朱砂叶螨几丁质酶TecCht1基因的原核表达与纯化
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摘要
【目的】朱砂叶螨是世界范围广泛分布的重要经济害螨,害螨防治十分困难。而几丁质酶是昆虫体内几丁质代谢中分解几丁质的关键酶,在更替新旧表皮和降解围食膜中起着重要的作用,几丁质酶是潜在的螨害生物防治靶标。本研究对朱砂叶螨几丁质酶TecCht1进行原核表达和纯化。【方法】克隆朱砂叶螨TecCht1基因的催化结构域,连接到pColdⅡ载体上,构建pColdⅡ/TecCht1重组表达载体,在大肠杆菌中进行高效表达,采用镍柱纯化目的蛋白,并进行Western Blot检测。【结果】TecCht1在pColdⅡ载体上成功高水平表达,确定最佳诱导表达时间为24h。对TecCht1进行大量表达和纯化,经Western Blot验证,获得纯度较高的目的蛋白。【结论】实现TecCht1体外高效原核表达,获得大量的TecCht1重组蛋白。
【Objective】The carmine spider mite,Tetranychus cinnabarinus(Boisduval),is an important agricultural worldwide pest,and it is an increasing challenge to controlling mites.Chitinase is a key enzyme in insect chitin metabolism,and plays a critical role in molting and degrading peritrophic membranes.Chitinase can be potential target for pest biological control.This study is to express and purify the mite chitinase TecCht1 in E.Coli.【Methods】The catalytic domain of TecCht1 was cloned and ligated to pColdII vector,and then the pColdⅡ/TecCht1 recombinant expression vector was constructed.TecCht1 was highly expressed by IPTG induction,and then was purified using Ni-NTA column.The purified protein was verified by Western Blot.【Results】The TecCht1 protein was successfully expressed in pColdⅡ,and the optimal induction time was 24 h.High quality TecCht1 protein was obtained,which were verified by Western Blot.【Conclusion】In the study,TecCht1 was highly expressed by IPTG induction and purified TecCht1 protein was obtained.
【Objective】The carmine spider mite,Tetranychus cinnabarinus(Boisduval),is an important agricultural worldwide pest,and it is an increasing challenge to controlling mites.Chitinase is a key enzyme in insect chitin metabolism,and plays a critical role in molting and degrading peritrophic membranes.Chitinase can be potential target for pest biological control.This study is to express and purify the mite chitinase TecCht1 in E.Coli.【Methods】The catalytic domain of TecCht1 was cloned and ligated to pColdII vector,and then the pColdⅡ/TecCht1 recombinant expression vector was constructed.TecCht1 was highly expressed by IPTG induction,and then was purified using Ni-NTA column.The purified protein was verified by Western Blot.【Results】The TecCht1 protein was successfully expressed in pColdⅡ,and the optimal induction time was 24 h.High quality TecCht1 protein was obtained,which were verified by Western Blot.【Conclusion】In the study,TecCht1 was highly expressed by IPTG induction and purified TecCht1 protein was obtained.
