Abstract:

This study aims to express housefly acetylcholinesterase (AChE) using gene recombination technology and a baculovirus-mediated Sf9 insect cell expression system, to provide a scientific basis for the development of AChE as a reagent for insecticide residue detection and insect resistance analysis. Using the pcDNA3.1-Md-ache plasmid containing the housefly AChE gene (Md-ache) as a template, EcoRⅠ and XhoⅠ restriction sites were introduced through PCR amplification to construct the recombinant plasmid Peasy-T1-Md-ache, which was further ligated with the pFastBac1 vector to obtain the recombinant plasmid pFastBac1-Md-ache. After transfection into DH10Bac competent cells, recombinant bacmid plasmids were screened and subsequently transfected into Sf9 cells for protein expression and identification. The results demonstrated that (1) the recombinant bacmid plasmids were successfully expressed in the Sf9 cells, with transfected cells exhibiting typical cytotoxic effects including cell enlargement and proliferation inhibition; (2) the recombinant housefly AChE exhibited catalytic activity, with enzyme activity reaching 44.7 ± 2.3 nmol/(min·mg protein) in cell lysates and 10.1 ± 2.1 nmol/(min·mL) in the culture medium, indicating secretory expression; (3) the Km value of the recombinant AChE was measured to be 15.69 ± 1.75 μmol/L using the Lineweaver-Burk double-reciprocal plot method, suggesting high substrate affinity; (4) the organophosphorus insecticide chlorpyrifos inhibited the recombinant AChE activity in vitro, with a half-maximal inhibitory concentration (IC₅₀) of 11.18 ± 0.68 μmol/L calculated by using the modified Karber method. This study confirms that the baculovirus-mediated Sf9 cell expression system can efficiently express housefly AChE with both catalytic activity and sensitivity to insecticide. The expression product provides a key material for the development of the reagent which is expected to be used for the insecticide residue detection and the insect resistance analysis.

Key words: housefly, acetylcholinesterase, AChE gene, gene recombination, baculovirus expression system, Sf9 cell, organophosphorus insecticide, carbamate insecticide