NADPH依赖的乙酰乙酰辅酶A还原酶基因phbB的克隆和在大肠杆菌中的表达

目的：在大肠杆菌中克隆和表达NADPH依赖的乙酰乙酰辅酶A还原酶基因。方法：通过PCR方法克隆来源于Alcali-genes eutrophus菌的NADPH依赖性的乙酰乙酰辅酶A还原酶基因phbB，将其克隆在大肠杆菌表达质粒pET28a中，进行PCR和DNA序列测定验证，并通过IPTG诱导表达。结果：通过PCR和DNA核酸序列分析，证实获得的基因片段为phbB全编码序列，phbB被克隆在大肠杆菌表达质粒pET28a中，重组表达质粒在大肠杆菌BL21(DE3)中经IPTG诱导获得表达。结论：获得了Al-caligenes eutrophus菌的NADPH依赖的乙酰乙酰辅酶A还原酶基因，将其克隆在pET28a中，经诱导获得PhbB的表达，为进一步研究PhbB的理化性质和酶动力学性质奠定了坚实的基础。

Cloning of NADPH-dependent acetoacetyl-Con A reductase gene phbB and its expression in Escherichia coli

Objective To clone and express acetoacetyl?Con A reductase gene (phbB) in Escherichia coli.Methods Using PCR methodology,NADPH?dependent acetoacetyl?Con A reductase gene from Alcaligenes eutrophus was amplified and cloned into Escherichia coli expression vector pET28a followed by verification with PCR amplification and DNA sequencing. PhbB was expressed under IPTG induction.Results PCR and DNA sequencing confirmed the obtained full length phbB gene and the cloning of phbB in expression vector pET28a.After IPTG induction,recombinant plasmid obviously expressed NADPH?dependent acetoacetyl?Con A reductase in Escherichia coli BL21(DE3).Conclusion We have cloned the NADPH?dependent acetoacetyl?Con A reductase gene of Alcaligenes eutrophus and cloned it into pET28a.The apparent expression of PhbB after induction has laid a solid foundation for further investigations of PhbB into its physical and chemical properties,and enzymatical dynamics.