【摘要】 根据已知的α-乙酰乳酸脱羧酶(α-acetolactatedecarboxylase,ALDC,EC.4.1.1.5)基因序列,利用PCR方法从产气大肠杆菌(Enterobacter aerogenes)中克隆到0.8 kb的DNA片段,经DNA序列分析表明该DNA片段为ALDC基因.将该片段插入到原核表达载体pBV220中,获得表达质粒pBVEDAD,转化大肠杆菌DH5α(Escherichia coli),经热诱导后,通过SDS-PAGE分析和酶活测定,结果表明ALDC获得了高效表达.重组细胞表达的ALDC酶活是出发菌产气肠杆菌(E.aerogenes)的1 100倍.DH5α(pBVEDAD)在无选择压力下37℃连续培养60代,在42℃下热诱导5 h未见质粒丢失.更多还原

【Abstract】 According to the reported sequences of α-acetolactate decarboxylase(ALDC,EC.4.1.1.5) gene,a 0.8 kb DNA fragment is obtained from Enterobacter aerogenes chromosome DNA by using PCR technique.The fragment is confirmed as ALDC gene by DNA sequencing,then is inserted into pBV220,to give the recombinant plasmid pBVEDAD.ALDC gene in pBVEDAD is overexpressed in Escherichia coli DH5α by heat induction.The α-acetolactate decarboxylase activity of recombinant bacterium is 1100-fold higer than that of E.aerogenes.Ecoli DH5α(pBVEDAD) is cultivated in LB broth at 37 ℃ for about 60 generations without antibiotic,then induced at 42 ℃ for 5 hours.The pBVEDAD in DH5α(pBVEDAD) is proved to be stable.更多还原