引文
[1]Li S,Peng W,Wang X,Shen G,Zhang J,Xiao W,Xu Z,Xu Q,Lin H.Functional analysis of EsteraseTCE2Gene from Tetranychus cinnabarinus(Boisduval)involved in acaricide resistance[J].Scientific Reports,2016,6:18646
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[4]韩晶玉,楼怡宁,师光禄,陈青,彭博,卜春亚.朱砂叶螨乙酰胆碱酯酶真核表达载体构建及在293T细胞中表达条件[J].北京农学院学报,2015,30(4):24-28
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[7]陈秋双,赵舒,邹晶,石力,何林.朱砂叶螨抗药性监测[J].应用昆虫学报,2012,49(2):364-369
[8]张东博.药用植物中生物碱类乙酰胆碱酯酶抑制剂的筛选[D].兰州:兰州理工大学,2010
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[14]李玉泉,宋占午,金祖荫.朱砂叶螨危害初期豇豆幼苗叶片PPO、PAL及POD的研究[J].西北师范大学学报:自然科学版,2003,39(3):61-64
[15]李瑶,范晓军.昆虫几丁质酶及其在害虫防治中的应用[J].应用昆虫学报,2011,48(5):1489-1494
[16]Clarke P H,Tracey M V.The occurrence of chitinase in some bacteria[J].Journal of General Microbiology,1956,14(1):188
[17]Zhu Q,Arakane Y,Banerjee D,Kramer K J,Muthukrishnan S.Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J].Insect Biochemistry&Molecular Biology,2008,38(4):452-466
[18]Fukamizo T.Chitinolytic enzymes:catalysis,substrate binding,and their application[J].Current Protein&Peptide Science,2000,1(1):105-124
[19]Zhu Q,Arakane Y,Beeman R W,Kramer K J,Muthukrishnan S.Functional Specialization among Insect Chitinase Family Genes Revealed by RNA Interference[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(18):6650-6655
[20]Zhu Q,Arakane Y,Beeman R W,Kramer K J,Muthukrishnan S.Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum[J].Insect Biochemistry&Molecular Biology,2008,38(4):467-77
[21]樊东.几种鳞翅目昆虫几丁质酶基因的克隆与表达研究[D].哈尔滨:东北农业大学,2004
[22]Lin S,Liu Z Q,Yi M,Wu H,Xu F,Zheng YG.Mining and characterization of two novel chitinases from Hirsutella sinensis using an efficient transcriptome-mining approach[J].Protein Expression&Purification,2017,133:81
[23]Mohammadzadeh R,Agheshlouie M,Mahdavinia G R.Expression of chitinase gene in BL21pET system and investigating the biocatalystic performance of chitinase-loaded AlgSep nanocomposite beads[J].International Journal of Biological Macromolecules,2017,104(Pt B):1664-1671
[24]张道伟,陈静,张正玲,曾燕玲,郭玉双.二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备[J].东北农业大学学报,2015,46(1):62-67
[25]Kramer K J,Muthukrishnan S.Insect chitinases:molecular biology and potential use as biopesticides[J].Insect Biochemistry&Molecular Biology,1997,27(11):887
[26]Assenga S P,You M,Shy C H,Yamagishi J,Sakaguchi T,Zhou J,Kibe MK,Xuan X,Fujisaki K.The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis,and its potential application as a bioacaricide for tick control[J].Parasitology Research,2006,98(2):111-118
[27]李宏伟,王吉平,张堤,张春林,赵文静,张晶,翟素珍.美洲大蠊几丁质酶基因的原核表达及蛋白纯化[J].基因组学与应用生物学,2016,35(9):2311-2316
[28]Shinoda T,Kobayashi J,Matsui M,Chinzei Y.Cloning and functional expression of a chitinase cDNA from the common cutworm,Spodoptera litura,using a recombinant baculovirus lacking the virus-encoded chitinase gene[J].Insect Biochemistry&Molecular Biology,2001,31(6-7):521-532
[29]魏巍.棉铃虫几丁质酶cDNA克隆及表达研究[D].北京:中国农业科学院植物保护研究所,2002
[2]忻介六.农业螨类学[M].北京:中国农业出版社,1988:237-238
[3]Ros V I,Breeuwer J A.Spider mite(Acari:Tetranychidae)mitochondrial COI phylogeny reviewed:host plant relationships,phylogeography,reproductive parasites and barcoding[J].Experimental&Applied Acarology,2007,42(4):239-262
