三疣梭子蟹P-gp蛋白在氟苯尼考代谢中的功能研究  

Study on the function of P-gp protein in florfenicol metabolism of Portunus tritructus

作　　者：徐垚 邵慧鑫 高保全[2,3] 李健 蔡月凤[1] 任宪云 XU Yao;SHAO Huixin;GAO Baoquan;LI Jian;CAI Yuefeng;REN Xianyun(Jiangsu Key Laboratory of Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology,College of Marine Science and Fisheries,Jiangsu Ocean University,Lianyungang Jiangsu 222005,China;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao Shandong 2660712,China;Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao Shandong 266071,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai 201306,China)

机构地区：[1]江苏海洋大学海洋科学与水产学院,江苏省海洋生物资源与环境重点实验室/江苏省海洋生物技术重点实验室,江苏连云港222005 [2]中国水产科学研究院黄海水产研究所,农业农村部海洋渔业可持续发展重点实验室,山东青岛266071 [3]青岛海洋科学与技术试点国家实验室-海洋渔业科学与食物产出过程功能实验室,山东青岛266237 [4]水产科学国家级实验教学示范中心,上海201306

出　　处：《海洋渔业》2022年第5期521-534,共14页Marine Fisheries

基　　金：国家自然科学基金面上项目(41876186);国家自然科学基金青年基金项目(41706135);国家虾蟹产业技术体系(CARS-48);中国水产科学研究院院级基本科研业务费“海水池塘生态养殖创新团队项目(2020TD46)”;江苏省研究生实践创新计划项目(SJCX20_1282)。

摘　　要：为研究三疣梭子蟹(Portunus trituberculatus)的P-糖蛋白(P-glycoprotein,P-gp)的转运功能,利用RACE扩增技术克隆得到abcb1(ATP-binding cassette transporter subfamily b1)基因全长序列,命名为Ptabcb1,cDNA全长为4923 bp,开放阅读框(ORF)为3522 bp,编码1172个氨基酸,具有2个疏水性的跨膜区(TMD)和2个核苷酸结合区(NBD),符合ABC转运蛋白的基本结构特征。序列比对结果显示,Ptabcb1基因与脊尾白虾(Exopalaemon carinicauda)abcb1基因同源性最高(73.32%)。系统进化树分析表明,Ptabcb1氨基酸序列与脊尾白虾紧密聚为一支。组织表达分布结果显示,Ptabcb1基因在所有组织中均有表达,其中在肝胰腺中的表达量最高。采用荧光原位杂交技术,利用合成目的基因Ptabcb1的杂交探针,通过荧光染色,追踪到Ptabcb1基因主要在肝胰腺的细胞质中表达。利用实时荧光定量技术探究肌注20 mg·kg^(-1)、40 mg·kg^(-1)和80 mg·kg^(-1)不同浓度的氟苯尼考(FLR)对Ptabcb1基因在三疣梭子蟹体内转录表达的影响;以及用10μM维拉帕米(verapamil,VER)注射三疣梭子蟹1 h后,再进行40 mg·kg^(-1) FLR的注射处理,探究FLR与VER联合注射给药对三疣梭子蟹Ptabcb1基因转录表达的影响以及转运活性的变化。结果显示,FLR在24 h内以浓度依赖的方式,通过促进Ptabcb1基因的转录上调,显著促进PtP-gp蛋白在转录水平上的转运活性;同时发现40 mg·kg^(-1) FLR与VER联合注射处理组与单独注射40 mg·kg^(-1) FLR处理组相比,在12 h之内强烈抑制FLR的外排,导致PtP-gp蛋白转运活性降低;12 h之后,PtP-gp蛋白转运活性逐渐恢复到对照组水平。此外,还发现三疣梭子蟹具有保守的P-gp蛋白介导的防御系统。研究结果可为进一步了解P-gp蛋白介导甲壳类动物解毒的分子防御机制提供依据。Portunus trituberculatus is a famous swimming crab with the highest output in the north of China.With the rapid development of aquaculture industry,the environment has become increasingly deteriorated,resulting in the increase of pathogens and high incidence of disease in cultivation environments,which may cause huge economic losses to the mariculture industry and seriously hinder the sustainable and healthy development of mariculture.At present,aquatic drugs are extensively applied as effective measures to prevent and control aquatic animal disease because of their remarkable efficacy,easy production and utilization,and low cost.Florfenicol(FLR)is one of the most commonly used antimicrobial agents in aquaculture.This chemical ingredient is characterized with broad spectrum,easy absorption,wide distribution and low residue in the body after administration.FLR can bind closely to the 50 S subunit of 70 S ribosome of bacteria,which plays a killing role by effectively inhibitting the proteins’biosynthesis in bacteria,and is widely used in the prevention and treatment of infections caused by Vibrio.In the actual breeding process,there are unreasonable uses of drug and even abuse of drugs.When entering the breeding environment,the antimicrobial agents will induce the opportunistic pathogens of the breeding organisms themselves;microorganisms in aquatic environments including aquaculture sediment can develop drug resistance;FLR will also remain in the cultured subjects,leading to a series of adverse responses of the body,including the induced oxidative stress,inhibited metabolism and growth process in organisms,disrupted behavior and immune response,altered transcriptional expression of key genes in related metabolic pathways,and destroyed balance of the ecological environment in aquaculture.ATP-binding cassette(ABC)transmembrane transporters(ABC transporters)are primarily involved in the active,ATP-dependent transport of biological molecules across plasma membranes.The most toxicologically relevant ABC transporter i

关 键 词：三疣梭子蟹 P-糖蛋白 氟苯尼考 转运活性 

分 类 号：S948[农业科学—水产养殖]