The Study Of Using SiRNA Technology To Silence Expression Of Outer Membrane Protein OprM Of Pseudomonas Aeruginosa

Objective This study was to investigate the inhibition of OprM that is an outer membrane protein of active efflux system of pseudomonas aeruginosa with siRNA technology.Methods The research uses genetic recombinanting technology,through the PCR amplificate oprM gene of pseudomonas aeruginosa,construct the prokaryotic expression model of OprM that is an outer membrane protein of active efflux system of pseudomonas aeruginosa,and induce expression of protein OprM,then cutting the expressive protein from SDS-PAGE to purificate them,and using it to make polyclonal antibody.Transformating PA competent bacteria with some siRNAs which was designed incording to oprM gene sequence, selected one siRNA which target to protein OprM using antimicrobial susceptibility testing, constructing vector with its sequence.Then,western blot technique was used to detect protein OprM to know the silence efficiency of vector.Results1.Through genetic recombinanting and purification technology,amplificating oprM gene, construct the prokaryotic expression model of OprM,and inducing expression the OprM protein with His.Tag.The test results of western blot showed that the expression protein was the insoluble OprM protein with His.Tag.Then making anti-OprM polyclonal antibody with purificated insoluble protein.2.PA were changed into competent cell with Cacl₂,statistics were used to verify drug resistance of PA were not changed in two conditions.3.Transformating PA competent cells with 3 siRNAs which was designed incording to oprM gene sequence,By selecting with antimicrobial susceptibility testing,the optimum transformation concentration and siRNA sequence were found that the transformation concentration is 25μl and the best siRNA sequences is 5' -AGAUCAACGUCGCGGAGUATT-3',3'-UACUCCGCGACGUUGAUCUTT-5'.4.The test results of western blot showed that the protein OprM was decreased after transformating vector. Conclusions Outer membrane protein OprM was inhibited by vector which was constructed based on siRNA sequences selected.