重组人乙酰胆碱酯酶基因在毕赤酵母中分泌表达及其对农药的敏感性评估
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摘要
乙酰胆碱酯酶是用酶抑制法检测有机磷和氨基甲酸酯类农药残留的关键用酶,本研究利用毕赤酵母表达系统分泌表达人乙酰胆碱酯酶,并评估其对8种农药的敏感性,为工业化大规模生产高敏感性酶源奠定基础。根据GenBank上已发表的人乙酰胆碱酯酶(human acetylcholinesterase, hAChE)基因核酸序列设计引物,从pReceiverM02-h AChE-ORF质粒中扩增出hAChE的开放阅读框(open reading frame, ORF)片段,接着利用SnaBⅠ和AvrⅡ双酶切将其克隆至载体pPIC9K中,构建pPIC9K-hAChE表达载体,然后将SalⅠ线性化的pPIC9K-hAChE电转化至毕赤酵母GS115中,在含4.00 mg/mL G418抗性平板上筛选获得阳性克隆,最后经0.5%甲醇诱导分泌表达144 h,发现其发酵上清液的十二烷基磺酸钠-聚丙烯酰胺凝胶电泳图谱在65 kDa处有一条明显的蛋白条带,大小与预期的hAChE蛋白一致。利用Q-Sepharose FF柱层析法对其总蛋白进行纯化,获得了较纯的人乙酰胆碱酯酶。用Bradford法测得发酵上清液中hAChE蛋白质量浓度为1.330 mg/mL,占总蛋白的5.58%,纯化后比酶活达到1 080nmol/(min·mL)。通过初步计算比对重组工程菌发酵上清液对8种常见农药的半抑制浓度,得出敌百虫>敌敌畏>克百威>马拉硫磷>乐果>甲胺磷>甲萘威>毒死蜱,即人乙酰胆碱酯酶对毒死蜱最敏感。
Acetylcholinesterase(AChE) is a key enzyme for the detection of organophosphorus and carbamate pesticide residues by enzyme inhibition method. In this study, human acetylcholinesterase(hAChE) was secreted and expressed by Pichia pastoris expression system, and its sensitivity to eight pesticides was evaluated, which laid the foundation for large-scale industrial production of highly sensitive enzyme sources. Primers F1 and R1 were designed according to the published gene sequence of hAChE in GenBank. Then the hAChE gene coding region sequence was amplified by high fidelity PCR using pReceiver-M02-hAChE-ORF plasmid as the template, and the recombinant expression vector pPIC9 K-hAChE was constructed by inserting into the vector pPIC9K through double enzyme digestion of SnaB Ⅰ and Avr Ⅱ. The transformant was selected to sequence and verify the accuracy of the sequence. The linearized pPIC9 K-hAChE by Sal Ⅰ was electro-transformed into P.pastoris GS115. The recombinant strains were screened by minimal dextrase medium(MD) plate and G418 resistance gradient plate. Finally, the positive strain colony was screened on the yeast extract peptone dextrose medium(YPD) plate with 4.00 mg/mL G418 resistance and was identified by polymerase chain reaction(PCR).The expression of the recombinant strain was induced by 0.5% methanol for 144 h. The sodium dodecyl sulfonate(SDS)-polyacrylamide gel electrophoresis map of the fermentation supernatant showed an obvious protein band at 65 kDa, and the size was consistent with the expected hAChE protein. The purified hAChE was obtained using a QSepharose FF chromatography. The content of hAChE protein in the fermentation supernatant was 1.330 mg/m L and accounted for 5.58% in total protein by Bradford method. The highest enzyme activity reached 1 080 nmol/(min·mL)after purification. By preliminary calculation of the 50% inhibitory concentration(IC50) of the eight common pesticides of the fermentation supernatant, it was found that trichlorfon>dichlorvos>arbofuran>malathion>dimethoate>Methamidophos>Carbaryl>chlorpyrifos, suggesting that hAChE is most sensitive to chlorpyrifos.
