Antigenicity analysis of the recombinant outer membrane NlpI protein of Mannheimia haemolytica

Abstract

To explore the antigenic characteristics of the NlpI protein. This study screened a potential antigen of Mannheimia haemolytica using NCBI Blast analysis and investigated its immunogenicity using online software. The recombinant NlpI protein (rNlpI) was expressed in prokaryotic cells, and its reactogenicity was detected by western blot. The transcription level of nlpI mRNA in M. haemolytica after various culture durations was determined by RT-qPCR. Mice were challenged with M. haemolytica strain 95 at an LD₅₀ dose, and their serum was collected at various times. The rNlpI protein in M. haemolytica and western blot were used to detect the expression of antibodies specific to rNlpI. BALB/c mice were immunized with various protein doses and white oil adjuvant on days 0, 7, and 14, and the antibody titer was detected by indirect ELISA. Cytokines were detected by double antibody sandwich ELISA. Seven days after the third immunization, M. haemolytica strain 95 was injected intraperitoneally at 3 × 10⁸ CFU/mouse. After 48 h, the surviving mice were dissected and the bacterial load in their lungs was assessed. NlpI is a hydrophilic and soluble protein with a transmembrane domain, 11 antigenic determinants, and 27 B cell epitopes. It was speculated that the protein has strong immunogenicity. The expressed NlpI protein could bind to bovine serum positive for M. haemolytica. The transcription level of the nlpI gene was the highest in the stable phase, followed in decreasing order by the logarithmic, slow, and decline phases. The expression levels in the various phases differed insignificantly. The IgG antibody titers of mice immunized with 10, 20, and 30 µg NlpI + white oil adjuvant and 30 µg NlpI were measured. The antibody level of the 30 µg NlpI + white oil adjuvant group was higher than that in the other groups (P < 0.001). The immune protection rates of the 10 µg rNlpI + white oil adjuvant and 30 µg rNlpI groups were under 30%, while they were 50% and 60%, respectively, in the 20 and 30 µg rNlpI + white oil adjuvant groups. The bacterial load of all immunized groups was 99.99% lower than in the PBS and white oil adjuvant control groups. The protection rates differed significantly among immunized groups (P < 0.001), with the 30 µg rNlpI + white oil adjuvant group showing the lowest bacterial load in the lungs. The rNlpI protein can react with bovine M. haemolytica positive blood with good reactogenicity, and can induce mice to produce high titers of specific IgG antibodies, effectively resisting homologous strain invasion. The results lay a good foundation for developing new M. haemolytica vaccines.

Keywords: Mannheimia haemolytica; Antigenicity; Bioinformatics characteristic; NlpI protein; Prokaryotic expression; Transcription level.

Fig. 1

Partial bioinformatics results of the NlpI protein. A Hydrophilicity/hydrophobicity prediction results for the NlpI protein. B Prediction of the NlpI protein transmembrane structure. C ORFs analysis of the NlpI protein. D Prediction of the secondary structure of the NlpI protein. h, α-helix; e, β-sheet; t, β-turn; c, random coil. E The predicted tertiary structure of the NlpI protein.

Fig. 2

Prediction of antigenic determinants for the NlpI protein.

Fig. 3

Results of the double enzyme digestion, target gene homology, and protein purification. A Double digestion of M. haemolytica NlpI-32a. M: DL10, 000 DNA Marker; NlpI-32a: recombinant plasmid pET-32a-NlpI; N: negative control. B Phylogenetic tree of the nlpI gene of M.haemolytica. C Protein purification results. M: protein marker; 1: the purified protein.

Fig. 4

Western blot results of the rNlpI protein.

Fig. 5

Transcription level of the nlpI gene of M. haemolytica strain 95 at the four growth phases. The group means were compared using one-way ANOVA with Dunnett’s multiple-comparison test. **P < 0.01.

Fig. 6

The retention of anti-NlpI antibody of M. haemolytica in BALB/c mice (n = 3). A Mice infection and blood collection. B–E Western Blot was used to analyze the anti-NlpI protein antibody in the serum of mice at 7,14,21,28 days after infection with 95 strains of M. haemolytica. Images are created using GraphPad.Prism.9.5 (https://www.graphpad-prism.cn/) and combined using Adobe Illustrator 2020 (https://helpx.adobe.com/cn/download-install.html) software.

Fig. 7

Cytokine levels (IFN-γ, TNF-α, and IL-6) in mice serum at various time points post immunization (n = 4). A The immunization schedule for titer evaluation. B Cytokine levels (IFN-γ, TNF-α, and IL-6) in the serum at various time points after immunization. Two-way ANOVA was used for comparing the groups. ****P < 0.0001. Images are created using GraphPad.Prism.9.5 (https://www.graphpad-prism.cn/) and combined using Adobe Illustrator 2020 (https://helpx.adobe.com/cn/download-install.html) software.

Fig. 8

IgG antibody generation against M. haemolytica strain 95 in reaction to rNlpI protein immunization (n = 10). A Immunization schedule for titer evaluation. B IgG titers against M. haemolytica strain 95, measured in the serum of BALB/c mice immunized with 30 µg rNlpI and 10, 20, and 30 µg rNlpI + white oil adjuvant; PBS and white oil adjuvant acted as controls. Measurements started seven days post injection. C IgG subtype titers (IgG1, IgG2a, IgG2b, and IgG3) against M. haemolytica strain 95 in immunized mice were measured after the third immunization. D IgG subtype detection over time inthe four mouse rNlpI protein immunization groups. Two-way ANOVA with Dunnett’s multiple comparison test was used to compare the groups. **** P < 0.0001. Images are created using GraphPad.Prism.9.5 (https://www.graphpad-prism.cn/) and combined using Adobe Illustrator 2020 (https://helpx.adobe.com/cn/download-install.html) software.

Fig. 9

Survival rates of mice challenged with M. haemolytica strain 95 (n = 10).

Fig. 10

Detection of lung bacterial loads in immunized and non-immunized mice (n = 3). Mice were challenged with M. haemolytica strain 95 (3 × 10⁸ CFU per mouse). We assessed the bacterial loads in the lungs. The groups were compared using one-way ANOVA with Dunnett’s multiple-comparison test. ****P < 0.0001.