鸭疫里默氏杆菌Tet(X)基因原核表达及其功能鉴定  

The prokaryotic expression and function identification of Tet (X) gene in Riemerella anatipestifer

作　　者：黄雪龙 单敏 陈烨 刘玲俐 何晶 王小兰[2] 于圣青[2] 罗廷荣[3] 涂剑[1] 李涛[2] HUANG Xue-long;SHAN Min;CHEN Ye;LIU Ling-li;HE Jing;WANG Xiao-lan;YU Sheng-qing;LUO Ting-rong;TU Jian;LI Tao(Institute of Pharmacy and Pharmacology,University of South China,Hengyang,Hunan 421001,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]南华大学药物药理研究所,湖南衡阳421001 [2]中国农业科学院上海兽医研究所,上海200241 [3]广西大学动物科学技术学院,南宁530004

出　　处：《南方农业学报》2019年第4期844-850,共7页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2017YFD0500802013);上海市自然科学基金项目(18ZR1448800);中央级公益性科研院所基本科研业务费专项(2017JB12);湖南省分子靶标新药研究协同创新中心项目(2016-429)

摘　　要：【目的】确定鸭疫里默氏杆菌Tet(X)基因是否与四环素类耐药相关,寻找出抗鸭疫里默氏杆菌药物研发新靶点。【方法】克隆鸭疫里默氏杆菌Tet(X)基因,以大肠杆菌原核表达体系进行诱导表达,利用生物信息学软件分析原核表达蛋白的功能和特性,并采用实时荧光定量PCR检测四环素和多西环素对鸭疫里默氏杆菌Tet(X)基因的调控作用。【结果】从鸭疫里默氏杆菌基因组扩增获得的Tet(X)基因编码框全长1167 bp,编码388个氨基酸,其编码蛋白分子量约42.53 kD,理论等电点(pI)为10.073,与AS87_09615(Yb2)、RIA_0369(YA-GD)、RAYM_08235(RA-YM)、G148_1777(RA-CH-2)、RIA_0365(YA-GD)、G148-1767(RA-CH-2)、B739_0035(RA-CH-1)和B739_0030(RA-CH-1)等8个来源于不同种鸭疫里默氏杆菌的蛋白具有较高同源性(96%～100%)。利用原核表达体系诱导表达获得的Tet(X)融合蛋白分子量约45.40 kD,且主要以包涵体的形式表达。Tet(X)融合蛋白能有效提高宿主菌株对典型四环素类药物(四环素和多西环素)的最小抑菌浓度(MIC);实时荧光定量PCR检测结果显示,四环素和多西环素对鸭疫里默氏杆菌Tet(X)基因表达量的调控呈非线性。四环素浓度为0～4 mg/L时,Tet(X)基因相对表达量随药物浓度的增加而逐渐增加;浓度为4～12 mg/L时,Tet(X)基因相对表达量随药物浓度的增加而逐渐降低。多西环素药物浓度为0～3 mg/L时,Tet(X)基因相对表达量随药物浓度的增加而逐渐增加;浓度为3～6 mg/L时,Tet(X)基因相对表达量随药物浓度的增加而逐渐降低。【结论】Tet(X)可有效提高宿主菌株对四环素类药物的耐受性,在一定的四环素类药物剂量范围内可诱导Tet(X)基因表达。不同鸭疫里默氏杆菌Tet(X)基因具有不同来源,说明Tet(X)可能存在基因水平转移,且具有环境扩散的风险。【Objective】This study was conducted to determine whether the Riemerella anatipestifer Tet(X)gene was associated with tetracycline resistance,and to find new targets of drug development against R. anatipestifer.【Method】R. anatipestifer Tet(X)gene was cloned,and induced by prokaryotic expression in Escherichia coli. Its prokaryotic expression protein function and characters were analyzed by the bioinformatics software. The regulation effects of tetracycline and doxycycline on R. anatipestifer Tet(X)gene were analyzed by real-time fluorescence quantitative PCR.【Result】The full length of the Tet(X)gene obtained from the genome amplification of R. anatipestifer was 1167 bp,encoding 388 amino acids. The molecular weight of the encoded protein was about 42.53 kD,and the theoretical isoelectric point(pI)was 10.073. It had high homology(96%-100%)with eight proteins from different strains of R. anatipestifer,including AS87_ 09615(Yb2),RIA_0369(YA-GD),RAYM_08235(RA-YM),G148_1777(RA-CH-2),RIA_0365(YA-GD),G148- 1767(RA-CH-2),B739_0035(RA-CH-1)and B739_0030(RA-CH-1). The molecular weight of the fusion protein of Tet (X) obtained by prokaryotic expression induction was 45.40 kD,and mainly expressed as inclusion body in prokaryotic expression system. The Tet(X)fusion protein could effectively increase the minimal inhibitory concentration(MIC)of the host strain against typical tetracyclines(tetracycline and doxycycline). The results of real-time quantitative fluorescence PCR showed that a non-linear effect of the etracycline and doxycycline on the expression of Tet(X)gene. When tetracycline concentration was 0-4 mg/L,the relative expression of Tet(X)gene increased with the increase of drug concentration. At concentrations ranging from 4 to 12 mg/L,the relative expression of Tet(X)gene decreased with increasing drug concentration. When doxycycline concentration was 0-3 mg/L,the relative expression of Tet(X)gene increased with the increase of drug concentration. At concentrations of 3 to 6 mg/L,the relative expression o

关 键 词：鸭疫里默氏杆菌 Tet(X)基因 原核表达 功能鉴定 

分 类 号：S858.32[农业科学—临床兽医学]