摘要
利用Trizol法从尖音库蚊中提取总RNA,构建cDNA文库,并克隆出乙酰胆碱酯酶外显子序列;利用Bac-to-Bac杆状病毒表达系统对蚊虫乙酰胆碱酯酶进行真核表达,并利用Ni-琼脂糖对酶进行纯化。采用SDS-PAGE对纯化产物进行检测,结果表明得到了纯度较高的乙酰胆碱酯酶。参照Ellman法对酶的活性进行测定,结果显示纯酶的活力为2.219×10-4 mol/(min.g)。
The residues of the high toxic organophosphorous can cause chronic poisoning. At present,the detection of organophosphorous residue needs complicated operation, time consuming and high cost. Here,we harvested total RNA of mosquito by Trizol methods, constructed eDNA library, and got the exon of acetylcholinesterase. The acetylcholinesterase from culex was expressed by Bac-to-Bac system, then the enzyme was purified using Ni-sepharose. The crude and purified enzyme was tested and verified by SDS-PAGE. The enzyme activity was determined by gllman methods. It will be of great significance to biosensors in organophosphorous pesticides detection.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2012年第4期423-427,共5页
Journal of Huazhong Agricultural University
基金
国家"863"高技术研究发展计划项目(2009AA06A417)
