痤疮丙酸杆菌异构酶在小鼠细胞中异源表达及t10c12-共轭亚油酸的制备  

Heterologous Expression of Propionibacterium acnes Isomerase in Mouse(Mus musculus)Cells and Production of t10c12-conjugated Linoleic Acid

作　　者：王超 王洋洋 饶喻 李美娟 刘宗平 李世丽 苟克勉 WANG Chao;WANG Yang-Yang;RAO Yu;LI Mei-Juan;LIU Zong-Ping;LI Shi-Li;GOU Ke-Mian(Institute of Comparative Medicine,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;State Key Laboratory of Agribiotechnologies,China Agricultural University,Beijing 100193,China;Department of Molecular Genetics,University of Texas Southwestern Medical Center,Dallas 75390,USA)

机构地区：[1]扬州大学兽医学院比较医学研究院,扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]中国农业大学农业生物技术国家重点实验室,北京100193 [4]美国得克萨斯大学西南医学中心分子遗传学系,达拉斯75390

出　　处：《农业生物技术学报》2021年第12期2304-2311,共8页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFC1003800);江苏高校优势学科建设工程资助项目。

摘　　要：来源于痤疮丙酸杆菌异构酶(Propionibacterium acnes isomerase,PAI)能够将亚油酸转化成反式10,顺式12-共轭亚油酸(trans 10,cis 12-conjugated linoleic acid,t10c12-CLA),饲喂t10c12-CLA的动物会减少脂肪沉积和乳脂含量,能量的动态平衡受到影响。本研究用密码子优化的pai基因转染小鼠(Mus musculus)3T3细胞,经高压气相色谱分析证实,PAI将底物亚油酸高效催化成t10c12-CLA,t10c12-CLA含量最高达到了细胞内多不饱和脂肪酸总量的7.09%;与转染pIRES2-AcGFP1的对照细胞株比较,转染pai基因并合成t10c12-CLA的细胞株出现亚油酸积累(12.46%vs 14.38%~15.54%)(P<0.05)和花生四烯酸减少(13.31%vs 9.03%~11.13%)(P<0.05),同时硬脂酸(14.07%vs 11.85%~12.54%)(P<0.05)和油酸(20.29%vs 14.89%~16.87%)(P<0.05)同步减少。qPCR结果显示,pai转染细胞中生成的t10c12-CLA可上调硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,scd1)基因表达(P<0.05),可能是转基因细胞中硬脂酸含量下降的原因;但是,外源t10c12-CLA没有下调脂肪酸去饱和酶1(fatty acid desaturase 1,fads1)和fads2基因表达。高压气相色谱检测结果显示,外源添加的t10c12-CLA阻滞了亚油酸向下游花生四烯酸的代谢(P<0.05),即t10c12-CLA可能竞争性结合FADS2/FADS1酶系,从而抑制亚油酸向花生四烯酸的正常代谢。本研究证实微生物的pai基因能够在哺乳动物细胞中正常表达并生产t10c12-CLA,可为研制pai转基因动物提供参考依据。Trans 10,cis 12-conjugated linoleic acid(t10c12-CLA)can be obtained from linoleic acid by Propionibacterium acnes isomerase(PAI).Animals fed t10c12-CLA reduced fat deposition and milk fat content,and the dynamic balance of energy was affected.In this study,the codon optimized pai gene was transfected into mouse(Mus musculus)3 T3 cells.It was confirmed by gas chromatography that PAI could efficiently catalyze the substrate linoleic acid into t10c12-CLA.The content of t10c12-CLA was high,and reached 7.09%of the total amount of poly-unsaturated fatty acids in cells.When compared with the control cell line transfected with pIRES2-AcGFP1,the cell lines transfected with pai gene and synthesized t10c12-CLA showed linoleic acid accumulation(12.46%vs 14.38%~15.54%)(P<0.05)and arachidonic acid decrease(13.31%vs 9.03%~11.13%)(P<0.05),while stearic acid(14.07%vs 11.85%~12.54%)(P<0.05)and oleic acid(20.29%vs 14.89%~16.87%)(P<0.05)decreased simultaneously.T10c12-CLA produced in pai transfected cells could up-regulate the gene expression of stearoyl-CoA desaturase 1(scd1)(P<0.05),which may be the reason for the decrease of stearic acid content in transgenic cells.However,exogenous t10c12-CLA did not down-regulate the expression of fatty acid desaturase 1(fads1)and fads2 genes.The results of high pressure gas chromatography showed that the exogenous t10c12-CLA blocked the metabolism of linoleic acid to downstream arachidonic acid(P<0.05),that is,t10c12-CLA may competitively bind to FADS2/FADS1 enzyme system,so as to inhibit the normal metabolism of linoleic acid to arachidonic acid.This study confirmed that microbial pai gene could be normally expressed in mammalian cells and produce t10c12-CLA,which can provide a reference basis for the development of pai transgenic animals.

关 键 词：痤疮丙酸杆菌 异构酶 共轭亚油酸 脂肪酸 小鼠3T3细胞 

分 类 号：S188[农业科学—农业基础科学]