[4]韩晶玉,楼怡宁,师光禄,陈青,彭博,卜春亚.朱砂叶螨乙酰胆碱酯酶真核表达载体构建及在293T细胞中表达条件[J].北京农学院学报,2015,30(4):24-28
[5]刘波,桂连友.我国朱砂叶螨研究进展[J].长江大学学报:自科版,2007(3):9-12
[6]彭博,卜春亚,师光禄,李金玲,冯晓姣,韩晶玉,高品,王有年.朱砂叶螨乙酰胆碱酯酶的原核表达及其活性分析[J].应用与环境生物学报,2015,21(2):215-220
[7]陈秋双,赵舒,邹晶,石力,何林.朱砂叶螨抗药性监测[J].应用昆虫学报,2012,49(2):364-369
[8]张东博.药用植物中生物碱类乙酰胆碱酯酶抑制剂的筛选[D].兰州:兰州理工大学,2010
[9]Zhang J P,Wang J J,Zhao Z M,Dou W,Chen Y.Effects of simulated acid rain on the physiology of carmine spider mite,Tetranychus cinnabarinus(Boisduvals)(Acari:Tetranychidae)[J].Journal of Applied Entomology,2004,128(5):342-347
[10]郭凤英,赵志模.抗药性叶螨的种群参数和相对适合度[J].蛛形学报,2001(1):41-43
[11]唐振华.昆虫抗药性的遗传与进化[M].上海:上海科学技术文献出版社,2000
[12]Merzendorfer H,Zimoch L.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].Journal of Experimental Biology,2003,206(Pt 24):4393
[13]洪鹏,李博,沈宏亮,张鑫,师光禄,卜春亚.朱砂叶螨几丁质酶TecCht2基因的克隆及特征分析[J].北京农学院学报,2017,32(3):1-7
[14]李玉泉,宋占午,金祖荫.朱砂叶螨危害初期豇豆幼苗叶片PPO、PAL及POD的研究[J].西北师范大学学报:自然科学版,2003,39(3):61-64
[15]李瑶,范晓军.昆虫几丁质酶及其在害虫防治中的应用[J].应用昆虫学报,2011,48(5):1489-1494
[16]Clarke P H,Tracey M V.The occurrence of chitinase in some bacteria[J].Journal of General Microbiology,1956,14(1):188
[17]Zhu Q,Arakane Y,Banerjee D,Kramer K J,Muthukrishnan S.Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J].Insect Biochemistry&Molecular Biology,2008,38(4):452-466
[18]Fukamizo T.Chitinolytic enzymes:catalysis,substrate binding,and their application[J].Current Protein&Peptide Science,2000,1(1):105-124
[19]Zhu Q,Arakane Y,Beeman R W,Kramer K J,Muthukrishnan S.Functional Specialization among Insect Chitinase Family Genes Revealed by RNA Interference[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(18):6650-6655
[20]Zhu Q,Arakane Y,Beeman R W,Kramer K J,Muthukrishnan S.Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum[J].Insect Biochemistry&Molecular Biology,2008,38(4):467-77
[21]樊东.几种鳞翅目昆虫几丁质酶基因的克隆与表达研究[D].哈尔滨:东北农业大学,2004
[22]Lin S,Liu Z Q,Yi M,Wu H,Xu F,Zheng YG.Mining and characterization of two novel chitinases from Hirsutella sinensis using an efficient transcriptome-mining approach[J].Protein Expression&Purification,2017,133:81
[23]Mohammadzadeh R,Agheshlouie M,Mahdavinia G R.Expression of chitinase gene in BL21pET system and investigating the biocatalystic performance of chitinase-loaded AlgSep nanocomposite beads[J].International Journal of Biological Macromolecules,2017,104(Pt B):1664-1671
[24]张道伟,陈静,张正玲,曾燕玲,郭玉双.二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备[J].东北农业大学学报,2015,46(1):62-67
[25]Kramer K J,Muthukrishnan S.Insect chitinases:molecular biology and potential use as biopesticides[J].Insect Biochemistry&Molecular Biology,1997,27(11):887
[26]Assenga S P,You M,Shy C H,Yamagishi J,Sakaguchi T,Zhou J,Kibe MK,Xuan X,Fujisaki K.The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis,and its potential application as a bioacaricide for tick control[J].Parasitology Research,2006,98(2):111-118
[27]李宏伟,王吉平,张堤,张春林,赵文静,张晶,翟素珍.美洲大蠊几丁质酶基因的原核表达及蛋白纯化[J].基因组学与应用生物学,2016,35(9):2311-2316
[28]Shinoda T,Kobayashi J,Matsui M,Chinzei Y.Cloning and functional expression of a chitinase cDNA from the common cutworm,Spodoptera litura,using a recombinant baculovirus lacking the virus-encoded chitinase gene[J].Insect Biochemistry&Molecular Biology,2001,31(6-7):521-532
[29]魏巍.棉铃虫几丁质酶cDNA克隆及表达研究[D].北京:中国农业科学院植物保护研究所,2002