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通过PCR方法扩增达托霉素生物合成相关的酰基CoA连接酶DptE及其突变体dptE-296的基因片段,经限制性内切酶bamHⅠ和Xho Ⅰ双酶切后插入到原核表达载体pet22b,利用质粒自带的氨苄青霉素抗性筛选阳性克隆,并进行菌液PCR和测序鉴定;将测序正确的重组质粒通过热击法转入BL21(DE3)表达菌株,IPTG诱导蛋白表达,渗透压休克法提取周质蛋白,镍柱亲和纯化,通过SDS-PAGE,Western blot鉴定蛋白表达;连接生物素,通过Fortebio检测蛋白和底物癸酸的结合.测序结果显示成功构建重组载体DptE-pet22b和DptE-296-pet22b,SDS-PAGE和Western blot结果显示表达产生的蛋白大小与预期一致,Fortebio实验显示DptE及其突变体DptE-296对底物癸酸有结合,但结合的不强.“,”The gene fragment encoding acyl-CoA ligase DptE and its mutant DptE-296 were amplified using polymerase chain reaction(PCR) and were inserted into the prokaryotic expression vector pet22b through the restriction enzymes bamH Ⅰ and Xho L The recombinant plasmid was identified by screening positive clones using ampicillin,bacteria PCR and DNA sequencing.The correct plasmid was transformed into E.coliBL21 (DE3),induced by IPTG,extracting periplasmic proteins by osmotic shock method,purified by nickel affinity chromatography,identified by SDS-PAGE and Western blot.The purified protein was connected to biotin to assay the affinity with decanoic acid by fortebio.The recombinant vector DptE-pet22b and DptE-296-pet22b was successfully constructed.SDS-PAGE and Western blot showed that the expressed protein was consistent with the expected.Fortebio experiments showed that both DptE and DptE-296 can bind decanoic acid,but the combination is not strong.