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  

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 王丽华[1] 丁斓 翟晓虎[1] 杨俊花[2] HE Wei-hua;CHEN Shi-qi;WANG Li-hua;DING Lan;ZHAI Xiao-hu;YANG Jun-hua(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Institute for Agri-food Standards and Testing Technology,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China)

机构地区：[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

出　　处：《江苏农业学报》2023年第8期1722-1728,共7页Jiangsu Journal of Agricultural Sciences

基　　金：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)。

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。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.

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 

分 类 号：S852.23[农业科学—基础兽医学]