[1]贺卫华,陈诗琪,王丽华,等.猪UGT 1A1酶基因的克隆、表达及其对呕吐毒素特异性降解的作用[J].江苏农业学报,2023,(08):1722-1728.[doi:doi:10.3969/j.issn.1000-4440.2023.08.012]
　HE Wei-hua,CHEN Shi-qi,WANG Li-hua,et al.Expression and identification of porcine UGT 1A1 gene and its special effect of degradation for deoxynivalenol[J].,2023,(08):1722-1728.[doi:doi:10.3969/j.issn.1000-4440.2023.08.012]
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猪UGT 1A1酶基因的克隆、表达及其对呕吐毒素特异性降解的作用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年08期
页码:
1722-1728
栏目:
畜牧兽医·水产养殖
出版日期:
2023-12-31

文章信息/Info

Title:
Expression and identification of porcine UGT 1A1 gene and its special effect of degradation for deoxynivalenol
作者:
贺卫华1; 陈诗琪1; 王丽华1; 丁斓1; 翟晓虎1; 杨俊花2
(1.江苏农牧科技职业学院,江苏泰州225300；2.上海市农业科学院农产品质量标准与检测技术研究所,上海201403)
Author(s):
HE Wei-hua1; CHEN Shi-qi1; WANG Li-hua1; DING Lan1; ZHAI Xiao-hu1; YANG Jun-hua2
(1.Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300,China；2.Institute for Agri-food Standards and Testing Technology,Shanghai Academy of Agricultural Sciences, Shanghai 201403,China)
关键词:
呕吐毒素(DON); 葡萄糖醛酸缀合物; 尿苷二磷酸葡萄糖醛酸转移酶(UGT); 猪肝脏
Keywords:
deoxynivalenol; glucuronic acid conjugate; uridine diphosphate glucuronosyltrans ferase (UGT); porcine liver
分类号:
S852.23
DOI:
doi:10.3969/j.issn.1000-4440.2023.08.012
文献标志码:
A
摘要:
为了在Sf9昆虫细胞中表达猪尿苷二磷酸葡萄糖醛酸转移酶(UGT) 1A1蛋白并对其进行功能鉴定。首先,合成密码子优化的UGT 1A1基因,克隆到载体pFastBacⅠ中,构建杆状病毒重组转移载体,然后导入DH10Bac中,获得重组的穿梭质粒,再转染Sf9细胞得到重组杆状病毒,采用Western blotting方法鉴定重组后UGT 1A1蛋白的表达；对镍柱亲和层析纯化获得目的蛋白酶的动力学参数及其对呕吐毒素(DON)的代谢进行检测。结果表明:pFastBac-UGT 1A1质粒被成功构建,导入感受态细胞DH10Bac中获得重组穿梭质粒Bacmid-UGT 1A1,再转染昆虫细胞Sf9获得重组Bacmid-UGT 1A1蛋白,能够与多组氨酸标签单抗发生特异性反应。优化目的蛋白表达,并对其体外代谢DON进行了酶学性质和动力学参数的研究。本研究通过基因克隆、表达和代谢产物的分析等技术手段获得具有较好的生物活性且纯化的Bacmid-UGT 1A1蛋白,为揭示DON在肝脏中的代谢途径、代谢产物和关键调控因子提供参考。
Abstract:
To express the porcine uridine diphosphate glucuronosyltransferase (UGT) 1A1 protein in Sf9 insect cells and characterize its function, the codon-optimized UGT 1A1 gene was synthesized and cloned into pFastBacI vector to construct a recombinant baculovirus intermediate transfer vector. The vector was introduced into DH10Bac to obtain recombinant shuttling plasmid, which was used to transfect the Sf9 cells to get recombinant baculovirus. Expression of the recombinant UGT 1A1 protein was confirmed by Western blotting. Kinetic parameters of the target protease purified by nickel column affinity chromatography and its metabolism effect of deoxynivalenol (DON) were detected. The results showed that, the plasmid pFastBac-UGT 1A1 was successfully constructed and was transducted into competent cells DH10Bac to obtain recombinant shuttling plasmid Bacmid-UGT 1A1. The recombinant shuttling plasmid was used to transfect Sf9 insect cells and obtained recombinant protein Bacmid-UGT 1A1, which was capable of taking specific reactions with His-tagged monoclonal antibodies. The optimal conditions for the target protein expression was screened, and the metabolism of DON in vitro was studied from the aspects of enzymatic properties and kinetic parameters. In this study, purified Bacmid-UGT 1A1 protein with good biological activities was obtained by technical methods such as gene clone and expression, analysis of metabolites, which provided references for revealing the metabolic pathways, metabolites and key regulatory factors of DON in the liver.

