Secretory Expression Of Recombinant Human And Drosophila Melanogaster Acetylcholinesterase In Pichia Pastoris And Evaluation Of Their Sensitivity To Pesticides

Background:For the rapid detection of organophosphorus and carbamate pesticides,enzyme inhibition method is the most studied method at home and abroad,and it is also the most simple,sensitive,economical and fast detection method.The activity of acetylcholinesterase(AChE)is specifically inhibited by organophosphorus and carbamate pesticides,so it becomes a target enzyme for monitoring organophosphorus and carbamate pesticides.Based on the results of comparative analysis of the acetylcholinesterase sources which have been studied by Schulze et al.we found that the bimolecular rate constants of acetylcholinesterases from human and drosophila melanogaster were much higher than those from other sources,which proved that the acetylcholinesterase sources of human and drosophila melanogaster were more sensitive to pesticides.Acetylcholinesterases from human and drosophila melanogaster were used as the research object.At present,acetylcholinesterase is mainly extracted from insect tissues or animal blood,which is difficult to isolate and purify acetylcholinesterases and has such problems as its low yield,high cost and large sensitivity difference.Therefore,it is particularly important to find a low-cost and efficient production system.The proteins expressed in pichia pastoris expression system are generally protein-active,and have the characteristics of high-density fermentation,low cost of raw materials,high genetic stability,high expression efficiency,secretory expression and post-translational processing modification,which can meet the need of large-scale industrial production of acetylcholinesterase.At present,the system has been widely promoted and applied.Objective:The secretory expression system of pichia pastoris was used to secrete and express acetylcholinesterases from human and drosophila melanogaster respectively,whose sensitivity of eight common pesticides on the market was tested.The preliminary research on acetylcholinesterase from these two sources was carried out at laboratory level,which laid a foundation for large-scale industrial production.Methods:In order to realize the secretion and expression of human and drosophila melanogaster acetylcholinesterase in pichia pastoris,the primers F1/R1 ? F2/R2 were designed according to the nucleic acid sequence of human and drosophila melanogaster acetylcholinesterase genes published on Genbank·The ORF fragments of hAChE and dmAChE genes were amplified by high fidelity PCR and RT-PCR respectively.The expression vector pPIC9K of pichia pastoris was selected.The ORF fragments of hAChE and dmAChE genes were cloned into the vector pPIC9K by SnaB ? and Avr ?digestion.The recombinant plasmids were verified by PCR and double digestion and then the positive recombinants were sequenced.The recombinant plasmids were linearized by Sal I and then transformed into pichia pastoris GS115 by electroporation.HIS+positive transformants were screened by MD plate and multi-copy strains were screened by YPD plate containing G418 resistance gradient.Colony PCR was used to verify the positive strains.Finally,AChE-positive yeast expression strains of human and drosophila melanogaster were screened on 4.0 mg/mL G418 YPD resistance plate respectively.Mut phenotype was verified by universal primers,which showed that GS115/pPIC9K-hAChE was MutS type,GS115/pPIC9K-dmAChE was Mut+type.The recombinant GS115 strains were induced to express with 0.5%methanol,and the fermentation products were purified by Q-sepharose FF column chromatography.A relatively pure acetylcholinesterase was obtained.The gel analysis of SDS-PAGE protein was carried out on the expressed products.The modified Ellman method was used to detect the enzyme activity of the fermented expression products and the content of protein was determined by Bradford.The IC50 of fermentation supernatants of GS115/pPIC9K-hAChE and GS115/pPIC9K-dmAChE recombinant engineering strains for dimethoate,dichlorvos,methamidophos,trichlorfon,malathion,methamphetamine,chlorpyrifos and carbofiuran were compared by preliminary calculation.Finally,the sensitivity and validity of enzyme sources were evaluated.Results:The results showed that there was an obvious protein band at 65 kDa in GS115/pPIC9K-hAChE,which was consistent with the expected hAChE protein.The protein content of hAChE was 1.33 mg/mL,accounting for 5.58%of the total protein.The highest specific enzyme activity reached 1080 U/mL.GS115/pPIC9K-dmAChE had an obvious protein band at 67 kDa,the size of which was consistent with the expected dmAChE protein.The protein content of dmAChE was 2.52 mg/mL,accounting for 10.58%of the total protein.The highest specific enzyme activity of dmAChE protein reached 1914 U/mL.The results of GS115/pPIC9K-hAChE for eight pesticides IC50 were as follows:trichlorfon>dichlorvos>carbofuran>malathion>dimethoate>methamidophos>carbaryl>chlorpyrifos,that is,human acetylcholinesterase,was the most sensitive to chlorpyrifos.The sensitivity of GS115/pPIC9K-dmAChE to eight pesticides was also determined.The results of GS115/pPIC9K-dmAChE for eight pesticides IC50 were as follows:dimethoate>methamidophos>carbofuran>malathion>dichlorvos>carbaryl>trichlorfon>chlorpyrifos.Drosophila acetylcholinesterase is most sensitive to chlorpyrifos.