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作 者:宋学文[1] 赵崇[1] 周仁[1] 郭善一[1] 张镜宇[1]
机构地区:[1]天津医科大学内分泌研究所,天津医科大学总医院内分泌科,天津医科大学代谢病中心
出 处:《生物化学与生物物理进展》1996年第2期169-172,共4页Progress In Biochemistry and Biophysics
基 金:天津市21世纪青年科学基金
摘 要: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.
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