鸭疫里默氏杆菌Tet(X)基因原核表达及其功能鉴定
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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 ami-no acids. The molecular weight of the encoded protein was about 42.53 kD,and the theoretical isoelectric point(pI)was10.073. 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 Te(tX)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 Te(tX)gene decreased with increasing drug concentration. When doxycycline concentration was 0-3 mg/L,the relative expression of Te(tX)gene increased with the increase of drug concentration. At concentrations of 3 to 6 mg/L,the relative expression of Tet(X)gene decreased with increasing drug concentration.【Conclusion】Te(tX)gene can enhance the tetracycline resistance of host bacteria,and its expression level can be induced by tetracycline drugs within a certain dose range. The gene Te(tX)of R. anatipestifer has different origins,suggesting that Te(tX)may have horizontal gene transfer and risk of environmental spread.
【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 ami-no acids. The molecular weight of the encoded protein was about 42.53 kD,and the theoretical isoelectric point(pI)was10.073. 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 Te(tX)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 Te(tX)gene decreased with increasing drug concentration. When doxycycline concentration was 0-3 mg/L,the relative expression of Te(tX)gene increased with the increase of drug concentration. At concentrations of 3 to 6 mg/L,the relative expression of Tet(X)gene decreased with increasing drug concentration.【Conclusion】Te(tX)gene can enhance the tetracycline resistance of host bacteria,and its expression level can be induced by tetracycline drugs within a certain dose range. The gene Te(tX)of R. anatipestifer has different origins,suggesting that Te(tX)may have horizontal gene transfer and risk of environmental spread.
引文
陈阳,杨涵超,刘生,魏哲洋,王天帅,潘圆.2018.四环素类抗生素耐药机制研究进展[J].广东化工,45(3):89-90.[Chen Y,Yang H C,Liu S,Wei Z Y,Wang T S,Pan Y.2018.Research progress in antibiotic resistance mechanism of tetracycline antibiotics[J].Guangdong Chemical Industry,45(3):89-90.]
程龙飞,傅光华,林群群,刘荣昌,陈翠腾,傅秋玲,万春和,施少华,陈红梅,黄瑜.2018.同时检测水禽3种常见病原菌的环介导间接PCR方法的建立[J].中国兽医学报,38(1):96-99.[Cheng L F,Fu G H,Lin Q Q,Liu R C,Chen C T,Fu Q L,Wan C H,Shi S H,Chen H M,Huang Y.2018.Development of loop-mediate indirect PCR for simultaneously detecting 3 pathogenic bacteria of waterfowl[J].Chinese Journal of Veterinary Science,38(1):96-99.]
程增青,申洪银,范根成,李明义,常顺志,王传禹,许琳,郭玉广,马爽.2009.鸭疫里默氏杆菌分离鉴定及药敏试验[J].中国动物检疫,26(12):32-34.[Cheng Z Q,Shen HY,Fan G C,Li M Y,Chang S Z,Wang C Y,Xu L,Guo Y G,Ma S.2009.Isolation and identification and drug sensitivity test of Riemerella anatipestifer[J].Chinese Journal of Animal Health Inspection,26(12):32-34.]
范忠军,许保疆,翟崇凯,郁川,王欢莉.2017.鸡源肠炎沙门氏菌快速检测方法的建立及其分离株的耐药性分析[J].河南农业科学,46(10):137-142.[Fan Z J,Xu B J,Zhai C K,Yu C,Wang H L.2017.Establishment of rapid detection method of Salmonella enteritidis and drug resistance analysis of isolates from chickens[J].Journal of Henan Agricultural Sciences,46(10):137-142.]
刘洪明.2017.鸭疫里默氏杆菌分离株外膜蛋白(ompA)基因的克隆、表达及其单克隆抗体制备[D].兰州:甘肃农业大学.[Liu H M.2017.Gene cloning,prokaryotic expression of isolated R.anatipestifer outer membrane protein(ompA)and the preparation of monoclonal antibody[D].Lanzhou:Gansu Agricultural University.]
任晓梅,王小兰,韩文龙,张雪梅,陈宗超,李涛,丁铲,于圣青.2018.鸭疫里默氏杆菌的分离鉴定与生物学特性研究[J].中国动物传染病学报,26(3):47-51.[Ren X M,Wang X L,Han W L,Zhang X M,Chen Z C,Li T,Ding C,Yu S Q.2018.Isolation,identification and biological characterization of Riemerella anatipestifer[J].Chinese Journal of Animal Infectious Diseases,26(3):47-51.]
单敏.2017.鸭疫里默氏杆菌耐药相关基因M949_0011和M949_0459的鉴定及其功能研究[D].南宁:广西大学.[Shan M.2017.Identification and functional analysis of the drug resistance genes of Riemerella anatipestifer M949_0011 and M949_0459[D].Nanning:Guangxi University.]
