Effects Of Different Signal Stimulation On Transcription And Expression Of PhoP Gene In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)has a serious hazard to poultry farming in various countries,becasue of its high incidence rate and a strong susceptibility pathogens.The PhoP-PhoQ two-component system in APEC is mainly involved in regulating the expression of bacterial virulence genes,participating in the adaptation process of bacteria to Mg2+-restricted growth environment,participating in epithelial cell invasion and anti-infection of antibacterial peptides.The PhoP-PhoQ two-component system plays an important role in the pathogenesis of avian pathogenic E.coli.Studies have shown that acidic pH,low Mg2+ and antimicrobial peptides can promote the phosphorylation of PhoP,and activate the downstream target genes regulated by PhoP protein to adapt the bacteria to environmental changes.Therefore,the AE17,?phoP,?phoQ and ?phoPQ deletion strains were used as the research object,and the growth of bacteria in vitro was detected by Mg2+ concentration,low pH and avian beta defensin(AvBD9).By preparing PhoP rabbit polyclonal antibody,RT-qPCR and Western blot were used to detect the changes of phoP gene transcription and expression in different pH,Mg2+concentration and antimicrobial peptide culture environment,in order to provide a theoretical basis for further study on the regulation of PhoP in APEC.The main research contents are as follows:1.Optimization of PhoP protein expression conditions and preparation and detection of PhoP rabbit polyclonal antibodyIn this experiment,the optimal conditions for the highest expression of the target protein were screened by optimizing the induction temperature,IPTG induction concentration,induction time and bacterial concentration.The results showed that IPTG was added to the final concentration of 0.75 mmol/L when the OD600 nm 1.0,and the highest expression of PhoP protein was induced in the shaker at 25? for 11 h.The concentration of the protein was 3.621 mg/ml by BAC protein assay.Rabbits were immunized four times with purified PhoP protein as immunizing antigen,and the titer of immune serum was determined by two-way agar diffusion method and ELISA method;the specificity of immune serum was detected by Western blot.The two-way agar diffusion results showed that the rabbit anti-PhoP polyclonal antibody had a titer of 1:16;the ELISA results showed that the rabbit anti-PhoP polyclonal antibody titer was 1:209715200;the results of Western blot showed that the PhoP recombinant protein specifically binds to antibodies in rabbit serum.2.Effect of acidic pH on the expression of phop gene in AE17 strainIn order to investigate whether acidic pH affects the in vitro growth of avian pathogenic Escherichia coli and changes in the amount of phop gene expression,this experiment compares AE17 and AphoP under normal and acidic pH conditions by culturing bacteria with LB broth medium of different pH.The in vitro growth rate of ?phoQ and ?phoPQ deletion strains,and the changes of phoP mRNA transcriptional expression and protein expression were detected by RT-qPCR and Western blot,respectively.The in vitro growth rate assay showed that the acidic pH affected the growth of AE17 strain in vitro,and the growth rate of AphoPQ deletion strain was higher than that of AE17 wild strain under the conditions of pH 5.0 and pH 4.5;the growth rate of AphoP and AphoQ deletion strain was lower than AE17 Wild strain.The results of RT-qPCR and Western blot showed that the transcription level and protein expression level of phoP gene of AE17 strain were up-regulated and down-regulated under different pH culture conditions.3.Effect of Mg2+ concentration and acidic pH on the expression of phop in AE17 strainIn order to investigate whether the concentration of Mg2+affects the growth of avian pathogenic Escherichia coli and the change of the expression of the phop gene,this experiment determined whether the concentration of Mg2+ affects the growth rate of APEC in vitro by culturing APEC with different Mg2+concentrations.The results showed that the growth of the AE17 strain was significantly reduced at a Mg2+concentration of less than 250 ?M.The concentration of Mg2+at 250 was then selected to investigate whether the growth of the AE17 strain was affected at different pH.The results showed that the AE17 strain grew normally at pH 5.0,5.5 and 7.0,grew slowly at pH 4.5,and did not grow at pH 4.0.The results of RT-qPCR and Western blot showed that the transcription level and protein expression level of phoP gene of AE17 strain were up-regulated and down-regulated under different pH culture conditions with Mg2+concentration of 250 ?M.4.Effect of AvBD9 on the expression of phop of AE17 strainIn order to study whether antibacterial peptides affect the transcription and expression levels of APEC gene phop,this study was based on the co-culture of AvBD9 and AE17,and then the mRNA transcription level and protein expression level of phoP were detected by RT-qPCR and Western blot.The results showed that the transcript level of phoP mRNA decreased with time.Compared with the treatment without AvBD9,the mRNA transcription level of phoP decreased after the addition of the antimicrobial peptide,and the expression level decreased after 30 min of culture.After 120 min of culture,the expression level tended to be gentle and the difference was significant.This result indicates that the antimicrobial peptide regulates the transcription and expression of phoP.The results showed that pH and Mg2+ concentrations and antimicrobial peptides affected the in vitro growth of AE17 by culturing AE17 under different culture conditions of pH,Mg2+ and antimicrobial peptides,and these signals stimulated the transcription and expression of phoP gene.The study of APEC has different effects on the transcription and expression levels of phoP gene under different acidic pH,Mg2+ concentration and antimicrobial peptides,which provides a theoretical basis for further study on the regulation of transcriptional regulator PhoP in avian pathogenic Escherichia coli.