摘要
从小鼠肝脏中克隆羧酸酯酶(CES)基因家族成员Ces1f的cDNA,在大肠杆菌中表达,并验证重组酶对农药的作用。采用RT-PCR克隆Ces1f cDNA,与pUCm-T载体连接测序后,亚克隆至表达载体pET-32a,构建重组表达质粒pET-32a-Ces1f,转化到宿主菌BL21(DE3)后经IPTG诱导表达,经SDS-PAGE鉴定,采用Ni柱对表达产物进行纯化并测重组CES的酶比活,利用HPLC验证重组酶对不同农药的作用。结果显示:成功获得纯化的77kDa的重组CES蛋白,其酶液的比活为55.37U/mg,HPLC检测发现重组CES对西维因、呋喃丹及甲基对硫磷等具有降解作用。成功表达重组CES,其对农药具有降解作用,为环境中农药残留的降解奠定了基础。
Qbjective：The Ces1f cDNA coding carboxylesterase was cloned from Mouse liver.Methods：The Ces1f cDNA was amplified from mouse liver by RT-PCR, and inserted in pUCm-T to determine the DNA sequence. the Ces1f cDNA was subcloned into expression vector pET-32a to construct the recombinant expression vector pET-32a-Ces1f, and transformed into the host strain E. coli BL21 （DE3） . Recombinant CES expression was induced by IPTG, and purified by Ni-NTA affinity chromatography. SDS-PAGE method were used to identify the expression and its speci-fied enzyme activity were assayed.Results：A 77kDa recombinant CES was successfully expressed, The specified en-zyme activity of purified enzyme were 55.37U/mg. HPLC detection showed Carbaryl、carbofuran and Metaphos were degraded by the recombinant.Conclusion：A method for expression and purification of mouse recombinant CES was suc-cessfully established.The method laid the foundation for the degradation of pesticide residues in the environment.
出处
《长春理工大学学报（自然科学版）》
2014年第5期159-163,共5页
Journal of Changchun University of Science and Technology(Natural Science Edition)
基金
吉林省科技厅资助项目(20120963)
关键词
羧酸酯酶
克隆表达
农药降解
RT-PCR
HPLC
carboxylesterase
RT-PCR
clonging expression
HPLC
pesticide degradation
