Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (11): 2189-2198.doi: 10.3864/j.issn.0578-1752.2012.11.007

• PLANT PROTECTION • Previous Articles     Next Articles

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

 HE  Yong-Sheng, CHEN  Shu-Chi, WANG  Hong, HAN  Shuang-Yan, YANG  Qiong, LIU  Xi-Xia, DONG  Jie-Xian, YANG  Wu-Ying, SUN  Yuan-Ming   

  1. 1.广东省食品质量与安全重点实验室/华南农业大学食品质量与安全研究所，广州 510642
    2.华南理工大学生物科学与工程学院，广州 510006
    3.广东省农业科学院蚕业与农产品加工研究所，广州510610
  • Received:2011-08-15 Online:2012-06-01 Published:2011-11-29

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Abstract: 【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.

Key words: Pichia pastoris, recombinant Bombyx mori acetylcholinesterase, expression, bioactivity assay, organophosphorus pesticides, carbamate pesticides

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