Abstract
Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis.
 Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentation
Highlights
Mastitis is a common disease in the animal breeding industry, especially in cows and dairy goats [1]
The ompA gene recombinant plasmid was transformed into E. coli Bl-21 strain to create the outer membrane protein A (OmpA) protein-expressing strain
We detected a single band corresponding to the expected molecular weight of OmpA protein, indicating that the OmpA antiserum was specific (Figure 3 A)
Summary
Mastitis is a common disease in the animal breeding industry, especially in cows and dairy goats [1]. Outer membrane proteins (OMPs) are the main extracellular proteins of E. coli They play an important role in drug resistance, substance transport, immune recognition of the bacteria and host, and improve pathogenicity [8]. The experimental and control groups were immunized with OmpA protein (50 μg per mouse) and PBS solution, respectively. Pathogens of animal mastitis of E. coli and S. aureus were collected at logarithmic growth phaseby centrifugation and washed two times with a 0.85% NaCl solution. Purified OmpA protein (50 μg per mouse) was injected three times into the experimental group while the control group received a PBS solution. OmpA protein and the control of PBS solution were emulsified with Freund’s Complete Adjuvant (Sigma-Aldrich, USA) for the first immunization.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