Acetylcholinesterase(AChE) is a key enzyme for the detection of organophosphorus and carbamate pesticide residues by enzyme inhibition method. In this study, human acetylcholinesterase(hAChE) was secreted and expressed by Pichia pastoris expression system, and its sensitivity to eight pesticides was evaluated, which laid the foundation for large-scale industrial production of highly sensitive enzyme sources. Primers F1 and R1 were designed according to the published gene sequence of hAChE in GenBank. Then the hAChE gene coding region sequence was amplified by high fidelity PCR using pReceiver-M02-hAChE-ORF plasmid as the template, and the recombinant expression vector pPIC9 K-hAChE was constructed by inserting into the vector pPIC9K through double enzyme digestion of SnaB Ⅰ and Avr Ⅱ. The transformant was selected to sequence and verify the accuracy of the sequence. The linearized pPIC9 K-hAChE by Sal Ⅰ was electro-transformed into P.pastoris GS115. The recombinant strains were screened by minimal dextrase medium(MD) plate and G418 resistance gradient plate. Finally, the positive strain colony was screened on the yeast extract peptone dextrose medium(YPD) plate with 4.00 mg/mL G418 resistance and was identified by polymerase chain reaction(PCR).The expression of the recombinant strain was induced by 0.5% methanol for 144 h. The sodium dodecyl sulfonate(SDS)-polyacrylamide gel electrophoresis map of the fermentation supernatant showed an obvious protein band at 65 kDa, and the size was consistent with the expected hAChE protein. The purified hAChE was obtained using a QSepharose FF chromatography. The content of hAChE protein in the fermentation supernatant was 1.330 mg/m L and accounted for 5.58% in total protein by Bradford method. The highest enzyme activity reached 1 080 nmol/(min·mL)after purification. By preliminary calculation of the 50% inhibitory concentration(IC50) of the eight common pesticides of the fermentation supernatant, it was found that trichlorfon>dichlorvos>arbofuran>malathion>dimethoate>Methamidophos>Carbaryl>chlorpyrifos, suggesting that hAChE is most sensitive to chlorpyrifos.
引文
[1]THOMAS S S.Characterization of human metabolism and exposures to organophosphorus and pyrethroid insecticides.New York,US:Faculty of the Graduate School of the State University of New York,University at Buffalo,2015:2-18.
[2]ZHANG F N,WU J,YING B Y,et al.Carbon nanomaterialenabled pesticide biosensors:design strategy,biosensing mechanism,and practical application.Trends in Analytical Chemistry,2018,106:62-83.
[3]WEI J C,WEI B,YANG W,et al.Trace determination of carbamate pesticides in medicinal plants by a fluorescent technique.Food and Chemical Toxicology,2018,119:430-437.
[4]伍小红,李建科,惠伟.农药残留对食品安全的影响及对策.食品与发酵工业,2005,31(6):80-84.WU X H,LI J K,HUI W.The effect of pesticide residues on food safety and resolve strategies.Food and Fermentation Industries,2005,31(6):80-84.(in Chinese with English abstract)
[5]柴丽月,常卫民,陈树兵,等.食品中农药残留分析技术研究.食品科学,2006,27(7):260-264.CHAI L Y,CHANG W M,CHEN S B,et al.Review on analytical technology of pesticide residues in food.Food Science,2006,27(7):260-264.(in Chinese with English abstract)
[6]李太平.食品中农药最大残留限量标准的安全漏洞分析.食品科学,2011,32(3):266-271.LI T P.Safety trap analysis of maximal residue limits of pesticide in foods.Food Science,2011,32(3):266-271.(in Chinese with English abstract)
[7]赵建庄,康国瑞.快速测定果蔬中农药残留量的方法研究.农业环境保护,2002,21(1):70-71.ZHAO J Z,KANG G R.Methodology for rapid determination of pesticide residues in vegetables and fruits.Agro-Environ‐mental Protection,2002,21(1):70-71.(in Chinese with English abstract)
[8]刘永杰,张金振.酶抑制法快速检测农产品农药残留的研究与应用.现代农药,2004,3(2):25-27.LIU Y J,ZHANG J Z.Study and application of enzyme inhibition method for rapid detection of pesticide residues in agricultural products.Modern Agrochemicals,2004,3(2):25-27.(in Chinese)
[9]林春绵,胡晓燕,张安平.酶抑制法快速检测有机磷农药残留的研究进展.浙江工业大学学报,2009,37(4):386-391.LIN C M,HU X Y,ZHANG A P.Development of enzyme inhibition for rapid detection of organophosphorus pesticide residues.Journal of Zhejiang University of Technology,2009,37(4):386-391.(in Chinese with English abstract)
[10]MIROSLAV P,KAMIL M,KAMIL K.Progress of biosen‐sors based on cholinesterase inhibition.Current Medicinal Chemistry,2009,16(14):1790-1798.
