Preliminary Study On Expression Of Recombinant Acetylcholine-Sterase Based On Mammalian Cells(CHO-S、Expi293F)

Enzyme inhibition method based on acetylcholinesterase is widely used in the field of rapid detection of pesticide residues in food and plays a very important role.However,at present,the source of acetylcholinesterase is mainly based on the extraction of natural animals and plants and microbial samples,and the enzyme sources used are different and the quality is uneven,which leads to low reproducibility of detection results and lack of uniform standards.Compared with natural extraction method,recombinant expression preparation has the advantages of single product component,good stability and large-scale preparation,which is of great significance for standardized production and uniformity of detection results.This study focused on the exploration of recombinant acetylcholinesterase expression in mammalian cells.Firstly,the activity and pesticide inhibition rate of natural acetylcholinesterase which is widely used in the market were evaluated,and the species with higher activity and inhibition rate were optimized.On this basis,the eukaryotic expression vector of recombinant acetylcholinesterase was constructed,and the effects of core factors such as expression vector type,signal peptide type,mammalian cell type and transfection conditions on the expression of recombinant acetylcholinesterase were systematically analyzed.The expression conditions of soluble recombinant acetylcholinesterase were optimized,which provided the preliminary research basis for recombinant expression of acetylcholinesterase based on mammalian cells and its application in pesticide residue detection.The main research results were as follows:(1)The enzyme activity and pesticide inhibition rate of seven kinds of natural acetylcholinesterases widely used in the market were evaluated.The results showed that there were significant differences in activity,specific activity and pesticide inhibition rate of acetylcholinesterases from different species and brands,and the performance of acetylcholinesterases from electric eel was significantly better than that from flies.The enzyme activity and specific activity of electric eel were 0.26～0.87U/m L and 0.25～217 U/mg respectively,while those of fly were 0.24～0.28 U/m L and0.25～0.30 U/mg respectively.Dichlorvos in organophosphorus can generally inhibit acetylcholinesterase from flies and electric eels,and its inhibition rate is 83.6～91.4%;Carbamate pesticides generally inhibit acetylcholinesterase from flies and electric eels.The highest inhibition rate of carbofuran and methomyl on acetylcholinesterase is 98.9%and 97.5%,respectively.The inhibition effect of carbamate pesticides on acetylcholinesterase is significantly stronger than that of organophosphorus pesticides.(2)Taking acetylcholinesterase from Musca domestica as the research object,the coding gene of acetylcholinesterase from Musca domestica(mdAChE)was designed and synthesized by codon optimization,and four expression vectors of recombinant acetylcholinesterase from Musca domestica(pCMV3-Humaninsulin-mdAChE,pc DNA3.1(+)-Humaninsulin-mdAChE,pc DNA3.4-Humaninsulin-mdAChE and pCMV3-Human CD33-mdAChE)were successfully constructed.On this basis,transient transfection of recombinant acetylcholinesterase from Musca domestica was studied in CHO-S cells and Expi293 F cells,respectively.The results showed that pc DNA3.1(+)-Humaninsulin-mdAChE,pc DNA3.4-Humaninsulin-mdAChE and pCMV3-Human CD33-mdAChE were soluble expressed in CHO-S cells with activity of 0.085～0.152 U/m L;pc DNA3.1(+)-Humaninsulin-mdAChE and pc DNA3.4-Humaninsulin-mdAChE were expressed in Expi293 F cells.The activity of recombinant Musca domestica acetylcholinesterase was 0.113～0.141 U/m L.(3)Taking the acetylcholinesterase from Electrophorus electricus as the research object,the coding gene of Electrophorus electricus acetylcholinesterase(eleel AChE)was designed and synthesized by codon optimization,and three kinds of recombinant expression vectors of electric eel acetylcholinesterase(pCMV3-mouse heavy chaineleel AChE,pc DNA3.1(+)-mouse heavy chain-eleel AChE and pc DNA3.4-mouse heavy chain-eleel AChE)were successfully constructed.On this basis,transient transfection of recombinant acetylcholinesterase from Electrophorus electricus was studied in CHO-S cells and Expi293 F cells,respectively.The results showed that pc DNA3.1(+)-mouse heavy chain-eleel AChE and pc DNA3.4-mouse heavy chaineleel AChE were soluble expression of recombinant acetylcholinesterase derived from Electrophorus electricus in two kinds of mammalian cells.In CHO-S cells,the concentration of recombinant acetylcholinesterase from Electrophorus electricus was4.37 mg/m L,and the enzyme activity was 0.085～0.130 U/m L;In Expi293 F cells,the concentration of purified product of recombinant acetylcholinesterase from Electrophorus electricus was 7.19 mg/m L,and the enzyme activity was 0.107～0.152U/m L.However,the activity of recombinant acetylcholinesterase from Electrophorus electricus was low by enzyme activity test,and the key factors affecting its enzyme activity should be further studied.(4)The exploration of whether natural acetylcholinesterase is produced in the culture product of Expi293 F cells after natural apoptosis was carried out.After purifying the supernatant of Expi293 F cells cultured for 10 days,we found a product suspected of acetylcholinesterase with a molecular weight of about 67 k Da and a concentration of 24.12 mg/m L after ultrafiltration.The enzyme activity and specific activity were 0.464 U/m L and 0.019 U/mg,respectively.Further pesticide inhibition experiments showed that the suspected acetylcholinesterase product showed good sensitivity to organophosphorus and carbamate pesticides,and the inhibition rates of dichlorvos,chlorpyrifos and monocrotophos were 94.3%,60.6% and 57.1%,respectively.The inhibition rates of carbofuran,carbaryl,methomyl and methomyl in carbamate pesticides were 89.8%,86.7%,86.0% and 80.7%,respectively.This discovery provides a new way to obtain enzyme sources in the field of rapid detection of pesticide residues.In the future,we can continue to explore the function and structure of this enzyme,and provide a new way to obtain acetylcholinesterase enzyme sources for pesticide residue detection.