Bioinformatics, reactogenicity, and immunogenicity of the outer membrane protein ChaN of Mannheimia haemolytica

Abstract:

[Background] Bovine respiratory syndrome seriously affects the health of cattle and has a serious impact on the breeding industry. Mannheimia haemolytica, as the main pathogenic bacteria of bovine respiratory tract, often causes bovine respiratory disease (BRD) when the immunity of cattle is low. At present, Mannheimia haemolytica is mostly controlled by vaccine. [Objective] To analyze the bioinformatics function and immunogenicity of ChaN protein and lay a foundation for the development of subunit vaccines. [Methods] NCBI BLAST was used to screen the potential antigens of Mannheimia haemolytica, and online tools such as Expasy ProtParam, Expasy Protscale, and TMHMM-2.0 were used to analyze their antigenic characteristics. RT-qPCR was conducted to determine the mRNA level of ChaN in M.haemolytica cultured for different time periods. The recombinant ChaN (rChaN) was obtained by prokaryotic expression, and the reactivity of rChaN was examined by Western blotting with the M.haemolytica- positive bovine serum. Mice were vaccinated with different doses of rChaN emulsified with white oil as the adjuvant, and the immunogenicity of rChaN was evaluated. Indirect ELISA was employed to measure the antibody titer. Seven days after the third vaccination, mice were subjected to intraperitoneal injection of the pathogen strain Mh95, and the protective effect of rChaN was tested. [Results] ChaN was a hydrophilic soluble protein with no transmembrane domain, an open reading frame, 16 epitopes, and 23 B cell epitopes. The mRNA level of ChaN was the highest in the logarithmic phase, moderate in the stationary phase and lag phase, and the lowest in the decline phase, showing differences in the four phases (P<0.001). ChaN reacted with the M.haemolytica-positive bovine serum. After immunization with 40 μg ChaN+white oil, the IgG level kept rising. IgG subtyping results showed that ChaN mainly caused the body to produce IgG1 and IgG2b. After mice were injected intraperitoneally with Mh95 strain on day 7 after the third immunization, the survival rate of mice in the 40 μg ChaN+white oil group was 90%, which was higher than those in the commercial vaccine, blank control, PBS, and white oil groups. The survival rates of mice in the blank control, PBS, and white oil groups were less than 20% within 7 days. [Conclusion] ChaN has good reactogenicity and immunogenicity and thus can be used as a potential immunogenic protein of M.haemolytica for subsequent research. The results provide a scientific basis for developing subunit vaccines against M.haemolytica.

Key words:Mannheimia haemolytica;ChaN;bioinformatics;prokaryotic expression;reactivity;immunogenicity