[11]宋国林,王宁华.乙酰胆碱酯酶法检测果蔬农药残留的研究.生物防护防疫装备,2005,26(4):31-32.SONG G L,WANG N H.The inspection of the pesticide remains on the fruit and vegetable by acetylcholinesterase.Biological Protective and Anti-epidemic Equipment,2005,26(4):31-32.(in Chinese with English abstract)
[12]何东海,孟范平,朱小山.有机磷农药对海洋动物乙酰胆碱酯酶(ACh E)的毒性效应研究进展.海洋通报,2003,22(6):71-78.HE D H,MENG F P,ZHU X S.Advances in the study on toxic effect of organophosphorus pesticides on acetylcholinesterases(ACh E)of marine animals.Marine Science Bulletin,2003,22(6):71-78.(in Chinese with English abstract)
[13]LI F,HAN Z J.Purification and characterization of acetylcholinesterase from cotton aphid(Aphis gossypii Glove).Archives of Insect Biochemistry and Physiology,2002,51(1):37-45.
[14]王金斌,谭芙蓉,王利刚,等.乙酰胆碱酯酶酶抑制法快速检测农药残留的研究进展.上海农业学报,2009,25(4):131-135.WANG J B,TAN F R,WANG L G,et al.Advances on rapid detection of pesticide residues by the method of restraining acetylcholinesterase activity.Acta Agriculturae Shanghai,2009,25(4):131-135.(in Chinese with English abstract)
[15]朱美财,辛颜彬,孙曼霁,等.人脑乙酰胆碱酯酶的分离纯化及特性.中国科学:B辑,1992,22(8):835-841.ZHU M C,XIN Y B,SUN M J,et al.Purification and characterization of acetylcholinesterase from human brain.Science in China:Series B,1992,22(8):835-841.(in Chinese)
[16]王旭,高希武,郑炳宗,等.有机磷药剂对棉铃虫羧酸酯酶的抑制作用.昆虫学报,1998,41(增刊1):12-18.WANG X,GAO X W,ZHENG B Z,et al.Inhibition of organophosphorus insecticides to carboxylesterase in Helicoverpa armigera.Acta Entomologica Sinica,1998,41(Suppl.1):12-18.(in Chinese with English abstract)
[17]SCHULZE H,MUENCH S B,VILLATE F,et al.Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.Analytical Chemistry,2005,77(18):5823-5830.
[18]彭宇,王荫长,韩召军,等.二化螟体内乙酰胆碱酯酶的分布及纯化方法.昆虫学报,2002,45(2):209-214.PENG Y,WANG Y C,HAN Z J,et al.Distribution and purification of acetylcholinesterase in Chilo suppressalis.Acta Entomologica Sinica,2002,45(2):209-214.(in Chinese with English abstract)
[19]IVANOV A N,EVTUGYN G A,GYURCSAYI R E,et al.Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination.Analytica Chimica Acta,2000,404(2):55-59.
[20]张健慧,王首锋.膜荚黄芪苯丙氨酸解氨酶基因在毕赤酵母中的分泌表达.浙江大学学报(农业与生命科学版),2014,40(1):1-8.ZHANG J H,WANG S F.Expression of Astragalus membranaceus phenylalanine ammonia-lyase gene in Pichia pastoris.Journal of Zhejiang University(Agriculture and Life Sciences),2014,40(1):1-8.(in Chinese with English abstract)
[21]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,1976,72(1):248-254.
[22]LAYNE E.Spectrophotometric and turbidimetric methods for measuring proteins.Methods in Enzymology,1957,3:447-455.
[23]GORUN V,PROINOV I,BALTESCU V,et al.Modified Ellman procedure for assay of cholinesterase in crude enzymatic preparation.Analytical Biochemistry,1978,86(1):324-326.
[24]高希武.Gorun等改进的Ellman胆碱酯酶活性测定方法介绍.昆虫知识,1987,24(4):245-246.GAO X W.Gorun’s introduction of improved Ellman cholinesterase activity determination method.Insect Knowledge,1987,24(4):245-246.(in Chinese)
[25]XU S C,WU A B,CHEN H D,et al.Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues.Biomolecular Engineering,2007,24(2):253-261.
