鸡肝脏解毒AFB1代谢的关键GSTs基因的鉴定及其酶学特性的研究

黄曲霉毒素B1（AFB1）对畜禽生产危害极大，雏鸡对其较为敏感。AFB1-外-8,9-环氧化物（AFBO）是AFB1毒性极强的代谢产物，肝脏谷胱甘肽S-转移酶系（GSTs）能催化AFBO与谷胱甘肽结合而起到关键的解毒作用。目前，鸡体内共鉴定出20余种GSTs酶系，但是，哪些关键GSTs基因在鸡肝脏中解毒AFB1起作用尚不清楚。本项目拟通过基因过表达和敲除技术在鸡肝细胞中筛选解毒AFB1的候选关键GSTs酶系；然后，运用毕赤酵母表达和镍柱亲和层析纯化上述候选关键GSTs蛋白；随后，运用酶促反应动力学等技术研究上述候选关键GSTs的酶学特性，确定高活力解毒AFB1的关键GSTs酶系；最后，探究营养素调控上述关键GSTs酶系而解毒AFB1致鸡肝毒性的可行性。研究结果有助于揭示鸡肝脏解毒AFB1代谢的关键GSTs基因及其酶学特性，可为营养素防治鸡AFB1中毒提供新的调控靶点。

Dietary exposure to aflatoxin B1 (AFB1) is harmful to the production of domestic animals. Chickens are sensitive to the toxic actions of AFB1. Exo-AFB1-8,9-epoxide (AFBO) is the most toxic metabolite of AFB1. It can be detoxified through conjugation with glutathione, which is mainly catalyzed by glutathione S-transferases (GSTs) in the liver. Although approximately 20 GSTs isozymes have been identified in the liver of chickens, which GSTs isozymes play key role in detoxification of AFB1 in chickens and the enzyme characteristics remained unknown. This project intends to use gene overexpression and knockout technologies to screen the candidate pivotal GSTs isozymes that involved in the detoxification of AFB1 in chicken hepatocytes. Moreover, the candidate pivotal GSTs isozymes will be expressed by Pichia pastoris and purified by Ni Sepharose affinity column. Furthermore, the characterization of the candidate pivotal GSTs isozymes activity will be analyzed by kinetics enzyme-catalyzed reactions. Based on the results, the GSTs isozymes with highly AFB1 detoxification activity will be identified as the key enzymes. Finally, we will explore whether nutrients could mitigate aflatoxicosis in chickens through the regulation of these crucial GSTs isozymes. This study will reveal the pivotal GSTs isozymes that responsible for the bioactivation of AFB1 in chickens and their enzyme characteristics, which can provide the new regulatory targets for the development of nutritional strategies to prevent aflatoxicosis in chickens.