Immunogenicity Of Stx2B-Tir-Stx1B-Zot Protein From Escherichia Coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic enteric pathogen of worldwide importance, and is one of Shiga toxin-producing Escherichia coli (STEC). The type III secretion system of E. coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E.coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXcp of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery.In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-StxlB-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST'Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Our aim is to determine whether antibodies specific for Stx2B-Tir-Stx1B-Zot could prevent O157:H7 colonization, to determine the role of Zot in the blocking colonization as well as to develop the subunit vaccine for EHEC O157:H7.1. Construction and Expression of Recombinant Protein Tir from Escherichia coli O157:H7The objective of the experiment is to clone, express and study the antigenicity of translocation intimin receptor(tir) gene from EHEC O157:H7. The vector pET28 and BL21(DE3) were used to construct and overexpress the recombinant protein of tir gene by prokaryotic expression. The recombinant Tir protein was used to immunize rabbits to obtain high-titer polyclonal antibodies,which was used to analyze the antigenicity of Tir by Western blot. We obtain successfully high-level expression of recombinant Tir protein and the molecular weight is 48kD.The polyclonal antibody against Tir protein have good a reactive fragment with the native Tir from Escherichia coli O157:H7. In conclusion, tir gene was cloned and expressed successfully in Escherichia coli, and recombinant Tir protein has a good antigenicity.2. Biological Characterization of Recombinant Tir Protein from Escherichia coli O157:H7HEp-2 cell and the polyclonal anti-Tir were used to evaluate adherence, adherence inhibition and attaching and effacing lesion (A/E) of EHEC O157:H7. Balb/c mice were inoculated with purified 100μg Tir protein subcutaneously and the protection rate and fecal shedding were analyzed after orally challenged by 1010CFU EHEC O157:H7. Anti-Tir IgG titers were detected by indirect ELISA. Balb/c mice immunized twice showed survival rate up to 100% compared to the 50% survival rate. Moreover, no EHEC O157:H7 in the fecal samples was detected after 8d challenged with O157:H7,but non-immunized mice still sheded the organism on 16th day.The above-mentioned results indicated that Tir protein has a good antigenicity and could be used for EHEC 0157 genetic engineering vaccines.3. Expression Stx2B-Tir-StxlB-Zot Multivalent fusion protein from EHEC O157:H7EHEC O157:H7 mainly colonize the intestine tissue, therefore, developing the genetic engineering vaccine enhancing the mucosal immunity has a significant dominance. In this study, we amplified tir, stx1b and stx2b genes and contructed the recombinant plasmid of pGEX-stx2b-tir-stx1b. The positive pGEX-stx2b-tir-stx1b were digested with Xho I, dephosphorylated with alkaline phosphatase and ligated with the zot gene to construct the pGEX-stx2b-tir-stx1b-zot. BL21(pGEX-stx2b-tir-stx1b-zot) was induced with IPTG and analysize with SDS-PAGE. All the results indicated that the recombinant Stx2B-Tir-Stx1B-Zot was successfully expressed, the molecular weight is 107kD and the anti-Stx2B-Tir-Stx1B-Zot antibody are able to react with the native Stx2B,Tir and Stx1B from EHEC 0157:H7. 4. Immunogenicity Analysis of Stx2B-Tir-StxlB-Zot from EHEC O157:H7Balb/c mice were immunized subcutaneously and intranasally with Stx2B-Tir-StxlB-Zot protein, respectively at week 0,3. After the second immunization, the blood from the immunized and naive mice were collected to prepare sera for detecting the IgG and IgA titers. The feces form the immunized and naive mice were collected to detect the secretory IgA titers. Balb/c mice immunized intranasally have 100% survival rate, no detectable bacteia on the 7th after challenged, the highest shedding of 3×103CFU/0.1g and 105 IgG titers in sera and aroud102IgA titers in feces. Balb/c mice immunized subcutanously have 88% survival rate, no detectable bacteia on the 9th after challenged, the highest shedding of 1.1×105CFU/0.1g and 105 IgG titers in sera and no IgA titers in feces. Stx2B-Tir-Stx1B-Zot immunized intranasally is capable to play better immogenicity and able to be a preferred antigen to develop the subunit vaccine for EHEC O157:H7.