大鼠脑cDNA文库的构建

Construction of a Wistar Rat Brain cDNA Library

采用简单高效的ｃＤＮＡ合成技术制备Ｗｉｓｔａｒ大鼠脑ｃＤＮＡ基因文库，以纯化的ｐｏｌｙ（Ａ）＋－ＲＮＡ为模板，含ＮｏｔＩ切点的ｏｌｉｇｏ－（ｄＴ）１５为引物，在反转录酶的作用下，合成第一股单链ｃＤＮＡ；用Ｅ。ｃｏｌｉＲＮａｓｅＨ除去模板ＲＮＡ，并以Ｅ．ｃｏｌｉＤＮＡ聚合酶Ｉ，Ｅ．ｃｏｌｉＤＮＡ连接酶和Ｔ４ＤＮＡ聚合酶催化合成ｃＤＮＡ第二条链，即成为双链ｃＤＮＡ；此双股ｃＤＮＡ除０．５μｇ用于插入ｐＳＰＯＲＴＩ载体，转入Ｅ．ｃｏｌｉＤＨ５α，建成ｃＤＮＡ文库外，其余保存在－２０℃，以此ｃＤＮＡ为模板，应用ＰＣＲ方法，先后克隆了谷氨酸脱羧酶（ＧＡＤ，１８００ｂＰ）、神经元特异性烯醇化酶（ＮＳＥ，１３４０ｂＰ），甲状腺激素受体（Ｔ３－ｒｅｃｅｐｔｏｒ，１２３０ｂｐ）、胆囊收缩素（ＣＣＫ，３４５ｂｐ）的全编码基因．

A cDNA library of Wistar rat brain was constructed with a highly effcient and simplified method. The template POly (A) + -RNA was extracted from Wistar rat brain. The first strand of cDNA was synthesized by SuperScript RNase H- reverse transcriptase with the oligo (dT)15 primer which contains Not I site. After the poly (A) + -RNA was removed with E. coliRNase H, the second strand was synthesized by means of E. coli DNA polymerase I, E. colt DNA ligase and T4 DNA polymerase.Then Sal I adapter was added and the cDNA digested with Not I. Half of the cDNA was inserted into plasmid pSPORT I and transformed E. coli DH 5a. The other half was stored at -20℃. It was shown that the cDNA length ranged from 100 to 10 000 bp. Using this cDNA library as a template, four genes have been amplified successfully. They are glutamic acid decarboxylase (GAD, 1800 bp),neuron-specific enolase (NSE,1340 bp),T3 receptor (1230 bp) and cholecystokinin (CCK, 345 bp). The method for storage of the cDNA library which can be kept for a longer period was also developed.