Cloning Of Resistant Genes AcrA,acrR In E.coli And Preliminary Screening Expression Strains

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.multidrug resistant of Escherichia coli with related active efflux system of overexpression,overexpression of active efflux system may cause bacterium resistance for the high-frequency,the high level.AcrAB-TolC pump was the most main active efflux system of Escherichia coli.If AcrAB-TolC pump was inactivation, Escherichia coli was from multidrug resistant to sensitive.AcrAB-TolC contains drug proton AcrB,interstitial substance fusion protein AcrA and membrane channel protein TolC.Express level of AcrAB are regulated by mang regulating factor,as AcrR,MarA,MppA,SdiA,Rob,SoxS and so on.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 acrA and acrR gene of multiple drug resistance of Escherichia coli were amplified by PCR from DNA of Escherichia coli and fragments were ligated with the pMD18-T simple Vector.The ligated products were cloned into Escherichia coli JM109.Positive plasmid were sequenced.The sequence result indicated:Homology analysis of the acrA and acrR genes nucleotides sequence and deduced amino acids sequence revealed that the acrA and acrR of different Escherichia coli had most similarity with that gene of GeneBank.Homology analysis of the acrA gene nucleotides sequence and deduced amino acids sequence revealed that nucleotide sequence homology in 99.1%～100%,amino acid homology in 99.7%～100%;Homology analysis of the acrR gene nucleotides sequence and deduced amino acids sequence revealed that nucleotide sequence homology in 98.2%～100%,amino acid homology in 98.6%～100%.On the basis of acrA andacrR gene clone and the homology comparative analysis,double enzyme digested the positive cloning plasmid and the expression vector pPICZaA,ligate the reclaimed productions,ligation was transferred into E.coli TOP10 competence cells,the recombinant plasmid pPICZaA- acrA and pPICZaA-acrR were linearized with SaeI 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 acrA and acrR gene may integrate into the GS115 yeast cells,the PCR productions' molecular weight is 1.8Kb,1.2Kb respectively. This experiment obtains nucleotide sequence homology comparative analytic result of the clinical isolated E.coli's MDR gene acrA and acrR.it will provide the references to research regulative mechanism of AcrAB-TolC efflux pump;Constructing the Pichia pastoris expression vectors of acrA and acrR 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.