昆明鼠乙酰胆碱酯酶基因克隆与表达  

Cloning and expression of acetylcholinesterase in brain of mice

作　　者：张立媛[1] 李娜[1] 张建华[1] 杨俊忠[1] 张华[1] 杨雪婷[1] 袁永泽[1] 刘德立[1,2] 

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]华中师范大学湖北省遗传调控与整合生物学重点实验室,武汉430079

出　　处：《华中师范大学学报（自然科学版）》2008年第4期597-601,共5页Journal of Central China Normal University：Natural Sciences

基　　金：国家重大基础研究项目(2004CCA00100);教育部博士点基金(20060511002);"211"重点学科建设项目;湖北省科技攻关项目(2007AA201C50;2007AA301C26);武汉市重点科技攻关项目(20062001018).

摘　　要：乙酰胆碱酯酶(AChE)作为有机磷和氨基甲酸酯类农药的最适反应底物,在农药残留的快速检测中得到广泛应用,本实验根据家鼠AChE全基因序列设计并合成一对特异性引物,对昆明鼠脑组织AchE进行RT-PCR扩增,扩增产物经T-载体克隆、酶切鉴定和测序分析,结果表明:昆明鼠AChE编码序列长1 845 bp.经BLAST比对发现该序列与已报道序列(No.BC046327)的同源性为99.78%.构建原核重组表达质粒pET-AChE,并在E.coli中得到高效表达,表达蛋白的分子量约为68 000.运用改进的Ellman法成功检测到重组表达蛋白的活性.As the important target enzyme for organophosphate and carbamate pesticides, acetylcholinesterase has been widely used in the rapid detection of those pesticide residues. The cDNA of AChE was amplified with the specific primers designed according to the gene of acetylcholinesterase registed in GenBank （No. BC046327）. The amplified products were cloned into pMD18-T to construct the recombinant plasmid pMD- AchE. The cloned AChE gene was identified by EcoR 1 and Sal Ⅰ , sequenced by Ying Jun Biotechnology Company in Shanghai and blasted analysis in NCBI. The result showed that the length of the gene is 1845bp and the homology between the cloned gene and that resisted in GenBank （No. BC046327） is about 99.78%. The recombinant prokaryotic expression plasmid pET-AchE was constructed and expressed in E. coli with lmmol/L IPTG induction. The SDS-PAGE analysis showed that the specific band about 68 000 appeared. The activity of AChE was successfully detected by using improved Ellman＇s method.

关 键 词：乙酰胆碱酯酶 RT-PCR CDNA克隆 表达 

分 类 号：Q556[生物学—生物化学] S852.65[农业科学—基础兽医学]