Cloning Of Resistant Genes AcrB,marR In E.coli And Preliminary Screening Expression Strains

Antibiotics frequent,widespread and unreasonable application caused the Esherichia coli mulitiple antiobiotics resistance seriously day by day,the mulitiple antiobiotics resistance were concerned with efflux pumps existving in cellular membrane.Generally thought that AcrAB-TloC efflux pump played a major role in the phenotype of E.coli mulitiple antiobiotics resistance,if the system was deactivated,the MDR Esherichia coli may become from resistant to sensitive.the multidrug resistant efflux transporting gene acrB and inhibiting gene marR are important for studying the AcrAB-TolC system.The paper uses the conventional method to extract E.coli's acrB,marR chromatosome DNA of ATCC25922 and 5 kinds of different animal sources,different resistant level from clinical isolates,designing the specificity primers,amplificated by PCR,ligated the PCR production and the pMD 18-T cloning vector with T4 DNA ligase,the ligation was transformed into JM109 competence cells,and then determinated sequence of positive cloning plasmid that was dentified by enzyme digesting and PCR amplification,that obtained acrB, marR entire gene orders.Results showed that homology of 3 different animal sources between acrB gene order and GeneBank published is 100%;Besides one marR gene have not the mutation,others partial basic groups have some mutations,few of them lead the amino acid changed.nucleotide sequence homology is between 97.5%and 98.2%,amino acid sequence homology is between 97.3%and 98.6%,including two com-mutations: 307site G→A,amino acid Gly→Ser;409site T→C,amino acid Try→His.Above mentioned the marR mutations of persisters whether to affect the regulatiion of AcrAB-TolC system,and then affected the MDR level,Also waited for further studies.On the basis of acrB and marR gene clone and the homology comparative analysis,double enzyme digested the positive cloning plasmid and the expression vector pPICZαA,ligate the reclaimed productions, ligation was transferred into E.coli TOP10 competence cells,the recombinant plasmid pPICZαA -marR and pPICZαA -acrB were linearized with SacI enzyme,and then transformed into GS115 yeast competence cells by electroporation,using the method of boiling-freezing- boiling to extract yeast genome,bolting gene integration recombinant GS115 by PCR,the result of 1%electrophoretic indicate that acrB and marR gene may integrate into the GS115 yeast cells,the PCR productions' molecular weight is 1.6Kb,1.0Kb respectively.This experiment obtains nucleotide sequence homology comparative analytic result of the clinical isolated E.coli's MDR genes acrB and marR.it will provide the references to research regulative mechanism of AcrAB-TolC efflux pump;Constructing the Pichia pastoris expression vectors of acrB and marR gene will establish the foundation for inducting Pichia pastoris expression,preparing antibodies,establishing the fast effective method to detect genes expression level,elucidating the multidrug resistant mechanism of clinical isolated E.coli.