| Enterohemorrhagic Escherichia coli (EHEC) is one of the significant foodborne pathogenic bacteria, which was first isolated in 1975, and then confirmed as pathogenic bacterium. It can lead to hemorrhagic or nonhemorrahagic diarrhea, even diseases such as hemorrhagic enteritis (HC), hemolytic-uremicsyndrome (HUS), thrombotic thrombocytopenicpurpura (TTP) in human and animals. There were hundreds of outbreaks of EHEC all around the world including China, O157 is the major serotype. One of the toxiferous characters of EHEC O157 is to cause attaching and effacing (AE) lesion in human enterocyte, and eae is one of the most important genes that encode AE lesion, which are all located in the LEE pathogenicity island in bacterial chromosomes. Eae encodes an outer membrane protein called intimin. Another very importannt virulence factor of EHEC O157 is Shiga toxic, which is encoded by the prophage inserted into bacterial chromosomes. Pathological changes in organs and life-threatening diseases are proposed to be mediated by Stxs in human. It has been proved that intimin and Stxs are the principle candidates to develop vaccine for EHEC infecions because of their good immunogenicity.In this study, Stx1B, Stx2B and eae genes were amplified by PCR and cloned into pGEX-6P-1 expression vector after sequencing and comparing with that of reference strain, respectively. The recombinant plasmids were named as pGEX-Stx1B, pGEX-Stx2B, pGEX-eae, and were transformed into E.coli BL21. All of the three recombinant proteins were expressed efficiently in form of inclusion bodies after inducing, and occupied 27%, 27.7%, 19.5% of total cell protein respectively.The proteins were purified by cutting gels and used to immunize BALB/c mice. SP2/0 cells were fused with spleen cells from the immunized BALB/c mice. Supernatants of hybridoma cells were screened by indirect ELISA and the screened cells were subcloned at least three times by limiting dilution. 5, 5, and 2 hybridoma cell lines secreting specific monoclonal antibodies (McAbs) against Stx1B, Stx2B, eae were obtained and named 1G11, 2H8, 1B10, 3A6, 4B1; 1B3, 4B4, 2C3, 3D1, 1C2; and 2D4, 3B4, respectively. The subclasses of the twelve McAbs were mainly IgG, except 2D4 of IgM. The results of counting chromosome number showed that the chromosome numbers of the twelve hybridoma cells were 103±5, much more than that of SP2/0 cell (65～75). Western blotting showed that 1G11, 2H8, 1B10; 1B3, 4B4, 2C3; 2D4, 3B4 could react specifically against Stx1B, Stx2B, and eae, separately, but GST. Ascites of 1G11 and 2H8 were purified by caprylic acid-saturate ammoniumsulphate method, SDS polyacrylamide gelelectrophoresis indicated that there were H line and L line obviously without visible mixed protein. The results of additional ELISA displayed that 1G11, 2H8, 1B10 recognized the same antigenic epitope; 1B3, 4B4, 2C3 recognized the same antigenic epitope; 2D4, 3B4 recognize different antigenic epitopes. The affinity constants of 1G11, 2H8, 1B10, 1B3, 4B4, 2C3, 2D4, 3B4 were all between 107～109M-1, showed that they have high affinity to their corresponding antigen. All of the twelve hybridoma series could steadily secrete McAbs after freezing and thawing, and all of them had no cross reaction to Salmonella, Escherichia coli, Mycobacterium bovis, Listeria monocytogenes, Streptococcus A, Staphylococcus aureus. It was concluded that these monoclonal antibodies processed high degree of specificity and could be used as useful reagents to detect EHEC O157. This study provided a material basis for the diagnosis, prevention and treatment of EHEC-infected disease. |
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