Prokaryotic Expression And Immunogenicity Analysis Of E.coli Outer Membrane Protein OmpC

Escherichia coli, a Gram-negative bacteria, is widely exist in nature and infect both human and animal. E. coli OmpC protein, an outer membrane protein, play a crucial role in resisting the E. coli infection which was. Therefore, the E. coli OmpC protein has good prospects in E. coli vaccine producing.In this study, the E. coli outer membrane proteins OmpC was expressed by prokaryotic expression by the molecular clone; the culture and protein expression of OmpC recombinant strain were optimized by orthogonal design; the expressed OmpC protein was purified by washing inclusion body and SDS-PAGE electrophoresis gel; polyclonal antibody was produced from immunized mice and its function was evaluated by detecting the protective effects in mice; the OmpC protein was analyzed by bioinformatics method and the recombinant epitope was designed.The main findings were as follows:(1) Prokaryotic expression strain construction: the recombinant plasmid of OmpC was constructed by molecular cloning and identified. The expressed OmpC protein was identified by SDS-PAGE electrophoresis confirmed.(2)The optimized conditions for OmpC protein induced-expression: the optimal culture condition was optimized by the method of orthogonal experimen: the glucose concentration was 0%, rotation rate was 230 r/min, and medium volume was 50 mL, the OmpC optimal induced-expressing condition: strain OD600 value was 0.8, IPTG final concentration was 0.3 mmol/L for 8 h at 32?.(3) OmpC polyclonal antibody preparation: the good specificity of antiserum was proved by western blotting. And the OmpC antibody titer was 1: 6400 examined by ELISA.(4) OmpC protein immunogenicity analysis: the mice was immuned by the pathogenic bacteria of E.coli for the antibody preparation. The immunprotection ratio can reach up to 63.64%.(5) Bioinformatics analysis of OmpC and the recombinant epitope vaccine: a higher OmpC protein homology was identified between different bacterial species. The OmpC antibodies might play a cross immune protective effect among different E.coli strains. 1-21 amino acids of OmpC was the signal peptide sequence with which was located on the cell outside membrane with no transmembrane structure. OmpC might have 5 B-cell epitopes, 1 CTL epitopes, and 1 Th epitopes; and OmpC protein epitope vaccine with good immunogenicity was obtained by. The OmpC protein epitope vaccine was obtained by recombinant splicing and its immunogenicity was good.