王良,刘思路,代光胜,奎磊,李双菊,田宇,陈建军.2017.洱海流域畜禽饲料·粪便·土壤中四环素类抗生素残留分析[J].安徽农业科学,45(18):70-72.[Wang L,Liu S L,Dai G S,Kui L,Li S J,Tian Y,Chen J J.2017.Analysis of residue of tetracyclines antibiotics in livestock manures and soil in Erhai-Lake basin[J].Journal of Anhui Agricultural Science,45(18):70-72.]
闫琦,刘培培,张娇娇,王跃华,任斌,丁路明.2018.畜禽粪便中残留四环素类抗生素的研究概况[J].家畜生态学报,39(5):80-86.[Yan Q,Liu P P,Zhang J J,Wang Y H,Ren B,Ding L M.2018.Research survey of tetracycline in animal manure[J].Acta Ecologiae Animalis Domasitici,39(5):80-86.]
叶凤梅.2015.贡山八角枝叶中化学成分及抗菌抗肿瘤活性研究[D].上海:上海交通大学.[Ye F M.2015.Chemical constituents from the branches and leaves of Illicium wardii and anti-bacterial and anti-tumor activity[D].Shanghai:Shanghai Jiaotong University.]
叶胜强,陈洁,何斌,童新红,杨文海,金尔光,钱运国,鲍伯胜.2013.鸭疫里默氏杆菌对主要抗菌药物敏感性分析及合理用药[J].养禽与禽病防治,(1):10-12.[Ye S Q,Chen J,He B,Tong X H,Yang W H,Jin E G,Qian Y G,Bao B S.2013.The analysis of the drug sensitivity and rational use of the main antimicrobial agents against Riemerella anatipestife[J].Poultry Husbandry and Disease Control,(1):10-12.]
雍燕,刘国昌,方炳虎,乔妍红.2017.不同地区鸭疫里氏杆菌的分离鉴定与药敏试验[J].中国畜牧兽医,44(1):242-247.[Yong Y,Liu G C,Fang B H,Qiao Y H.2017.Isolation,identification and drug-sensitivity test of Riemerella anatipestifer from different areas[J].China Animal Husbandry&Veterinary Medicine,44(1):242-247.]
Aminov R I.2009.The role of antibiotics and antibiotic resistance in nature[J].Environmental Microbiology,11(12):2970-2988.
Andrasevic A T,Dowzicky M J.2012.In vitro activity of tigecycline and comparators against Gram-negative pathogens isolated from blood in Europe(2004-2009)[J].International Journal of Antimicrobial Agents,39(2):115-123.
Chen Y P,Lee S H,Chou C H,Tsai H J.2012.Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese[J].Veterinary Microbiology,154(3-4):325-331.
Chu C Y,Liu C H,Liou J J,Lee J W,Cheng L T.2015.Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant[J].Vaccine,33(1):92-99.
Deng M,Zhu M H,Li J J,Bi S,Sheng Z K,Hu F S,Zhang JJ,Chen W,Xue X W,Sheng J F,Li L J.2014.Molecular epidemiology and mechanisms of tigecycline resistance in clinical isolates of Acinetobacter baumannii from a Chinese university hospital[J].Antimicrobial Agents and Chemotherapy,58(1):297-303.
Gyurisé,Wehmann E,Czeibert K,Magyar T.2017.Antimicrobial susceptibility of Riemerella anatipestifer strains isolated from geese and ducks in Hungary[J].Acta Veterinaria Hungarica,65(2):153-165.
Hua X,Zhou H,Jiang Y,Feng Y,Chen Q,Ruan Z,Yu Y.2012.Genome sequences of two multidrug-resistant Acinetobacter baumannii strains isolated from a patient before and after treatment with tigecycline[J].Journal of Bacteriology,194(24):6979-6980.
Leski T A,Bangura U,Jimmy D H,Ansumana R,Lizewski SE,Stenger D A,Taitt C R,Vora G J.2013.Multidrugresistant tet(X)-containing hospital isolates in Sierra Leone[J].International Journal of Antimicrobial Agents,42(1):83-86.
Li T,Shan M,He J,Wang X L,Wang S H,Tian M X,Qi J J,Luo T R,Shi Y H,Ding C,Yu S.2017.Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline[J].Oncotarget,8(57):96615-96626.
Reid G E,Grim S A,Aldeza C A,Janda W M,Clark N M.2007.Rapid development of Acinetobacter baumannii resistance to tigecycline[J].Pharmacotherapy,27(8):1198-1201.
