山梨糖脱氢酶在乙酸钙不动杆菌中的表达

目的:在乙酸钙不动杆菌Y2004中表达山梨糖脱氢酶。方法:将酮古龙酸菌山梨糖脱氢酶基因sdh以及从pWH1266质粒上扩增的复制原点ori先后酶切连接到pBBR1MCS2质粒上,构建pBBR1MCS2-ori-sdh穿梭质粒;再以pBBR1MCS2-ori-sdh/DH5α为供体菌、乙酸钙不动杆菌Y2004为受体菌、pRK2013/HB101为辅助菌进行三亲本接合转移;从氨苄青霉素和卡那霉素双抗平板上挑取转化子进行培养,通过菌落PCR和提取质粒复转筛选阳性克隆,再通过活性电泳和体外糖酸转化实验检测阳性克隆的山梨糖脱氢酶活性。结果:构建了pBBRMCS2-ori-sdh质粒并转入乙酸钙不动杆菌Y2004中,活性电泳和体外实验证实阳性克隆具有山梨糖脱氢酶活性。结论:实现了山梨糖脱氢酶在乙酸钙不动杆菌Y2004中的表达,为单菌糖酸转化的进一步研究奠定了基础。

Expression of Sorbose Dehydrogenase in Acinetobacter calcoaceticus

Objective： To express the sorbose dehydrogenase in Acinetobacter calcoaceticus.Methods： The plas-mid pBBR1MCS2-ori-sdh was constructed by ligating replicate origin ori which was cloned from pWH1266 plasmid and sdh gene from Ketogulonigenium vulgare to pBBR1MCS2 plasmid.With the help of pRK2013 / HB101,plasmid pBBR1MCS2-ori-sdh was transferred from E.coli DH5α into A.calcoaceticus strain Y2004 by triparental conjugal transfer.Then the transformed clone was identified by PCR and plasmid retransformation and the sorbose dehydro-genase activity was detected by native electrophoresis and transformation in vitro.Results： The pBBR1MCS2-ori-sdh plasmid was successfully constructed and tranferred into A.calcoaceticus Y2004.The transformed clone was proved to be positive in sorbose dehydrogenase activity by active electrphoresis and transformation in vitro.Con-clusion： The sorbose dehydrogenase was successfully expression in A.calcoaceticus,which will lay the foundation for further research of the sorbose-ketogulonic acid transformation by single bacteria.