Expression, Purification And Virulence Regulation Analysis Of PhoP/Q Proteins On Avian Pathogenic Escherich Coli

Avian pathogenic Escherichia coli(APEC)causes high mortality and significant harm to the development of poultry.The PhoP/Q binary regulation system exist in APEC and its outer membrane protein of PhoQ to feeling changes from the external environment,and then transmits the signal to PhoP,PhoP can regulation the expression of virulence genes and play a key role in pathogenicity through invasion epithelial cell.The exploration of PhoQ mediated PhoP to regulation APEC virulence genes is to detected outer membrane protein,endotoxin virulence genes by regulated of PhoP,it is foundation to the virulence of APEC that subsequent further clarify the impact of PhoP/Q regulation system.In order to study the regulation functions of PhoQ mediated to PhoP,in this experiment we start with genes to get Pho P/Q proteins product by prokaryotic expression and detected virulence genes by PhoP protein regulated;while the PhoQ protein has been provided materials for the laboratory's research.This is a reference for PhoP/Q regulatory function about virulence and mechanism.The results are as follws: 1 Expression of avian pathogenic Escherichia coli PhoP/Q proteinsBased on the genes sequence of E.coli PhoP/Q in NCBI(GenBank registration number CP000468.1),we design phoP(5?,3? were added EcoR I and Xho I restriction sites)and phoQ(5?,3? were added EcoR I and Hind III restriction sites,PhoQ are ectodomain)for PCR phoP/Q sequence.The phoP/Q sequence were cloned into the pMD19-Tsimple Vector and adapted into pET32 a after double digestion and sequencing verifying,construction prokaryotic expression vector and subsequently transformed into the E.coli BL21(DE3).The sequencing of E.coli BL21(DE3)-pET32a-phoP/Q inoculated in liquid LB,the recombinant proteins PhoP/Q were induced by IPTG,SDS-PAGE detection molecular weight is about 42.9 KDa and 34.43 KDa,it is consistent with the theoretical molecular weight.2 Purification and identification of avian pathogenic Escherichia coli PhoP/Q proteinsWe confirmed the best expression of the recombinant PhoP protein is soluble intracellular expression after induction.A well condition of expression to recombinant proteins were obtained after for one night with 0.5 mmol/L IPTG at 30?.The recombinant PhoP protein was purified by a column packed with Ni Resin after the supernatant of ice bath and sonicated,use SDS-PAGE and 1D-LC-MS to analysis recombinant PhoP and it was found that recombinant PhoP protein was successfully to express.The PhoQ is a membrane protein after two transmembrane,it was difficult to express the full sequence,and then we selected to express the ectodomain PhoQ.In this study,we learn expression and purification methods of PhoP,a well condition of expression the recombinant PhoQ protein was obtained after induction for one night with 0.5 mmol/L IPTG at 37? and the recombinant PhoQ protein was purified in LB supernatant,and then prepare for the follow-up experiments after SDS-PAGE verification.3 Avian pathogenic E.coli PhoP protein regulation analysis about virulence genesWe found several genes sequences from Genebank.The binding motifs of PhoP protein and DNA were predicted by bioinformatics analysis,then,the electrophoretic mobility shift assay(EMSA)were applied to analyze the capable of binding to promoter about the PhoP protein,any regulation genes about outer membrane protein,endotoxin were verified by electrophoretic mobility shift assay,this genes were iss serum virulence gene,mgtA magnesium transporter-related gene,chuA hemoglobin outer membrane receptor gene,uspA stress response gene,slyB outer membrane lipoprotein gene,hlyF hemolysis gene.In summary,this study successfully constructed the recombinantant engineered E.coli strain expressing pET32a-phoP/Q and get the soluble intracellular PhoP and secretory PhoQ.The biological activity of PhoP protein binding genes promoter region was verified by electrophoretic mobility shift assay(EMSA),the detecting methods was built about PhoP regulation of host bacteria DNA.At the same time,the successful expression of ectodomain PhoQ was provided some reference to research PhoQ.These results suggest that PhoP/Q has an important role in the regulation virulence of E.coli.This study provides some theory reference for further clarify the regulation function of PhoP/Q on E.coli.