[26]陈浩德.重组黑腹果蝇乙酰胆碱酯酶在甲醇酵母中表达的改善和基于此的新型农药残留快速检测传感器的改进.上海:上海交通大学,2008.CHEN H D.Improvement of the expression of recombinant Drosophila melanogaster acetylcholinesterase in P.pastoris and of the derived new biosensor for fast detection of pesticide residues.Shanghai:Shanghai Jiao Tong University,2008.(in Chinese with English abstract)
[27]张春媚.重组人乙酰胆碱酯酶治疗有机磷(敌敌畏)中毒初步研究.长春:吉林大学,2011.ZHANG C M.The initial study of the recombinant human acetylcholinesterase treatment of organophosphorous(dichlorvos)poisoning.Changchun:Jilin University,2011.(in Chinese with English abstract)
[28]韩雪清,刘湘涛,尹双辉.毕赤酵母表达系统.微生物学杂志,2003,23(4):35-36.HAN X Q,LIU X T,YIN S H.Pichia pastoris expression system.Journal of Microbiology,2003,23(4):35-36.(in Chinese with English abstract)
[2]ZHANG F N,WU J,YING B Y,et al.Carbon nanomaterialenabled pesticide biosensors:design strategy,biosensing mechanism,and practical application.Trends in Analytical Chemistry,2018,106:62-83.
[3]WEI J C,WEI B,YANG W,et al.Trace determination of carbamate pesticides in medicinal plants by a fluorescent technique.Food and Chemical Toxicology,2018,119:430-437.
[4]伍小红,李建科,惠伟.农药残留对食品安全的影响及对策.食品与发酵工业,2005,31(6):80-84.WU X H,LI J K,HUI W.The effect of pesticide residues on food safety and resolve strategies.Food and Fermentation Industries,2005,31(6):80-84.(in Chinese with English abstract)
[5]柴丽月,常卫民,陈树兵,等.食品中农药残留分析技术研究.食品科学,2006,27(7):260-264.CHAI L Y,CHANG W M,CHEN S B,et al.Review on analytical technology of pesticide residues in food.Food Science,2006,27(7):260-264.(in Chinese with English abstract)
[6]李太平.食品中农药最大残留限量标准的安全漏洞分析.食品科学,2011,32(3):266-271.LI T P.Safety trap analysis of maximal residue limits of pesticide in foods.Food Science,2011,32(3):266-271.(in Chinese with English abstract)
[7]赵建庄,康国瑞.快速测定果蔬中农药残留量的方法研究.农业环境保护,2002,21(1):70-71.ZHAO J Z,KANG G R.Methodology for rapid determination of pesticide residues in vegetables and fruits.Agro-Environ‐mental Protection,2002,21(1):70-71.(in Chinese with English abstract)
[8]刘永杰,张金振.酶抑制法快速检测农产品农药残留的研究与应用.现代农药,2004,3(2):25-27.LIU Y J,ZHANG J Z.Study and application of enzyme inhibition method for rapid detection of pesticide residues in agricultural products.Modern Agrochemicals,2004,3(2):25-27.(in Chinese)
[9]林春绵,胡晓燕,张安平.酶抑制法快速检测有机磷农药残留的研究进展.浙江工业大学学报,2009,37(4):386-391.LIN C M,HU X Y,ZHANG A P.Development of enzyme inhibition for rapid detection of organophosphorus pesticide residues.Journal of Zhejiang University of Technology,2009,37(4):386-391.(in Chinese with English abstract)
[10]MIROSLAV P,KAMIL M,KAMIL K.Progress of biosen‐sors based on cholinesterase inhibition.Current Medicinal Chemistry,2009,16(14):1790-1798.
[11]宋国林,王宁华.乙酰胆碱酯酶法检测果蔬农药残留的研究.生物防护防疫装备,2005,26(4):31-32.SONG G L,WANG N H.The inspection of the pesticide remains on the fruit and vegetable by acetylcholinesterase.Biological Protective and Anti-epidemic Equipment,2005,26(4):31-32.(in Chinese with English abstract)
[12]何东海,孟范平,朱小山.有机磷农药对海洋动物乙酰胆碱酯酶(ACh E)的毒性效应研究进展.海洋通报,2003,22(6):71-78.HE D H,MENG F P,ZHU X S.Advances in the study on toxic effect of organophosphorus pesticides on acetylcholinesterases(ACh E)of marine animals.Marine Science Bulletin,2003,22(6):71-78.(in Chinese with English abstract)
[13]LI F,HAN Z J.Purification and characterization of acetylcholinesterase from cotton aphid(Aphis gossypii Glove).Archives of Insect Biochemistry and Physiology,2002,51(1):37-45.
