长春理工大学学报（自然科学版）
長春理工大學學報（自然科學版）
장춘리공대학학보（자연과학판）
JOURNAL OF CHANGCHUN UNIVERSITY OF SCIENCE AND TECHNOLOGY(NATURAL SCIENCE EDITION)
2014年
5期
159-163
,共5页
羧酸酯酶%RT-PCR%克隆表达%HPLC%农药降解
羧痠酯酶%RT-PCR%剋隆錶達%HPLC%農藥降解
최산지매%RT-PCR%극륭표체%HPLC%농약강해
carboxylesterase%RT-PCR%clonging expression%HPLC%pesticide degradation
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。
從小鼠肝髒中剋隆羧痠酯酶（CES）基因傢族成員Ces1f的cDNA，在大腸桿菌中錶達，併驗證重組酶對農藥的作用。採用RT-PCR剋隆Ces1f cDNA，與pUCm-T載體連接測序後，亞剋隆至錶達載體pET-32a，構建重組錶達質粒pET-32a-Ces1f，轉化到宿主菌BL21（DE3）後經IPTG誘導錶達，經SDS-PAGE鑒定，採用Ni柱對錶達產物進行純化併測重組CES的酶比活，利用HPLC驗證重組酶對不同農藥的作用。結果顯示：成功穫得純化的77kDa的重組CES蛋白，其酶液的比活為55.37U/mg，HPLC檢測髮現重組CES對西維因、呋喃丹及甲基對硫燐等具有降解作用。成功錶達重組CES，其對農藥具有降解作用，為環境中農藥殘留的降解奠定瞭基礎。
종소서간장중극륭최산지매（CES）기인가족성원Ces1f적cDNA，재대장간균중표체，병험증중조매대농약적작용。채용RT-PCR극륭Ces1f cDNA，여pUCm-T재체련접측서후，아극륭지표체재체pET-32a，구건중조표체질립pET-32a-Ces1f，전화도숙주균BL21（DE3）후경IPTG유도표체，경SDS-PAGE감정，채용Ni주대표체산물진행순화병측중조CES적매비활，이용HPLC험증중조매대불동농약적작용。결과현시：성공획득순화적77kDa적중조CES단백，기매액적비활위55.37U/mg，HPLC검측발현중조CES대서유인、부남단급갑기대류린등구유강해작용。성공표체중조CES，기대농약구유강해작용，위배경중농약잔류적강해전정료기출。
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.
