家蚕乙酰胆碱酯酶基因bmace在毕赤酵母GS115中的表达及其分析

Expression of Bombyx mori Acetylcholinesterase Gene bmace in Pichia pastoris GS115 and Analysis of Its Bioactivity

【目的】构建表达家蚕乙酰胆碱酯酶(BmAChE)的毕赤酵母菌株,实现BmAChE在毕赤酵母中分泌表达并对其活性进行分析。【方法】从家蚕头部提取总RNA,经RT-PCR反转录成cDNA,通过PCR扩增出家蚕乙酰胆碱酯酶基因bmace并进行序列测定。采用PCR及重叠延伸PCR去除原bmace中的信号肽和疏水肽,并对序列中存在的2个A+T富含区进行同义密码子替换的改造。将改造后的bmace与表达载体pPIC9K连接,转化毕赤酵母GS115,甲醇诱导表达。SDS-PAGE和Western blotting鉴定重组蛋白,以Ellman法测定酶活力,毒扁豆碱和有机磷及氨基甲酸酯类农药抑制试验分析重组BmAChE(rBmAChE)的生物活性。【结果】改造后的bmace经重组毕赤酵母菌株分泌表达了1个分子量约为70 kD的蛋白,具有AChE活性,酶比活力约为1 187 U.mg-1。抑制试验显示,该rBmAChE能够被毒扁豆碱强烈抑制,并且可被皮蝇磷等10种有机磷农药和克百威等5种氨基甲酸酯类农药抑制,最低抑制浓度IC50值可达2.78×10-10mol.L-1。【结论】成功构建分泌表达BmAChE的毕赤酵母菌株,其表达了具有良好活性的rBmAChE,可用于进一步制备有机磷及氨基甲酸酯类农药快速检测分析制剂。

【Objective】The objective of this study is to construct a recombinant Pichia pastoris strain expressing Bombyx mori acetylcholinesterase（BmAChE） and analyze its bioactivity.【Method】cDNA encoding AChE was isolated from the silkworm,B.mori,then N-terminal signal peptide and C-termianl hydrophobic peptide were truncated by PCR and two AT-rich regions were replaced by splicing overlap extension.The modified AChE gene bmace was recombined into the pPIC9K vector and expressed in P.pastoris GS115.The recombinant BmAChE（rBmAChE） was identified by SDS-PAGE and Western blotting analysis.The enzyme activity was detected according to Ellman method.In addition,the bioactivity was verified by inhibition test with eserine and organophosphorus or carbamate pesticides.【Result】The modified AChE gene bmace could be expressed in P.pastoris GS115.The resulting 70 kD recombinant protein exhibited AChE activity and the specific enzyme activity is up to 1 187 U.mg-1.An inhibition assay indicated that the AChE was strongly sensitive to eserine as well as ten kinds of organophosphorus pesticdes and five kinds of carbamate pesticides.The lowest IC50 value for methiocarb was 2.78×10-10 mol.L-1.【Conclusion】BmAChE was expressed in P.pastoris strains GS115 successfully and possessed well bioactivity.