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The Expressing and Cloning of Multiple Drug Resistance Regulation Gene rob of Escherichia Coli

Author: LiuYuTang
Tutor: MaHongXia
School: Jilin Agricultural University
Course: Basic Veterinary Science
Keywords: Escherichia coli Multiple Drug Resistance rob clone Pichia prutoris
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 57
Quote: 0
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Abstract


The antibiotics in preventing and treating Escherichia coli diseases is indispensable.However,the person and the animal infectious diseases caused by resistant strains increase year by year because of the abuse of antibiotics.The problem of Escherichia coli resistance and multiple resistance to antimicrobial agents is extremely serious.Active efflux system of bacterial may evoke many antibiotic to resistance.AcrAB,AcrEF, EmrAB,Ecr,QacE are found in Escherichia coli.Efflux pump AcrAB-TolC is the most important active efflux system for Escherichia coli.AcrAB-TolC contain drug proton AcrB,interstitial substance fusion protein AcrA and membrane channel protein TolC.Express level of AcrAB are regulated by mang regulation factor, as acrR,marA,mppA,sdiA,rob,soxS and so on.rob is a positive regulation factor.5 strain of Escherichia coli from different animal species and quality control strain ATCC25922 were selected.Based upon drug resistance detection to main therapeutic drugs,the rob gene of multiple drug resistance of Escherichia coli was amplified by PCR from DNA of Escherichia coli and fragments was ligated with the pMD18-T simple Vector.The ligated products was cloned into Escherichia coli JM109.Positive plasmid was sequenced.The sequence result indicated.Homology analysis of the rob gene nucleotides sequence and deduced amino acids sequence revealed that the rob of different Escherichia coli had most similarity with that gene of GeneBank.Homology analysis of the rob gene nucleotides sequence and deduced amino acids sequence revealed that nucleotide sequence homology in 98.7%-99.9%,amino acid homology in 98.6%-100%.Gene mutation of rob gene of Escherichia coli from different animal species maybe relating to animal species and the level of drug resistance.The expression vector pPICZαA and the recombinant plasmid digested by KpnI and XbaI.The target gene was subcloned into vector pPICZαA.Positive clones named as pPICZαA- rob with interest gene was identified by restriction endonuclease analysis and PCR.Then the positive recombinant plasmid was transformed into yeast GS115 for Rob expression.The positive recombinant plasmid for induction expression ues methanol,yeast expression condition optimization(methanol density,induction time,induction outset OD600 value).The culture liquid of bacteria containing pPICZαA- rob was collected at different time and was subsequently examined by SDS-PAGE.Results showed that the rob gene of Escherichia coli could express successfully in yeast.The expression product was identified by SDS-PAGE and found to be 33 kDa as expected.This experiment obtains the clinical separation Escherichia coli multiple drug resistance regulation gene rob nucleotide sequence homology analysis result,may provide the reference about mechanism for AcrAB-TolC pump;Obtains rob gene expression protein to further prepare its immune body,establishes faste ffective gene expression level examination method and expounded that our country clinical separation animal source Escherichia coli multiple medicine mechanism to lay the foundation.

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