[14]王金斌,谭芙蓉,王利刚,等.乙酰胆碱酯酶酶抑制法快速检测农药残留的研究进展.上海农业学报,2009,25(4):131-135.WANG J B,TAN F R,WANG L G,et al.Advances on rapid detection of pesticide residues by the method of restraining acetylcholinesterase activity.Acta Agriculturae Shanghai,2009,25(4):131-135.(in Chinese with English abstract)
[15]朱美财,辛颜彬,孙曼霁,等.人脑乙酰胆碱酯酶的分离纯化及特性.中国科学:B辑,1992,22(8):835-841.ZHU M C,XIN Y B,SUN M J,et al.Purification and characterization of acetylcholinesterase from human brain.Science in China:Series B,1992,22(8):835-841.(in Chinese)
[16]王旭,高希武,郑炳宗,等.有机磷药剂对棉铃虫羧酸酯酶的抑制作用.昆虫学报,1998,41(增刊1):12-18.WANG X,GAO X W,ZHENG B Z,et al.Inhibition of organophosphorus insecticides to carboxylesterase in Helicoverpa armigera.Acta Entomologica Sinica,1998,41(Suppl.1):12-18.(in Chinese with English abstract)
[17]SCHULZE H,MUENCH S B,VILLATE F,et al.Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.Analytical Chemistry,2005,77(18):5823-5830.
[18]彭宇,王荫长,韩召军,等.二化螟体内乙酰胆碱酯酶的分布及纯化方法.昆虫学报,2002,45(2):209-214.PENG Y,WANG Y C,HAN Z J,et al.Distribution and purification of acetylcholinesterase in Chilo suppressalis.Acta Entomologica Sinica,2002,45(2):209-214.(in Chinese with English abstract)
[19]IVANOV A N,EVTUGYN G A,GYURCSAYI R E,et al.Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination.Analytica Chimica Acta,2000,404(2):55-59.
[20]张健慧,王首锋.膜荚黄芪苯丙氨酸解氨酶基因在毕赤酵母中的分泌表达.浙江大学学报(农业与生命科学版),2014,40(1):1-8.ZHANG J H,WANG S F.Expression of Astragalus membranaceus phenylalanine ammonia-lyase gene in Pichia pastoris.Journal of Zhejiang University(Agriculture and Life Sciences),2014,40(1):1-8.(in Chinese with English abstract)
[21]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,1976,72(1):248-254.
[22]LAYNE E.Spectrophotometric and turbidimetric methods for measuring proteins.Methods in Enzymology,1957,3:447-455.
[23]GORUN V,PROINOV I,BALTESCU V,et al.Modified Ellman procedure for assay of cholinesterase in crude enzymatic preparation.Analytical Biochemistry,1978,86(1):324-326.
[24]高希武.Gorun等改进的Ellman胆碱酯酶活性测定方法介绍.昆虫知识,1987,24(4):245-246.GAO X W.Gorun’s introduction of improved Ellman cholinesterase activity determination method.Insect Knowledge,1987,24(4):245-246.(in Chinese)
[25]XU S C,WU A B,CHEN H D,et al.Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues.Biomolecular Engineering,2007,24(2):253-261.
[26]陈浩德.重组黑腹果蝇乙酰胆碱酯酶在甲醇酵母中表达的改善和基于此的新型农药残留快速检测传感器的改进.上海:上海交通大学,2008.CHEN H D.Improvement of the expression of recombinant Drosophila melanogaster acetylcholinesterase in P.pastoris and of the derived new biosensor for fast detection of pesticide residues.Shanghai:Shanghai Jiao Tong University,2008.(in Chinese with English abstract)
[27]张春媚.重组人乙酰胆碱酯酶治疗有机磷(敌敌畏)中毒初步研究.长春:吉林大学,2011.ZHANG C M.The initial study of the recombinant human acetylcholinesterase treatment of organophosphorous(dichlorvos)poisoning.Changchun:Jilin University,2011.(in Chinese with English abstract)
[28]韩雪清,刘湘涛,尹双辉.毕赤酵母表达系统.微生物学杂志,2003,23(4):35-36.HAN X Q,LIU X T,YIN S H.Pichia pastoris expression system.Journal of Microbiology,2003,23(4):35-36.(in Chinese with English abstract)