Immuno-Protection Of Intimin And Tir-Tccp Recombination Protein Of Ehe O157:H7to Balb/C Mice And Animals Infection Experiment

Enterohaemorrhagic Escherichia coli(EHEC) O157:H7is an important zoonotic pathogens,causing diarrhea, haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura (TTP)and, in some cases,hemolytic uremic syndrome (HUS) leading to death in infants and young children and the elderly.Because the bacteria is presented in intestinal tract of calves, sheep, pigs, chickens and other domestic animals for a long time and the calves is the main reservoir,so it easily results in diarrhea in young animals and contaminates animal products and water and crops et al. Moreover, it has brought huge losses to the agricutural zone, and simultaneously poses a huge threat to the health of people. Furthermore, since the food-poisoning caused by bacteria discovered in the United States for the first time,the epidemic continued to spread to other countries one after another.In addition,the outbreaks of epidemic was also presented in some regions of China in2000and the harmful levels may be the most serious one so far. There is still no ideal vaccine able to control infections caused by bacteria at present,so the preparation of vaccine which can decrease the shedding of organism in reservoir to reduce the threat to the health of people would be the main research directions in this field.1. Construction of pCoId Ⅰ-tir-tccp and expression of fusion protein from Escherichia coli O157:H7tir-tccp fusion gene were amplicated by PCR and fused in frame with the multiple cloning sites of pCold Ⅰ-tir-tccp. The recombinant plasmid of pCold Ⅰ-tir-tccp was transfered to host cells E.coli BL21strain (DE3). Then, the protein was induced and expressed with induction of IPTG. The style of fusion protein were determined by SDS-PAGE and the result showed that most of fusion protein existed in supernatant. The Western blotting of recombinant protein Tir-TccP indicted that fusion protein has reactino genicity.2. Immuno-protection of Intimin and Tir-Tccp recombination protein of EHEC O157：H7to BALB/C miceTo evaluate the protective efficacy against1×1010CFU of EHEC O157:H7after subcutaneous immunized with Intimin and Tir-Tccp recombination protein at different concentration. The BALB/C mice were subcutaneous immunized twice (on days0and14)with Intimin and Tir-Tccp recombination protein.On the14th day following the second immunization,all the mice were orally infected with1×1010CFU of EHEC O157:H7.Serum samples were collected via cutting off the tail on every14days after every immunization and IgG antibody was determined by ELISA.The mortaily was recorded and feces were monitored for shedding after challenge.The result of ELISA demonstrated that the Tir-Tccp protein had elicited high titre antibody but the levels of antibody induced by Intimin was low.After challenge,the suvival rate of immunized groups was obviously higher than control group.Moreover,there was no EHEC O157:H7in the fecal samples of two immunized groups on the6th day after challenged,but control group mice still sheded high level of the organism on13th day.Intimin and Tir-Tccp recombination protein significantly reduced shedding of EHEC O157:H7from experimentally infected mice.3. Animals infection erperiment of O157:H7Calves and goats were inoculated intragastrically with1×1010CFU of EHEC O157:H7to detect shedding level of the organism and bacterial shedding time.All of the animals were afforded with5g/L Streptomycin solution for3days to enhance the colonization of O157:H7in the gut.Before challenge, animals were prohibited to drink and feed for12hours and12hours after challenge,animals were provided with0.5g/L Streptomycin solution and food.The shedding level of the organism were detected from24hours after challenge. The experiment determinated that bacterial shedding time of calves and goats was17days and13days,respectively;the highest shedding level was5×105CFU and1×1O5CFU,respectively. This model could be used to study the pathogenesis of EHEC O157:H7and established the foundation for the effective prevention methods.