| The dairy cow mastitis, a common and frequently-occurring diseases caused by a number of pathogens, is one of the main diseases of milk cow industry. More than 91% of cases are caused by pathogens including coliforms, Staphylococcus aureus and environmental Streptococcus and among of them 33.6% of cases are caused by coliforms. While Escherichia coli cases accounted for 76.1% of the coliform cases. Multidurg-resistant bacterial infections are increasing seriously, and some of them even do not respond in conventional antibiotic therapies at all. Hence, interest in immunotherapeutic strategies has seen resurgence in recent years. In the past, studies showed that the outer membrane protein A (OmpA) exist widespreadly on the gram-negative bacteria surface. OmpA play a vital role in gram-negative bacteria. OmpA can function as an adhesin and invasin, serve as a receptor for several bacteriophages, participate in biofilm formation, and act as an immune target for the innate immune system. But no report on immunogenicity and immunoprotection of OmpA has been seen. For the further study on genetic engineering vaccine, the protection of recombinant proteins OmpA against E.coli, Shigella flexneri and Klebsiella pneumonia was investigated.Firstly, ompA genes were amplified by PCR from E.coli 308-2, C83919, O157, J96, 2002-1, Shigella flexneri and Klebsiella pneumonia and sequenced. After sequence analysis, ompA gene from E.coli 308-2 was introduced into pET-32a vector. The recombinant plasmid, ompA-pET, was transformed into E. coil strain Rosetta, and was induced with IPTG to express recombinant OmpA protein. The recombinant OmpA protein was purified with His-tag by Ni2+ resin. The immunogens were prepared by emulsifying ingredients in each group either with freund's complete or incomplete adjuvant, and emulsifying PBS with freund's complete or incomplete adjuvant as a control group. Immunization and challenge schedule were carried out as followed: mice received boosting immunization three weeks after the primary inoculation, and were challenged with E.coli C83919, O157, J96, 2002-1, S. flexneri and K. pneumonia respectively two weeks after boost immunization. At the same time, blood samples were drawn on 0, 7, 14, 21, 28, 35, 42, 49, 56 day after the primary immunization, and the antibody titres in mice serum were detected with Enzyme-Linked Immunosorbnent Assay (ELISA) and the concentration of IFN-γand IL-4 cytokines secreted from lymphocytes were detected with Elispot. A phylogenetic analysis showed that the nucleotide sequences of the E.coli 308-2, C83919, O157, J96, 2002-1, S. flexneri and K. pneumonia exhibited 99.9%, 100%, 99.9%, 97.7%, 96.0%, 94.6% and 86.5% homology to the published ompA sequence (Gene ID: 7437746). Wsetern blot results showed that the recombinant OmpA was correctly expressed in E. coli Rosetta. The IgG antibody titers of the OmpA in serum from immunized mice at 14d after boosted immunization were 1:64 000. The spot forming cells of IL-4 and IFN-γin mice lymphocytes from immunized mice increased significantly (p < 0.05). In a week after challenge, the immunized mice had certain immunoprotection against different coliforms. The results suggest that the recombinant OmpA protein can be used as a vaccine candidate.
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