Rumbo C,Gato E,López M,Ruiz de Alegría C,FernándezCuenca F,Martínez-Martínez L,Vila J,Pachón J,Cisneros J M,Rodríguez-Ba?o J,Pascual A,Bou G,Tomás M.2013.Contribution of efflux pumps,porins,andβ-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii[J].Antimicrobial Agents and Chemotherapy,57(11):5247-5257.
Santajit S,Indrawattana N.2016.Mechanisms of antimicrobial resistance in ESKAPE pathogens[J].BioMed Research International,2016:2475067.doi:10.1155/2016/2475067.
van Berkel W J,Kamerbeek N M,Fraaije M W.2006.Flavoprotein monooxygenases,a diverse class of oxidative biocatalysts[J].Journal of Biotechnology,124(4):670-689.
Yang F F,Sun Y N,Li J X,Wang H,Zhao M J,Su J,Zhang Z J,Liu H J,Jiang S J.2012.Detection of aminoglycoside resistance genes in Riemerella anatipestifer isolated from ducks[J].Veterinary Microbiology,158(3-4):451-452.
程龙飞,傅光华,林群群,刘荣昌,陈翠腾,傅秋玲,万春和,施少华,陈红梅,黄瑜.2018.同时检测水禽3种常见病原菌的环介导间接PCR方法的建立[J].中国兽医学报,38(1):96-99.[Cheng L F,Fu G H,Lin Q Q,Liu R C,Chen C T,Fu Q L,Wan C H,Shi S H,Chen H M,Huang Y.2018.Development of loop-mediate indirect PCR for simultaneously detecting 3 pathogenic bacteria of waterfowl[J].Chinese Journal of Veterinary Science,38(1):96-99.]
程增青,申洪银,范根成,李明义,常顺志,王传禹,许琳,郭玉广,马爽.2009.鸭疫里默氏杆菌分离鉴定及药敏试验[J].中国动物检疫,26(12):32-34.[Cheng Z Q,Shen HY,Fan G C,Li M Y,Chang S Z,Wang C Y,Xu L,Guo Y G,Ma S.2009.Isolation and identification and drug sensitivity test of Riemerella anatipestifer[J].Chinese Journal of Animal Health Inspection,26(12):32-34.]
范忠军,许保疆,翟崇凯,郁川,王欢莉.2017.鸡源肠炎沙门氏菌快速检测方法的建立及其分离株的耐药性分析[J].河南农业科学,46(10):137-142.[Fan Z J,Xu B J,Zhai C K,Yu C,Wang H L.2017.Establishment of rapid detection method of Salmonella enteritidis and drug resistance analysis of isolates from chickens[J].Journal of Henan Agricultural Sciences,46(10):137-142.]
刘洪明.2017.鸭疫里默氏杆菌分离株外膜蛋白(ompA)基因的克隆、表达及其单克隆抗体制备[D].兰州:甘肃农业大学.[Liu H M.2017.Gene cloning,prokaryotic expression of isolated R.anatipestifer outer membrane protein(ompA)and the preparation of monoclonal antibody[D].Lanzhou:Gansu Agricultural University.]
任晓梅,王小兰,韩文龙,张雪梅,陈宗超,李涛,丁铲,于圣青.2018.鸭疫里默氏杆菌的分离鉴定与生物学特性研究[J].中国动物传染病学报,26(3):47-51.[Ren X M,Wang X L,Han W L,Zhang X M,Chen Z C,Li T,Ding C,Yu S Q.2018.Isolation,identification and biological characterization of Riemerella anatipestifer[J].Chinese Journal of Animal Infectious Diseases,26(3):47-51.]
单敏.2017.鸭疫里默氏杆菌耐药相关基因M949_0011和M949_0459的鉴定及其功能研究[D].南宁:广西大学.[Shan M.2017.Identification and functional analysis of the drug resistance genes of Riemerella anatipestifer M949_0011 and M949_0459[D].Nanning:Guangxi University.]
王良,刘思路,代光胜,奎磊,李双菊,田宇,陈建军.2017.洱海流域畜禽饲料·粪便·土壤中四环素类抗生素残留分析[J].安徽农业科学,45(18):70-72.[Wang L,Liu S L,Dai G S,Kui L,Li S J,Tian Y,Chen J J.2017.Analysis of residue of tetracyclines antibiotics in livestock manures and soil in Erhai-Lake basin[J].Journal of Anhui Agricultural Science,45(18):70-72.]
闫琦,刘培培,张娇娇,王跃华,任斌,丁路明.2018.畜禽粪便中残留四环素类抗生素的研究概况[J].家畜生态学报,39(5):80-86.[Yan Q,Liu P P,Zhang J J,Wang Y H,Ren B,Ding L M.2018.Research survey of tetracycline in animal manure[J].Acta Ecologiae Animalis Domasitici,39(5):80-86.]
