嗜麦芽寡养单胞菌D2株单加氧酶基因的克隆与表达

Cloning and Expression of Monooxygenase Gene in Stenotrophomonas maltophilia Strain D2

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。

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.