Preliminary Study On Purification And Crystallization Conditions Of Outer Membrane Protein Ompt Of Avian Pathogenic Escherichia Coli O2 Isolates

Avian colibacillosis is a general term for complex multicellular syndromes caused by avian pathogenic Escherichia coli(APEC).Avian colibacillosis has become one of the most serious bacterial diseases in chickens,mainly in the clinical manifestations of E.coli sepsis,pericarditis,perihepatitis,airsac inflammation,and lead to varying degrees of mortality,causing serious economic losses to the poultry industry,and there is growing evidence that APEC has the potential for zoonosis.The outer membrane protein T(OmpT)is localized in the outer membrane of E.coli,is a highly substrate-specific proteolytic enzyme that belongs to the Gram-negative bacteria omptin family.The OmpT protein in APEC is encoded by the chromosome ompT gene(c-ompT)and the ompT gene(p-ompT)located in the plasmid,but there are some differences in the performance of the function between them.The plasmid ompT gene(p-ompT)of the pAPEC-O2-ColV was found to be 79%homologous to the chromosome ompT gene(c-ompT)by sequence alignment.The aim of this study was to investigate the relationship between the structure and function of p-OmpT,to understand the structural differences between c-OmpT and p-OmpT,and to explore the functional differences between c-OmpT and p-OmpT fundamentally,to declare the pathogenesis of APEC and to find specificity control measures of avian colibacillosis.The recombinant expression vector containing the c-ompT or p-ompT gene was constructed and transferred into the host strain BL21(DE3)for expression.The results of the protamine inhibition test showed that the growth of the bacteria containing the p-ompT gene was inhibited while the growth of the bacteria containing c-ompT was not affected.In order to further study the relationship between the structure and function of p-OmpT,and clarify the hydrolysis mechanism of p-OmpT protein.in this study,the p-ompT gene fragment was amplified by PCR from the plasmid pAPEC-O2-ColV of avian pathogenic Escherichia coli E058,and ligated to the expression vector to construct recombinant expression plasmid.and then subjected to heterologous expression in E.coli BL21(DE3).Since the protein is overexpressed during the inducing process,the protein is restored by denaturation and renaturation,and then the protein is purified by ion exchange and gel filtration chromatography to obtain a pure p-OmpT protein sample with high purity and polymerization status.The purified protein was screened by gas diffusion method and the crystallization conditions were optimized.We obtained the protein crystals of p-OmpT and subjected to X-ray diffraction and collected data for analysis.Obtained p-OmpT crystal was helpful to further analyze and define the structure and function of this unique protein.The use of Escherichia coli OmpT to degrade antimicrobial peptides and to screen and modity new antimicrobial peptides can provide new ideas and create conditions for the development of anti-OmpT protease as new antimicrobial peptides.