猪肝羧酸酯酶控制猪常用抗菌药疗效及毒副作用机制的研究

Abusing of veterinary drugs results in the vicious cycle of series problems including drug resiudes, bacterial drug resistance and enviroment pollution,so understanding the mechanism of drug metabolizing enzyme is very crecial for clinical medication guidance.Human carboxylesterases control nearly one third drugs in their therapeutic effects and side effects by their hydrolysis activity, and the preliminary research results of this project suggest that porcine liver carboxylesterases may also control swine drug in therapeutic effects and side effects by the same pathway, so this project aims at studying on the mechanism of porcine liver carboxylesterases (also named pig liver esterases, PLEs) control antibiotics in therapeutic effects and side effects.Major isoenzymes of PLE will be obtained by molecular cloning and in　vitro expression, the antibiotics hydrolyzed by PLE, hydrolysis level and the hydrolyed products will be analyzed by LC-MS/MS, the antibacterial activity and the side effects of the hydrolyzed products of antibiotics will be evaluated by acteriostatic test and PLE overexpression cell line,the PLE expression profile and PLE expression level or enzyme activity differences among representative pig breeds will be revealed by real time PCR ,Western Blotting and universal substrate, thus the relationship between PLE and antibiotic hydrolysis or therapeutic effects and side effects would be revealed and lights would be shed on the relationship between PLE and antibiotics therapeutic effects or side effects , further on the correlation of PLE expression profile/breed differences and antibiotics therapeutic effects, finally,essential theory basis would be set up for swine reasonable clinical medication.

滥用兽药造成药物残留、环境污染、细菌耐药等问题的恶性循环，掌握动物药物代谢酶的作用机理，是指导合理用药的关键。人羧酸酯酶通过水解作用控制近三分之一临床用药的疗效及毒副作用，而本项目的前期研究结果提示：猪肝羧酸酯酶（PLE）也很有可能通过水解作用控制猪用药的疗效及毒副作用，因此，本项目拟研究猪肝羧酸酯酶(PLE)控制抗菌药疗效及毒副作用的机制，克隆和体外表达PLE主要同功酶，通过LC-MS/MS等方法对PLE水解抗菌药的种类、活性及产物进行分析，以体外抑菌试验及PLE过量表达的细胞系等分别评价PLE水解产物的抑菌活性及毒副作用，结合RT-PCR、WB及通用底物等研究PLE的表达谱和品种差异，揭示PLE与抗菌药水解、疗效及毒副作用的关系。阐明PLE与抗菌药的疗效及毒副作用的关联机制、阐明PLE表达谱及PLE品种差异与药物疗效的关联机制，为养猪业的合理用药提供必要理论依据。

申请人按计划高质量完成了项目设定的研究任务和研究目标。克隆和功能性表达了通城猪和大白猪肝脏中PLE主要同工酶，研究了PLE主要同工酶的水解特性；研制出高质量针对PLE各亚型的通用型多克隆抗体，为PLE的定性和定量打下必要的基础。选择我国地方优良品种通城猪和外来优良品种大白猪，研究了4个年龄段猪肝脏中PLE的mRNA水平、蛋白质水平、酶活力，不仅发现了103种PLE新亚型，且发现PLE的表达水平和酶活力存在显著品种差异和年龄差异；建立了LC/MS/MS方法，进行了可被PLE水解的常用兽药筛选，发现了可被PLE水解的兽药以及PLE水解这些兽药的产物及活力、水解前后药物的抑菌活性发现显著变化。为了给PLE在体水解药物提供有力证据，用猪肝原代细胞进行了药物水解试验，发现猪肝原代细胞培养上清中无PLE，但细胞内含有高丰度PLE，加入培养上清中的某些药物可被高效水解。提示存在于细胞内质网腔中的PLE可水解进入机体的这些药物。这些研究结果证明PLE是猪用兽药不可忽视的药物代谢酶，与某些药物的疗效及毒副作用密切相关联，在临床用药时要考虑PLE品种和年龄差异所致的疗效和毒副作用影响，PLE应成为用药方案制订的必要依据。基于前述研究结果，申请人进行了本项目的延伸研究，主要对PLE基因家族在染色体上的分布规律以及对影响PLE活性的关键氨基酸位点及氨基酸种类进行了探索。首次揭示了PLE基因的结构特点，阐明了PLE家族基因在染色体上是以基因簇的形式存在。研究结果不仅填补PLE基因序列信息空白，也为后续研究PLE的基因功能、调控机制等奠定了必要的基础；通过对PLE亚型的分析及定点突变，发现了对酶活力起到重要的关键氨基酸及其位点，为人工改造PLE酶活力提供了理论依据。