棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备

古新蓉; 罗生慧; 魏林昱; 姜岩; 刘小宁

doi:10.13568/j.cnki.651094.651316.2020.02.29.0001

您当前的位置：

首页 >

文章列表页 >

棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备

生命科学与技术 | 更新时间：2026-01-21

- 棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备
- 棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备
- 新疆大学学报（自然科学版中英文） 2021年38卷第2期页码：197-203
- 作者机构：
  
  新疆大学新疆生物资源和基因工程重点实验室
- 作者简介：
- 基金信息：
  
  国家自然科学基金(31972279);新疆维吾尔自治区2017年天山雪松计划(2017XS20)
- DOI：10.13568/j.cnki.651094.651316.2020.02.29.0001
  中图分类号： S435.622.3
- 纸质出版：2021
- 稿件说明：
移动端阅览
[1]古新蓉,罗生慧,魏林昱,等.棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备[J].新疆大学学报(自然科学版)(中英文),2021,38(02):197-203+212.
[1]古新蓉,罗生慧,魏林昱,等.棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备[J].新疆大学学报(自然科学版)(中英文),2021,38(02):197-203+212. DOI： 10.13568/j.cnki.651094.651316.2020.02.29.0001.

DOI：10.13568/j.cnki.651094.651316.2020.02.29.0001.

摘要

本课题组前期基于酵母单杂交技术筛选出响应2-十三烷酮诱导CYP6B6过表达的6个调控因子

乙醇脱氢酶5(HaADH5)是其中之一

乙醇脱氢酶是一种代谢乙醇的重要酶类

在哺乳动物中的研究很多

但在昆虫中的研究很少.为了进一步探究HaADH5是否参与P450 CYP6B6的过表达

进而参与棉铃虫对包括杀虫剂在内的外界有毒物质的代谢.本实验将pET32a-HaADH5转化至大肠杆菌Transetta中进行诱导表达

Western blot进一步验证融合蛋白HisHaADH5的表达

通过镍柱纯化获得融合蛋白His-HaADH5

且测定了酶活.用蛋白免疫法制备了鼠抗His-HaADH5的血清

Western blot分析了该抗血清的免疫特异性

继而用此抗体检测了HaADH5在不同组织中的表达.活性分析表明

在NAD+的存在下

融合蛋白可以对不同醇类和甲醛进行脱氢

尤其对异戊醇的脱氢活性最高

活性为1 333 U/mg.制备的抗血清效价为1∶409 600.Western blot结果表明

该抗血清既能与融合蛋白His-HaADH5结合

也能与棉铃虫体内的HaADH5结合

还发现HaADH5在脂肪体中的表达量最高.原核表达的融合蛋白能代谢异戊醇

制备的抗血清具有较好的免疫特性

这些结果为HaADH5蛋白水平的鉴定以及功能研究提供了重要的检测工具

也有助于我们更好地探索棉铃虫乙醇脱氢酶5的功能.

Abstract

On the basis of H. armigera CYP6 B6 overexpression response to 2-tridecanone

six regulators in response to 2-tridecan-one were selected by yeast one-hybrid

and ethanol dehydrogenation of Helicoverpa armigera(HaADH5) is one of them. Alcohol dehydrogenase is an important ethanol metabolic enzyme and has been studied mainly in mammals

and a little in insects. We aim to study whether alcohol dehydrogenase 5 is involved in the overexpression of P450 CYP6 B6

and further participates in the metabolism of external toxic substances including insecticides of H. armigera

these results will lay a foundation for effective control of H. armigera. In this study

pET32 a-HaADH5 was transformed into E. coli Transetta for induction expression

the fusion protein His-HaADH5 was further detected by Western-blot

purified using Ni-NTA

and its enzyme activity was measured. The antiserum of H. armigera His-HaADH5 was prepared by protein immunoassay. The titer of the polyclonal antibody was detected by enzyme-linked immune sorbent assay(ELISA). Western blot was used to analyze the immimu-specifity of prepared antiserum

and then used antiserum to detect the expression of HaADH5 in different tissues of H.armigera. Activity analysis showed that the fusion protein could catalyze the dehydrogenation of different alcohols and formaldehyde in the presence of NAD+condition. However

it had the highest metabolic capacity for isoamyl alcohol and its activity was 1 333 U/mg. The titer of the polyclonal antibody

which was detected by enzymelinked immune sorbent assay(ELISA)

was 1∶409 600. Immimu-specifity of polyclonal antibody showed that the prepared antiserum could not only bind to the fusion protein His-HaADH5

but also to HaADH5 in H. armigera

and then used it to detect the expression level of HaADH5 in different tissues of H. armigera

the results showed that the expression level of HaADH5 was highest in the fat body. The fusion protein expressed by prokaryote can metabolize isoamyl alcohol

and the prepared mouse anti-His-HaADH5 antiserum has better immune properties.These results will provide an important detection tool for the identification of HaADH5 protein. These results will help us better investigate the function of HaADH5 of H.armigera.

关键词

Keywords

references

MA C X,LI M,QIU X H,et al.Cloning and fusion expression of CYP6B7 gene from Helicoverpa armigera[J].Acta Entomologica Sinica,2006,49(6):944-949.

ZHAO J,WEI Q,GU X R,et al.Alcohol dehydrogenase 5 of Helicoverpa armigera interacts with the CYP6B6 promoter in response to 2-tridecanone[J].Insect Science,2019,1-14,DOI:10.1111/1744-7917.12720.

