E.coli DH10B碱性磷酸酶基因的克隆、表达及活性研究

Study on cloning,expression and activity of alkaline phosphatase from Escherichia coli DH10B

目的:克隆并表达E.coli DH10B碱性磷酸酶(AKP)成熟肽基因(phoAm),为AKP的定向改构及应用奠定基础。方法:采用PCR法从E.coli DH10B基因组扩增phoAm;将phoAm克隆入表达载体pET28a,再转入E.coli BL21(DE3)进行表达;用免疫金层析法和SDS-PAGE法检测表达的重组rAKP;采用对硝基酚磷酸(pNPP)法测定rAKP的活性。结果:rAKP单体分子量约47 K,活性分析比对照菌提高21.5倍。结论:获得AKP成熟肽基因并得了到具有较高活性的重组rAKP。

Objective： To clone and express alkaline phosphatase mature peptide gene of E.coli DH10B for the foundation of directed structural transformation and application of AKP.Methods： phoAm gene was amplified from E.coli DH10B genome by PCR;phoAm gene was cloned into pET28a and transformed into E.coli BL21（DE3）;rAKP was detected by immuno-gold chromatography and SDS-PAGE;the activity of rAKP was determined by pNPP method.Results： Molecular weight of rAKP monomer was about 47 K,and activity of rAKP increased 21.5 times compared with the control.Conclusion： AKP matural peptide gene and rAKP with higher activity is acquired.