假密环菌cDNA文库的构建及其阿拉伯糖苷酶基因的克隆(英文)

Construction of a cDNA Library and Cloning of an Arabinosidase cDNA from Armillariella tabescens

用SMART技术构建了载体为λTriplEx2的假密环菌的表达型cDNA文库。文库滴度为1.0×106pfu/ml,重组率约为98.3%,扩增后滴度为3.1×1 08pfu/ml,容量约为4.2×1010。从文库中随机挑选176个克隆进行5’端测序,得到147条表达序列标签(EST),并将测序结果提交到EMBL数据库。随机测序结果表明:插入片段长度均在200-800之间。测序结果经过生物信息学分析,发现有43 条序列与已知序列有明显同源性,其中序列AJ620046与多形拟杆菌的阿拉伯糖苷酶序列有很高的序列一致性。用SMART-RACE技术成功获得了AJ620046的全长cDNA,克隆了AJ620046的开放阅读框AF,并成功构建了重组质粒pHIL-S1-AF,在毕赤酵母菌中进行了初步表达。

The expression cDNA library of A . tabescens was constructed by SMART technique,which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0×106pfu/ml and 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1×108pfu/ml and 4.2×1010 . The library was used to provide expressed sequence tags (ESTs) . 147 Expressed Sequence Taqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. The sequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them were found that they have significant similarity with data in GenBank. EST AJ620046 was has significant similarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA of AJ620046 was successfully obtained. In order to initially characterize the biochemical properties of AJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast. Recombinant pHIL-S1- AF constructed by inserting AF into pHIL-S1 was transformed into Pichia Pastoris GS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinant yeast.