李笠坤,雒珺瑜,吴益东,等.转Cry1Ac/1Ab基因棉花对棉铃虫的控制效果及对甜菜夜蛾和斜纹夜蛾生长发育的影响[J].植物保护学报,2018,45(3):561-567.LI L K,LUO J Y,WU Y D,et al.The effects of transgenic Cry1Ac/1Ab cotton on cotton bollworm control and growth and development of non-target pests[J].Journal of Plant Protection,2018,45(3):561-567.(in Chinese)

PALRASU M,SIDDAVARAM N.Cytochrome P450 structure,function and clinical significance:a review[J].Current Drug Targets,2018,19(1):38-54.

GIRAUDO M,HILLIOU F,FRICAUX T,et al.Cytochrome P450s from the fall armyworm (Spodoptera frugiperda):responses to plant allelochemicals and pesticides[J].Insect Molecular Biology,2015,24(1):115-128.

GOFF GL,BOUNDY S,DABORN PJ,et al.Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila[J].Insect Biochemistry and Molecular Biology,2003,33(7):701-708.

GUO T T,HUI J,GAO X W.Diversity,evolution and expression regulation of cytochrome P450 monooxygenase genes in insects[J].Acta Entomologica Sinica,2009,52(3):301-311.

李芬,刘小宁.昆虫基因启动子及细胞色素P450基因启动子研究进展[J].生命科学,2012,24(5):77-81.LI F,LIU X N.Review on insect gene promoter and cytochrome P450 gene promoters[J].Chinese Bulletin of Life Sciences,2012,24(5):77-81.(in Chinese)

邱星辉,冷欣夫.昆虫细胞色素P450研究：P450酶系的可诱导性[J].昆虫知识,1999,36(1):48-53.QIU X H,LENG X F.The research of the Insect cytochrome P450:The induced ability of P450 enyme[J].Entomological Knowledge,1999,36(1):48-53.(in Chinese)

PETERSEN R A,ZANGERL A R,BERENBAUM M R,et al.Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (Lepidoptera:Papilionidae)[J].Insect Biochemistry and Molecular Biology,2001,31(6-7):679-690.

HATAICHANOKE P,BERENBAUM M R,SCHULER M A.Transcriptional regulation of the Papilio polyxenes CYP6B1 gene[J].Nucleic Acids Research,1994,22(15):3210-3217.

WANG L,ZHANG T,PAN Z,et al.The alcohol dehydrogenase with a broad range of substrate specificity regulates vitality and reproduction of the plant-parasitic nematode Bursaphelenchus xylophilus[J].Parasitology,2019,146(4):497-505.

常开霞.乙醇脱氢酶的外源表达及其改性的初步研究[D].无锡:江南大学,2017.CHANG K X.Heterologous expression of alcohol dehydrogenase and preliminary study on its modification[D].Wuxi:Jiangnan University,2017.(in Chinese)

LYU M,SUN H H,WAMG L H,et al.Effects of secondary metabolites on activities of glutathione s-transferases,carboxylesterase in aphid[J].Chinese Agricultural Science Bulletin,2012,28(3):253-256.

ABEL T,MICHELSON A M,MANIATIS T.A Drosophila GATA family member that binds to Adh regulatory sequences is expressed in the developing fat body[J].Development,1993,119(3):623-633

赵洁,任苏伟,马纪，等.棉铃虫乙醇脱氢酶的表达规律[J].新疆大学学报(自然科学版),2017,34(3):267-272.ZHAO J,REN S W,MA J,et al.Expression profile of alcohol dehydrogenase from Helicoverpa Armigera[J].Journal of Xinjiang University(Natural Science Edition),2017,34(3):267-272.(in Chinese)

HEINSTRA PWH,GEER BW,SEYKENS D,et al.The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase(EC1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae[J].Biochemistry Journal,1989,259(3):791-797.

HOOG J O,BRANDT M,HEDBERG J J,et al.Mammalian alcohol dehydrogenase of higher classes:analyses of human ADH5and rat ADH6[J].Chemico Biological Interactions,2001,130-132(1-3):395-404.

左其生,赵瑞峰,张文慧,等.脂代谢通路对鸡雄性生殖细胞分化的调控机制[J].中国农业科学,2015,48(22):4539-4550.ZUO Q S,ZHAO R F,ZHANG W H,et al.The regulatory mechanism of the lipid metabolism pathways during male germ cell differentiation in Chickens[J].Scientia Agricultura Sinica,2015,48(22):4539-4550.(in Chinese)

ZHU Q S,YASUYUKI A,DEBARSHI B.Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J].Insect Biochemistry and Molecular Biology,2008,38(4):0-466.

KOIVUSALO M,BAUMANN M,UOTILA L.Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase[J].Febs Letters,1989,257(1):105-109.

HORI T,YOKOMIZO T,AGO H,et al.Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop[J].The Journal of Biological Chemistry,2004,279(21):22615-22623.

申光茂,王晓娜,黄勇,等.橘小实蝇幼虫解毒酶系基因应对高效氯氰菊酯胁迫的组织特异性表达[J].中国农业科学,2015,48(19):3857-3865.SHEN G M,WANG X N,HUANG Y,et al.Tissue specific expression of genes encoding detoxification enzymes in the larvae of Bactrocera dorsalisunderβ-Cypermethrin stress[J].Scientia Agricultura Sinica,2015,48(19):3857-3865.(in Chinese)

浏览量

274

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

棉铃虫细胞色素P450CYP6B6基因启动子的活性分析

棉铃虫乙醇脱氢酶的表达规律