霍乱弧菌甘露醇-1-磷酸脱氢酶基因mtlD的克隆表达

梁未丽 陈保立 赵萌 周妍妍 阚飙

梁未丽, 陈保立, 赵萌, 周妍妍, 阚飙. 霍乱弧菌甘露醇-1-磷酸脱氢酶基因mtlD的克隆表达[J]. 疾病监测, 2014, 29(3): 219-222. doi: 10.3784/j.issn.1003-9961.2014.03.014
引用本文: 梁未丽, 陈保立, 赵萌, 周妍妍, 阚飙. 霍乱弧菌甘露醇-1-磷酸脱氢酶基因mtlD的克隆表达[J]. 疾病监测, 2014, 29(3): 219-222. doi: 10.3784/j.issn.1003-9961.2014.03.014
LIANG Wei-li, CHEN Bao-li, ZHAO Meng, ZHOU Yan-yan, KAN Biao. Cloning and expression of mtlD encoding mannitol-1-phosphate dehydrogenase of Vibrio cholerae[J]. Disease Surveillance, 2014, 29(3): 219-222. doi: 10.3784/j.issn.1003-9961.2014.03.014
Citation: LIANG Wei-li, CHEN Bao-li, ZHAO Meng, ZHOU Yan-yan, KAN Biao. Cloning and expression of mtlD encoding mannitol-1-phosphate dehydrogenase of Vibrio cholerae[J]. Disease Surveillance, 2014, 29(3): 219-222. doi: 10.3784/j.issn.1003-9961.2014.03.014

霍乱弧菌甘露醇-1-磷酸脱氢酶基因mtlD的克隆表达

doi: 10.3784/j.issn.1003-9961.2014.03.014
基金项目: 

国家自然科学基金(No. 81171640)

Cloning and expression of mtlD encoding mannitol-1-phosphate dehydrogenase of Vibrio cholerae

Funds: 

This study was supported by the National Natural Science Foundation of China (No. 81171640)

  • 摘要: 目的 克隆表达霍乱弧菌甘露醇特异性磷酸烯醇式丙酮酸依赖的磷酸转移酶系统操纵子中的甘露醇-1-磷酸脱氢酶基因mtlD。方法 以测序株N16961染色体为模板,聚合酶链反应(polymerase chain reaction,PCR)扩增mtlD基因,扩增产物经NdeⅠ和XhoⅠ酶切后克隆入表达载体pET-30a。0.1 mmol/L IPTG 诱导表达,超声裂解上清经Ni柱亲和层析纯化,比色法测定酶活性。结果 mtlD 基因成功克隆入表达载体pET-30a,在宿主菌BL21中诱导表达,利用Ni亲和层析柱获得较高纯度的羧基端带His标签的MtlD蛋白,并具有酶活性。结论 表达纯化了有活性的霍乱弧菌甘露醇-1-磷酸脱氢酶,为进一步的功能研究打下了基础。
  • [1] IwamotoK,KawanobeH,IkawaT,et,al.Characterizationofsalt-regulatedmannitol-1-phosphatedehydrogenaseintheredalgaCaloglossacontinua[J].PlantPhysiol,2003,133(2):893-900.
    [2] WolffJB,KaplanNO.D-Mannitol1-phosphatedehydrogenasefromEscherichiacoli[J].JBiolChem,1956,218(2):849-869.
    [3] JenningsDH.Polyolmetabolisminfungi[J].AdvMicrobPhysiol,1984,25:149-193.
    [4] StoopJM,MooibroekH.CloningandcharacterizationofNADP-mannitoldehydrogenasecDNAfromthebuttonmushroom,Agaricusbisporus,anditsexpressioninresponsetoNaClstress[J].ApplEnvironMicrobiol,1998,64(12):4689-4696.
    [5] SuvarnaK,BartissA,WongB.Mannitol-1-phosphatedehydrogenasefromCryptococcusneoformansisazinc-containinglong-chainalcohol/polyoldehydrogenase[J].Microbiology,2000,146(Pt10):2705-2713.
    [6] KumarS,SmithKP,FloydJL,etal.Cloningandmolecularanalysisofamannitoloperonofphosphoenolpyruvate-dependentphosphotransferase(PTS)typefromVibriocholeraeO395[J].ArchMicrobiol,2011,193(3):201-208.
    [7] WangHY,YanMY,ZhaoYW,etal.Transcriptionalrepressorgene-mtlRofmannitolPTSoperoninVibriocholerae[J].ActaMicrobiologicaSinica,2007,47(3):522-525.(inChinese)王海燕,闫梅英,赵英伟,等.霍乱弧菌甘露醇PTS操纵子中mtlR为转录抑制基因[J].微生物学报,2007,47(3):522-525.
    [8] MustachioLM,AksitS,MistryRH,etal.TheVibriocholeraemannitoltransporterisregulatedposttranscriptionallybytheMtlSsmallregulatoryRNA[J].JBacteriol,2012,194(3):598-606.
    [9] HorwitzSB,KaplanNO.HexitolDehydrogenasesofBacillusSubtilis[J].JBiolChem,1964,239:830-838.
    [10] WestPA,ColwellRR.IdentificationandclassificationofVibrionaceae-anoverview[M]//ColwellRR.Vibriosintheenvironment.Wiley:NewYork,1984:285-363.
    [11] DrakeSL,WhitneyB,LevineJF,etal.Correlationofmannitolfermentationwithvirulence-associatedgenotypiccharacteristicsinVibriovulnificusisolatesfromoystersandwatersamplesintheGulfofMexico[J].FoodbornePathogDis,2010,7(1):97-101.
    [12] WangHY,ZhengX,LouJ,etal.ComparisonofthetranscriptionallevelsofmannitolPTSoperonbetweenepidemicandnon-epidemicstrainsofVibriocholerae[J].ActaAcademiaeMedicinaeSinicae,2009,31(1):51-54.(inChinese)王海燕,郑霄,娄静,等.霍乱弧菌两类菌株甘露醇发酵过程中PTS操纵子转录水平差异分析[J].中国医学科学院学报,2009,31(1):51-54.
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出版历程
  • 收稿日期:  2014-02-10
  • 刊出日期:  2014-03-20

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