning and this complex was prepared for sequencing and other gene clone purpose. Result： A T-vector cloned with the interest BDNF gene was successfully constructed and confirmed by sequencing. Conclusion： The method of cloning BDNF gene with a pMD18-T simple vector is a successful and fast shortcut.

关 键 词：脑源性神经营养因子 T载体 基因克隆 

分 类 号：Q786[生物学—分子生物学]