The Effect Of Matrine On The Expression Of Outer Membrane Protein A In Escherichia Coli

Part Ⅰ The effect of different antimicrobial concentrations of matrine on the expression of outer membrane protein A in Escherichia coli.Object This experiment adopts the LB broth - peritoneal dialysis system external silicone tube to establish Escherichia coli bacterial biofilms(BBF)model in vitro .To investigate different inhibitory concentrations of matrine effects in biological membrane to the early, middle and late on the Escherichia coli outer membrane protein A (OmpA) expression.Method First,to measure the minimum inhibitory concentration (MIC) of matrine and levofloxacin against Escherichia coli.The high pressure sterilized peritoneal dialysis tube carrier is inserted into the 24-well plates, randomly divided into 6 groups: blank control group (negative control), 1/2MIC of levofloxacin group (positive control group), 1/2MIC、1/4MIC、1/8MIC、1/16MIC of matrine group.At the beginning of the establishment from Escherichia coli biofilm model, will be drug intervention group adding drugs to tune into the corresponding concentration of the drug.In Chapterl,3,7 days,using a bacterial protein extraction reagent to extract 24-well plates total bacterial protein respectively and detecting Escherichia coli outer membrane protein (ompA) expression levels of different drugs and concentrations at different times by Western blotting (Western blot).Result Under different inhibitory concentration of drug intervention, 1/2 MIC matrine group, 1/4 MIC matrine group ,1/8MIC matrine group, 1/16 MIC matrine group and 1/2 MIC levofloxacin group compared with those in the control group compared ,no matter in Escherichia coli biofilm formation of early,middle and late stage ,Escherichia coli outer membrane protein A (ompA)expression were reduced (P<0.05).And different concentration of matrine affects the quantity of ompA expression statistics has no obvious difference (P>0.05).Conclusion 1/2 MIC matrine group, 1/4 MIC matrine group , 1/8MIC matrine group, 1/16 MIC matrine group and 1/2 MIC levofloxacin group in Escherichia coli biofilm formation of early, middle and late can inhibit the expression of its outer membrane protein A (ompA).Part Ⅱ The construction of Escherichia coli OmpA prokaryotic expression vectorObject Escherichia coli outer membrane protein A gene (ompA) was cloned and identified, constructing prokaryotic gene expression vector, and expressed in prokaryotic system, to build a foundation for OmpA overexpression strain.Method Escherichia coli K12 strain genomic DNA as a template, ompA gene was amplified by PCR, followed by restriction enzyme digestion, ligation,transformation,and other methods,this gene was cloned into prokaryotic expression vector pET-32a (+) to construct recombinant plasmid ompA-pET-32a,and verified by PCR, preliminary verification of the correct recombinant plasmids were sequenced.The identification of the correct recombinant was transformed into the expression host strain of Escherichia coli Rosetta,follow-up of the transformed strain Rosetta prepare to IPTG induction.Result Identified by PCR, DNA sequencing recombinant plasmid was confirmed ompA gene was correctly inserted into pET-32a (+) to obtain recombinant plasmid ompA-pET-32a; Sequencing results show that the ompA is the complete coding region of the gene 1041 bp, relative molecular mass ofabout 36 kDa.Conclusion Successfully constructed the recombinant plasmid containing the original OmpA protein-coding genes, obtained positive recombinant plasmids Rosetta host strain, for the construction OmpA overexpression strain foundation.