The Immunoprotection And Function Of HtrA Protein In Pathogenic E.coli From Fox

In order to develop effective vaccine for Escherichia coli prevention,in this study,the htra gene was cloned from E.coli strain isolated from silver fox,double digested,and ligated with expression vector pET-32 a.The recombinattion plasmid pET-32a-htra was transformed into E.coli BL21 strain for protein expression,and purified with Ni-agrose and thereby used for mouse vaccine.Furtherly,we generated the htra gene deletion strain with Red homologous recombination technology to study the role of HtrA protein caused change in biological characteristics and pathogenicity of the E.coli.The contents of the study are listed as follows:1.Expression and immunogenicity identification on HtrA protein of E.coli isolate CLE-1In this study,we firstly designed the specific primers according to the published gene sequences of htra in NCBI GeneBank,and performed the samplificatioon by PCR with genosome of E.coli isolated CLE-1as template.The htra gene was then cloned into the pET-32 a vector,and transformed into E.coli BL21?DE3?cells,in which a recombinant HtrA protein?His-HtrA?was expressed by inducing with IPTG.SDS-PAGE analysis showed that the expressed His-HtrA was about 65 kD,and further confirmed by western blot.The mice were immunized intramuscularly three times with purified His-HtrA,and challenged with the E.coli isolated CLE-1 with 2LD50 dose.The data showed that the His-HtrA provided a 65%protection for immunized mice.The results indicated that the HtrA could be used as the antigen for developing the subunit vaccine.2.Construction and characterization of a mutant E.coli CLE-1?htra Strain To investigate the roles of HtrA protein in pathogenic processes of E.coli.?-Red homologous recombination was used for knockout of htra gene,and comparison of growth characteristics,biochemical characteristics were performed between E.coli wild type strain and htra knockout mutant.The results showed that the mutant showed no difference in growth characteristics and biochemical characteristics with wild type strains.3.Biological characteristics and bacterial virulence identification of htra gene knock-out mutant of E.coli strain In the study,we exa mined the growth characteristics,biochemical characteristics,exercise ability,biofilm formation ability,Iron-uptake ability,anti-phagocytosis ability and bacterial virulence of E.coli wild type strain and htra knockout mutant.The data showed that,compared to the wild type strains,exercise ability,biofilm formation ability,anti-phagocytosis ability were sharply impaired.htra gene deletion strain and wild type E.coli LD₅₀were assayed for pathogenty evaluation.The data showed that the LD₅₀of the mutant strain was 3.55×10⁷CFU,and the LD₅₀of the wild type E.coli was 5.01×10⁶CFU,indicating HtrA-inactivation leading to attenuated virulence of E.coli.