To establish a fluorescence-labeled Corynebacterium pseudotuberculosis(Cp),the super-folded green fluorescent protein gene(sfGFP)fused to the red fluorescent protein gene(mCher-ry)was cloned into pXMJ19 and expressed in Cp.The fluorescent signals of recombinant Cp cul-tured under different pH values,and the visualized detection of bacteria in the macrophages J774A.1 or mice infected with recombinant Cp were evaluated.The results showed that a double fluores-cence labeled XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)was successfully construc-ted by electrotransformation of Cp with pXMJ19-sfGFPmCherry,which containing sfGFP fused to mCherry.The colony morphology of XH02-sfGFPmCherry on fresh blood agar plates was pink.The XH02-sfGFP mCherry showed green and red fluorescence when observed under the fluo-rescence microscope.Compared with cultivation at pH7.0,XH02-sfGFPmCherry showed a signifi-cant decrease in green fluorescence intensity(fluorescence intensity/D600)at pH5.0,but signifi-cantly increased in red fluorescence intensity at pH5.0.Green and red fluorescences of Cp were ob-served in the XH02-sfGFPmCherry-infected J774A.1 in vitro or in the liver and kidney of XH02-sfGFPmCherry-infected mice in vivo,and with good overlap of two kinds of fluorescences.This study demonstrates that the constructed dual fluorescent labeled Cp can efficiently express both red and green fluorescent proteins,and express the dominant fluorescent signals under different pH value conditions,which can be used for Cp monitoring in vitro infection of macrophages and in vivo infection of mice by this pathogen,and providing potential tool for the localization and tracing research of Cp infection.

To establish a fluorescence-labeled Corynebacterium pseudotuberculosis(Cp),the super-folded green fluorescent protein gene(sfGFP)fused to the red fluorescent protein gene(mCher-ry)was cloned into pXMJ19 and expressed in Cp.The fluorescent signals of recombinant Cp cul-tured under different pH values,and the visualized detection of bacteria in the macrophages J774A.1 or mice infected with recombinant Cp were evaluated.The results showed that a double fluores-cence labeled XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)was successfully construc-ted by electrotransformation of Cp with pXMJ19-sfGFPmCherry,which containing sfGFP fused to mCherry.The colony morphology of XH02-sfGFPmCherry on fresh blood agar plates was pink.The XH02-sfGFP mCherry showed green and red fluorescence when observed under the fluo-rescence microscope.Compared with cultivation at pH7.0,XH02-sfGFPmCherry showed a signifi-cant decrease in green fluorescence intensity(fluorescence intensity/D600)at pH5.0,but signifi-cantly increased in red fluorescence intensity at pH5.0.Green and red fluorescences of Cp were ob-served in the XH02-sfGFPmCherry-infected J774A.1 in vitro or in the liver and kidney of XH02-sfGFPmCherry-infected mice in vivo,and with good overlap of two kinds of fluorescences.This study demonstrates that the constructed dual fluorescent labeled Cp can efficiently express both red and green fluorescent proteins,and express the dominant fluorescent signals under different pH value conditions,which can be used for Cp monitoring in vitro infection of macrophages and in vivo infection of mice by this pathogen,and providing potential tool for the localization and tracing research of Cp infection.