中华蜜蜂乙酰胆碱酯酶基因的克隆及其在毕赤酵母中的表达鉴定

doi:10.3969/j.issn.1673-1689.2016.12.016

首页 > 过刊浏览>2016年第35卷第12期 >1336-1343. DOI:10.3969/j.issn.1673-1689.2016.12.016

中华蜜蜂乙酰胆碱酯酶基因的克隆及其在毕赤酵母中的表达鉴定
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                        10.3969/j.issn.1673-1689.2016.12.016
                    
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Cloning，Expression and Identification of Apiscerana cerana Acetylcholinesterase Gene in Pichia pastoris

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有机磷污染是食品安全的重大隐患，乙酰胆碱酯酶（Acetyl cholinesterase，AChE）可用于有机磷农药残留的检测。中华蜜蜂是我国独有的蜜蜂当家品种，蜜蜂对农药的敏感性很高，中华蜜蜂来源的乙酰胆碱酯酶基因尚未被克隆并应用于食品安全监测。以中华蜜蜂头部组织总RNA为模板，经RACE方法获得了AChE基因cDNA全序列，全长约1.8 kb。构建重组表达载体pPIC3.5K-AChE，将目的基因表达盒整合入毕赤酵母GS115的基因组中获得重组菌株，0.5%甲醇诱导重组菌株表达AChE蛋白，经SDS-PAGE、酶活性分析表明，中华蜜蜂AChE基因首次克隆成功并首次在毕赤酵母中获得成功表达，筛选出一株酶活性较高的菌株，纯化的重组乙酰胆碱酯酶其活性为10 568 U/mg，可以用于有机磷农药残留的检测。

Abstract:

Organophosphate（OP） pollution is a major hidden danger to food safety，acetylcholinesterase can be used to detect the residual OP pesticide. Apiscerana cerana is a unique kind of Apoidea which has not been found in other countries except for China，and it has a high sensitivity to pesticides. On the other hand，acetylcholinesterase gene from Apiscerana cerana has not been cloned and applied in food safety detection. In this study，using the total RNA from Apiscerana cerana head tissue as a template，a 1.8kb AChE cDNA sequence was obtained by RACE method，then the target sequence was cloned into the yeast expression vector pPIC3.5K. AChE was expressed and located in Pichia pastoris GS115 after induced with 0.5% methanol. The SDS-PAGE and activity analysis showed that Apiscerana cerana AChE gene was successfully cloned and expressed in Pichia pastoris for the first time. A strain with higher activity AChE was selected to express AChE，and its AChE activity was 10 568 U/mg，which could be used to detect the residual OP pesticide.

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曹雪,陈占宽,白仲虎,杨艳坤,陈继峰.中华蜜蜂乙酰胆碱酯酶基因的克隆及其在毕赤酵母中的表达鉴定[J].食品与生物技术学报,2016,35(12):1336-1343.

CAO Xue, CHEN Zhankuan, BAI Zhonghu, YANG Yankun, CHEN Jifeng. Cloning，Expression and Identification of Apiscerana cerana Acetylcholinesterase Gene in Pichia pastoris[J]. Journal of Food Science and Biotechnology,2016,35(12):1336-1343.

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