叶凤梅.2015.贡山八角枝叶中化学成分及抗菌抗肿瘤活性研究[D].上海:上海交通大学.[Ye F M.2015.Chemical constituents from the branches and leaves of Illicium wardii and anti-bacterial and anti-tumor activity[D].Shanghai:Shanghai Jiaotong University.]
叶胜强,陈洁,何斌,童新红,杨文海,金尔光,钱运国,鲍伯胜.2013.鸭疫里默氏杆菌对主要抗菌药物敏感性分析及合理用药[J].养禽与禽病防治,(1):10-12.[Ye S Q,Chen J,He B,Tong X H,Yang W H,Jin E G,Qian Y G,Bao B S.2013.The analysis of the drug sensitivity and rational use of the main antimicrobial agents against Riemerella anatipestife[J].Poultry Husbandry and Disease Control,(1):10-12.]
雍燕,刘国昌,方炳虎,乔妍红.2017.不同地区鸭疫里氏杆菌的分离鉴定与药敏试验[J].中国畜牧兽医,44(1):242-247.[Yong Y,Liu G C,Fang B H,Qiao Y H.2017.Isolation,identification and drug-sensitivity test of Riemerella anatipestifer from different areas[J].China Animal Husbandry&Veterinary Medicine,44(1):242-247.]
Aminov R I.2009.The role of antibiotics and antibiotic resistance in nature[J].Environmental Microbiology,11(12):2970-2988.
Andrasevic A T,Dowzicky M J.2012.In vitro activity of tigecycline and comparators against Gram-negative pathogens isolated from blood in Europe(2004-2009)[J].International Journal of Antimicrobial Agents,39(2):115-123.
Chen Y P,Lee S H,Chou C H,Tsai H J.2012.Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese[J].Veterinary Microbiology,154(3-4):325-331.
Chu C Y,Liu C H,Liou J J,Lee J W,Cheng L T.2015.Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant[J].Vaccine,33(1):92-99.
Deng M,Zhu M H,Li J J,Bi S,Sheng Z K,Hu F S,Zhang JJ,Chen W,Xue X W,Sheng J F,Li L J.2014.Molecular epidemiology and mechanisms of tigecycline resistance in clinical isolates of Acinetobacter baumannii from a Chinese university hospital[J].Antimicrobial Agents and Chemotherapy,58(1):297-303.
Gyurisé,Wehmann E,Czeibert K,Magyar T.2017.Antimicrobial susceptibility of Riemerella anatipestifer strains isolated from geese and ducks in Hungary[J].Acta Veterinaria Hungarica,65(2):153-165.
Hua X,Zhou H,Jiang Y,Feng Y,Chen Q,Ruan Z,Yu Y.2012.Genome sequences of two multidrug-resistant Acinetobacter baumannii strains isolated from a patient before and after treatment with tigecycline[J].Journal of Bacteriology,194(24):6979-6980.
Leski T A,Bangura U,Jimmy D H,Ansumana R,Lizewski SE,Stenger D A,Taitt C R,Vora G J.2013.Multidrugresistant tet(X)-containing hospital isolates in Sierra Leone[J].International Journal of Antimicrobial Agents,42(1):83-86.
Li T,Shan M,He J,Wang X L,Wang S H,Tian M X,Qi J J,Luo T R,Shi Y H,Ding C,Yu S.2017.Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline[J].Oncotarget,8(57):96615-96626.
Reid G E,Grim S A,Aldeza C A,Janda W M,Clark N M.2007.Rapid development of Acinetobacter baumannii resistance to tigecycline[J].Pharmacotherapy,27(8):1198-1201.
Rumbo C,Gato E,López M,Ruiz de Alegría C,FernándezCuenca F,Martínez-Martínez L,Vila J,Pachón J,Cisneros J M,Rodríguez-Ba?o J,Pascual A,Bou G,Tomás M.2013.Contribution of efflux pumps,porins,andβ-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii[J].Antimicrobial Agents and Chemotherapy,57(11):5247-5257.
Santajit S,Indrawattana N.2016.Mechanisms of antimicrobial resistance in ESKAPE pathogens[J].BioMed Research International,2016:2475067.doi:10.1155/2016/2475067.
van Berkel W J,Kamerbeek N M,Fraaije M W.2006.Flavoprotein monooxygenases,a diverse class of oxidative biocatalysts[J].Journal of Biotechnology,124(4):670-689.
Yang F F,Sun Y N,Li J X,Wang H,Zhao M J,Su J,Zhang Z J,Liu H J,Jiang S J.2012.Detection of aminoglycoside resistance genes in Riemerella anatipestifer isolated from ducks[J].Veterinary Microbiology,158(3-4):451-452.
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