脑源性神经营养因子受体trkB基因扩增和真核表达载体的构建  

Amplification of brain-derived neurotrophic factor receptor trkB gene and construction of its eukaryotic expression vector

作　　者：黄涛[1] 徐如祥[1] 武雷[2] 姜晓丹[1] 邱晓忠[2] 秦建强[2] 余磊[2] 许忠[1] 

机构地区：[1]第一军医大学珠江医院神经外科,广东广州510282 [2]第一军医大学临床解剖学研究所,广东广州510515

出　　处：《第一军医大学学报》2003年第12期1283-1285,1289,共4页Journal of First Military Medical University

基　　金：广东省科技计划项目(2003A3020304);广东省团队项目[粤科基办2000(25)];军队十五重点项目(01ZD54)~~

摘　　要：目的构建大鼠脑源性神经营养因子受体trkB基因真核表达载体。方法根据trkB基因已知序列,设计合成一对引物,上、下游引物分别引入EcoRⅠ及BamHⅠ酶切位点,用反转录PCR(RT-PCR)方法从大鼠脑组织总RNA中扩增编码trkB的基因片断,插入克隆载体pMD 18-T,构建pMD 18-trkB载体。经EcoRⅠ、BamHⅠ双酶切出trkB基因片断,再克隆至载体pEGFP-C2中构建pEGFP-C2-trkB真核表达载体。结果trkB基因RT-PCR扩增产物大小约1 461 bp,真核表达载体经酶切鉴定表明为正确重组子,基因测序结果与已知序列基本吻合。结论成功扩增出完整trkB基因,构建了真核表达载体pEGFP-C2-trkB。Objective To construct a recombinant eukaryotic expression vector of rat brain-derived neurotrophic factor receptortrkB gene. Methods Using the total RNA extracted from rat brain tissue as the template, the trkB gene was amplified by reversetranscription-polymerase chain reaction (RT-PCR) with a pair of specific primers containing the restriction sites of EcoRⅠand BamHⅠ. The amplified fragment of trkB gene was digested with EcoRⅠand BamHⅠ, and then subcloned into cloningvector pMD18-T and then expression vector pEGFP-C2. The recombinant plasmid was identified by restriction endonucleaseanalysis and PCR. Results The amplified DNA fragment was about 1 461 bp in length, and enzyme digestion and PCRanalysis showed that trkB gene had been successfully cloned into the vectors pMD18-T and pEGFP-C2. Conclusion The trkB gene of rat has been successfully amplified and cloned into the eukaryotic expression vector pEGFP-C2.

关 键 词：脑源性神经营养因子受体 TRKB 基因扩增 载体 基因表达 

分 类 号：R346[医药卫生—基础医学]