嗜麦芽寡养单胞菌D2株单加氧酶基因的克隆与表达.PDF

微生物学杂志 2010 5 30 3 JOURNAL OF M ICROBIOLOGY M ay 2010 Vol. 30 No. 3 15 D2 * 辛晓妮, 闫志勇 , 杨 丽, 吴 瑶, 王 斌, 宋旭霞, 罗 玮 (, 266070) 构建嗜麦芽寡养单胞菌 D2株荧光素 单加氧酶基因表达克隆载体, 并进行融合表达PCR 扩增出 嗜麦芽寡养单胞菌 D2菌株基因组 DNA 中包含单加氧酶基因约 1 300 bp 的核酸片段, 将其克隆到 T 载体 pMD18 中进行序列测定, 所得序列申请并获得 GenBank登记号( GQ 122330)DNA star软件分析发现该基因 片段中含有 一个 6 bp的完整开放读码框架( ORF), 与 GenBank 中收录的 S. m altophilia R5513( CP001111/ Genom eProject17107)和 K27 a (AM74316 )的 MO 基因核酸序列同源性分别为 0% 和 8 % , 氨基酸序列同源 性分别为 3% 和 0% 根据该 ORF序列设计分别含有 BamH 和H ind 酶切位点的表达克隆扩增引物, PCR 扩增双酶切后将产物亚克隆到 pET32a载体 中, 经过双酶切验证, 证实成功获得了表达重组载体 pET32a/MO; 将其转化宿主菌E. coli BL21, IPTG 诱导后成功表达出 54. 2 ku的MO融合蛋白, 为该酶进一步的 功能研究和开发奠定了基础 嗜麦芽寡养单胞菌; 单加氧酶; 克隆; 原核表达 Q 33 A 1005- 7021( 2010) 03- 0015- 04 Cloning and Expression ofM onooxygenase Gene in Stenotrop hom onas ma ltop h ilia Strain D2 XIN X iaon,i YAN Zhiyong, YANG Li, WU Y ao, WANG Bin, SONG Xuxia, LUO W ei (M ed. Coll. , Qingdao Uni. Qingdao 266070) Abstract An expression clone vector of luciferaselike m onooxygenase ( MO) gene of Stenotrop homonas m altoph ilia (Sm ) strain D2w as constructed and carried out fusion expression. A nucleic acid fragm ent of about 1 300 bp genom e that contain ing MO w as amp lified by polym erase chain reaction (PCR) from Sm strain D2, and cloned it to T vector pMD18 to carry out sequence testing, the obtained sequence had applied and gained aG enBank registration num ber ( GQ 122330). The DNAstar softw are analysis had found that w ith in the gene fragm ent there was a 6 bp comp lete open reading fram e ( ORF) . And the homology of it was respectively 0% and 8 % hom ologousw ith MO gene nucle ic acid sequence of that Sm R5513 ( CP00111