Study On Biological Characteristics Of A Unique Gene Z3276 From EHEC O157:H7 And Development Specific PCR Based On Z3276 Gene

Enterohaemorrhagic Escherichia coli(EHEC)is a kind of zoonotic pathogen,of which E.coli O157:H7 is the most important serotype responsible for a number of outbreaks in animals,poultry,and humans worldwide,leading to serious public health cases.Ruminants,such as cattle and sheep,are major reservoirs of E.coli O157:H7 and these reservoir hosts are generally asymptomatic when carrying this microorganism.The most frequent route of transmission for E.coli O157:H7 infection is via consumption of contaminated food and water.E.coli O157:H7 infections can result in diarrhea ranging from mild to bloody and can induce hemorrhagic colitis,and some patients with hemorrhagic colitis develop a severe deadly complication known as the hemolytic uremic syndrome(HUS).Children and the elderly are at increased risk of it.There is no available vaccine product for preventing O157:H7 infection.Thus,early detection of this pathogen from easily contaminated food has become an important way to control disease outbreaks and it is necessary to develop a fast,sensitive and specific detection method.Recent study showed that a unique open reading frame(ORF),Z3276,was identified as a specific genetic marker of E.coli 0157:H7.The BLAST analysis suggested that z3276 nucleotides sequence,encoding a high homology amino acid sequence with other Escherichia coli type I pili,was only found in the genome of EHEC O157:H7.However,the precise role of z3276-encoding protein still needs to be defined.Type I fimbriae are the most important bacterial adhesion organelles that play a critical role in pathogenicity of most Gram-negative bacterial strains through facilitating bacterial colonization and being involed in activating inflammatory signaling pathways.Therefore,three parts of studies have been taken in this research.1.Prokaryotic expression and immunogenicity of the z3276 gene of EHEC O157:H7Protein prokaryotic expression vector has been built based on z3276 gene of O157:H786-24 strain.Then recombination protein has been expressed,and the immunogenicity has been confirmed positive by Western Blot.All of these laid the foundation of identification of o157:H7(Az3276)and analysis of its biological characteristics.2.Construction of EHEC o157:H7 z3726 gene knock-out mutant and its analysis of biological characteristicsUsing the genomic DNA of 86-24 strain as the template,343 bp(upstream z3726 arm)and 407 bp(downstream z3726 arm)fragments were amplified,respectively.The 811 bp Gm gene was amplified.The homologous arms and the Gm gene were linked to pMEG375 for constructing the vector pMEG375?Z3276.Candidate mutants were obtained through bacterial hybridization on plates,and then identified by PCR and Western blotting.The results showed that O157:H7(?z3276)was successfully constructed.The biological characteristics and pathogenicity of O157:H7(?z3276)and wild type(WT)were compared.WT entered the logarithmic growth phase after cultured 4 h,when it took 12 h under the same conditions for O157:H7(?z3276).Twitching motility assay showed twitching motility zone of O157:H7(?z3276)on the agar surface was smaller than WT.Biofilm formation ability of O157:H7(?23276)significantly decreased.Auto-aggregation assay showed that agg ability of O157:H7(?z3276)at all the times was lower than WT.The adhesion and invasion ability to Hep-2 cells of the mutant had not significant change,but the invasion ability to IPEC-J2 cells reduced.Mice pathogenicity tests showed that the LD50 of WT and O157:H7(?z3276)to BALB/c mice was 108 9 CFU and 109.5 CFU,respectively.In mice models of colonization,we observed shortened and lower fecal shedding in contrast with WT.All suggested that z3276 gene was associated with the growth,motility,biofilm formation,auto-aggregation,invasion to IPEC-J2 cells and mice pathogenicity of E.coli O157:H7.3.The development of z3276-PCR for detecting O157:H7According to sequence of z3276 gene of O157:H7 86-24 strain,we designed a pair of specific primers,target fragment size was expected as 608 bp.Tm was 60?.The tests results showed that the PCR assay was specific for O157:H7 detection,and the detection limit was 1 CFU and 10 CFU for cultures and clinical specimens inoculated E.coli O157:H7 respectively.The establishment of this method provided a specific,sensitive and rapid way for the detection of O157:H7 in clinical samples,which may contribute to the prevention and control of O157:H7 infection.