To evaluate the immunogenicity of the hemolysin HlyE protein of avian pathogenic Escherichia coli(APEC),this study performed prokaryotic expression and purification of the APEC hemolysin HlyE protein.The expressed recombinant protein was analyzed by SDS-PAGE and western blot.After purification,the protein was dialyzed at 4℃using a dialysis bag.The hemolytic activity of the dialyzed recombinant protein was then tested on blood agar plates.The results of SDS-PAGE and western blot analyses showed that the recombinant HlyE protein(rHlyE)with a molecular weight of approximately 36ku was successfully expressed and purified.The concentration of the purified protein was determined to be 0.65mg/mL using an ND-2000 ultra-micro nucleic acid and protein analyzer.The hemolytic activity test showed that rHlyE had hemolytic activity.The dialyzed rHlyE was used as an antigen to immunize mice at a dose of 50μg per mouse.The control group received an equal volume of PBS at the same time.An indirect ELISA method was used to detect the levels of specific antibodies in the serum of mice from both groups.On 18 days after the third immunization,the mice were challenged with APEC bacteria at 2LD50,and the survival of the mice was observed over 7 days.On 28 days after the first immunization,the mice from each group were sacrificed to obtain their spleens for preparing splenocytes.Flow cytometry was then used to detect the percentages of CD3+,CD4+,and CD8+ T lymphocytes,thereby assessing the immunogenicity of rHlyE.The results of the indirect ELISA showed that the protein was capable of inducing a humoral immune response in the organism,resulting in the secretion of high levels of IgG antibodies.The antibody level peaked 15 days after the third immunization.The rHlyE immunoprotective experiment indicated that the immunized mice exhibited no significant clinical symptoms and had a survival rate of 80%within 7 days;whereas,the control group mice showed evident clinical symptoms and all died within 3 days after challenge.Flow cytometry results demonstrated that,compared to the control group,the proportions of CD3+,CD4+,and CD8+ T lymphocyte subsets were all increased in the immunized mice.These findings suggest that rHlyE is partially soluble when expressed in Escherichia coli BL21,and immunizing mice with it induces a strong humoral immune response and provides effective immunoprotection.This study has clarified the immunogenicity of the APEC HlyE protein,offering a reference for the selection of immunoprotective proteins against APEC and for the development of cross-protective vaccines.
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