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嗜麦芽寡养单胞菌D2株单加氧酶基因的克隆与表达
引用本文:辛晓妮,闫志勇,杨丽,吴瑶,王斌,宋旭霞,罗玮.嗜麦芽寡养单胞菌D2株单加氧酶基因的克隆与表达[J].微生物学杂志,2010,30(3):15-18.
作者姓名:辛晓妮  闫志勇  杨丽  吴瑶  王斌  宋旭霞  罗玮
作者单位:青岛大学,医学院,山东,青岛,266070
基金项目:国家"973"基础研究前期研究专项项目,山东省科技公关项目,青岛市自然科学基金 
摘    要:构建嗜麦芽寡养单胞菌D2株荧光素样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1300bp的核酸片段,将其克隆到T载体pMD-18中进行序列测定,所得序列申请并获得GenBank登记号(GQ122330)。DNA star软件分析发现该基因片段中含有一个996bp的完整开放读码框架(ORF),与GenBank中收录的S.maltophilia R551-3(CP001111/GenomeProject17107)和K279a(AM743169)的MO基因核酸序列同源性分别为90%和89%,氨基酸序列同源性分别为93%和90%。根据该ORF序列设计分别含有BamHⅠ和HindⅢ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54.2ku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。

关 键 词:嗜麦芽寡养单胞菌  单加氧酶  克隆  原核表达

Cloning and Expression of Monooxygenase Gene in Stenotrophomonas maltophilia Strain D2
XIN Xiao-ni,YAN Zhi-yong,YANG Li,WU Yao,WANG Bin,SONG Xu-xi,LUO Wei.Cloning and Expression of Monooxygenase Gene in Stenotrophomonas maltophilia Strain D2[J].Journal of Microbiology,2010,30(3):15-18.
Authors:XIN Xiao-ni  YAN Zhi-yong  YANG Li  WU Yao  WANG Bin  SONG Xu-xi  LUO Wei
Institution:(Med. Coll., Qingdao Uni. Qingdao 266070)
Abstract:An expression clone vector of luciferase-like monooxygenase (MO) gene of Stenotrophomonas maltophilia (Sm) strain D2 was constructed and carried out fusion expression. A nucleic acid fragment of about 1 300 bp genome that containing MO was amplified by polymerase chain reaction (PCR) from Sm strain D2, and cloned it to T vector pMD-18 to carry out sequence testing, the obtained sequence had applied and gained a GenBank registration number (GQ122330). The DNAstar software analysis had found that within the gene fragment there was a 996 bp complete open reading frame (ORF). And the homology of it was respectively 90% and 89% homologous with MO gene nucleic acid sequence of that Sm R551-3 (CP001111/GenomeProject17107) and K279a (AM743169) included in GenBank, and the homology of amino acids sequence was respectively 93% and 90%. According to the sequence design the expression clone amplified primer respectively contained digestion sites of BamH I and HindIII, after PCR amplification and double enzyme digestion, and product was subcloned to pET32a vector, and proved through double enzyme digestion test and verification that recombinant vector pET32a/MO was successfully obtained. Subsequently, it was transformed into E. coli BL21. After induced by IPTG, the recombinant successfully expressed a fusion protein of 54.0 ku of MO, and this had laid a foundation for further study on the function of the enzyme.
Keywords:Stenotrophomonas maltophilia  monooxygenase  clone  prokaryotic expression
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