参考文献/References:

[1]贺烨宇,杨华,杨伟康,等. 谷物及其制品中真菌毒素污染和管控策略研究[J]. 浙江农业科学,2023,64(1):1-8.
[2]YAO Y Z, MIAO L. The biological detoxification of deoxynivalenol: a review[J]. Food Chem Toxicol,2020,145:111649.
[3]PIERRON A, ALASSANE-KPEMBI I, OSWALD I P. Impact of two mycotoxins deoxynivalenol and fumonisin on pig intestinal health[J]. Porcine Health Manag,2016,2:21.
[4]SUN Y, JIANG J, MU P Q, et al. Toxicokinetics and metabolism of deoxynivalenol in animals and humans[J]. Arch Toxicol,2022,96(10):2639-2654.
[5]MAUL R, WARTH B, KANT J S, et al. Investigation of the hepatic glucuronidation pattern of the fusarium mycotoxin deoxynivaleno in various species[J]. Chem Res Toxicol,2012,25(12):2715-2717.
[6]MACKENZIE P I, BOCK K W, BURCHELL B, et al. Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily[J]. Pharm Genom,2005,15(10):677-685.
[7]GUILLEMETTE C, LVESQUE E, HARVEY M, et al. UGT genomic diversity: beyond gene duplication[J]. Drug Metab Rev,2010,42(1):24-44.
[8]SCHWARTZ-ZIMMERMANN H, HAMETNER C, NAGL V, et al. Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows[J]. Arch Toxicol,2017,91(12):3857-3872.
[9]LU B J, TANG Q, WANG Q Y, et al. Recovery infectious enterovirus 71 by Bac-to-Bac expression system in vitro and in vivo[J]. Front Microbiol,2022,13:825111.
[11]LATTANZIO V M, SOLFRIZZO M, DE GIROLAMO A, et al. LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat[J]. J Chromatogr B,2011,879:707-715.
[12]FAN K, XU J J, JIANG K Q, et al. Determination of multiple mycotoxins in paired plasma and urine samples to assess human exposure in Nanjing, China[J]. Environmental Pollution,2019,248:865-873.
[13]ROWLAND A, MINERS J O, MACKENZIE P I. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification[J]. Int J Biochem Cell Biol,2013,45(6):1121-1132.
[14]BARBIER O,TROTTIER J,KAEDING J,et al. Lipid-activated transcription factors control bile acid glucuronidation[J]. Mol Cellbio Chem,2009,326(1):3-8.
[15]GALL W E, ZAWADA G, MOJARRABI B, et al. Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7[J]. J Steroid Biochem Mol Biol,1999,70(1/2/3):101-108.
[16]ZENG W, LIU X, WU Y, et al. Dysregulated hepatic UDP-glucuronosyltransferases and flavonoids glucuronidation in experimental colitis[J]. Frontiers in Pharmacology,2022,13:1053610.
[17]薛庆凯,黄玉政. 尿苷二磷酸葡萄糖醛酸转移酶在肝脏疾病中的作用及其转录调控机制[J]. 热带病与寄生虫学,2022,20(1):36-42.
[18]VAN OERS M M, PIJLMAN G P, VLAK J M. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology[J]. J Gen Virol,2015,96(1):6-23.

备注/Memo

备注/Memo:
收稿日期:2023-03-21基金项目:江苏农牧科技职业学院科研项目(NSF2023ZR06)；第二批国家级职业教育教师教学创新团队课题研究项目(ZI2021100104)作者简介:贺卫华(1982-),女,山西太谷人,硕士,副教授,主要从事动物毒理学研究。(E-mail)heweihua010@163.com通讯作者:翟晓虎,(E-mail)zhaixiaohu010@163.com；杨俊花,(E-mail)yangjunhua303@126.com
更新日期/Last Update: 2024-01-15