人β-NGF基因的体外扩增、克隆及其序列的分析

Amplification,Cloning in Vitro and Sequence Analysis of Human β-NGF Gene

目的 对人β－ ＮＧＦ基因进行体外扩增和克隆及其序列分析，为其在真核细胞中的表达及其临床应用打下基础。方法 本实验设计并合成一对特异性引物，采用聚合酶链反应分别从人脑ｃＤＮＡ 和人睾丸ｃＤＮＡ 以及人脑基因组ＤＮＡ中扩增出β－ 神经生长因子（β－ ＮＧＦ）基因，并和Ｔ４ 载体相连接，经筛选得到人β－ ＮＧＦ克隆，并用Ｓａｎｇｅｒ 双脱氧末端终止法进行序列分析。结果 凝胶电泳显示在预期的位置出现阳性条带。

Objective To amplificate,clone in vitro and analyse sequence of human β-NGF gene for its expression in the mammalian cells and the clinical application. Methods In this paper,a pair of specific primers has been designed and synthesizied. The cDNA encoding for mature β-NGF was successfully obtained from human brain cDNA、human testis cDNA library and human brain genomic DNA respectively by using polymerase chain reaction.Then,the cDNA of β-NGF gene was inserted into pGEM-T4 vector and did its sequencing analysis by Sanger's method. Results Gel electrophoresis shows that the positive bands appear in the prospective place.Conclusion right cloned gene sequence is confirmed by sequence analysis.