Construction and immune effect evaluation of recombinant M13 phage vaccine targeting outer membrane protein P6 of nontypeable Haemophilus influenzae
10.13200/j.cnki.cjb.004426
- VernacularTitle:靶向不可分型流感嗜血杆菌外膜蛋白P6重组M13噬菌体疫苗的构建及其免疫效果评价
- Author:
WANG Cai
- Publication Type:Journal Article
- Keywords:
Nontypeable Haemophilus influenzae(NTHi);
Outer membrane protein P6;
Phage display technique(PDT);
M13 phage vaccine;
Immunogenicity
- From:
Chinese Journal of Biologicals
2025;28(02):129-136+142
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant M13 phage vaccine targeting the outer membrane protein P6 of nontypeable Haemophilus influenzae(NTHi) and evaluate its immunogenicity in order to provide new ideas for further development of NTHi vaccines. Methods The NTHi P6 gene was fused with the vector pMECS Phagemid by gene recombination technique.After packaging and purification, the obtained recombinant P6-M13 phage was prepared into recombinant P6-M13 phage vaccine. The expression of P6-M13 PⅢ fusion protein in the vaccine was detected by Western blot, the vaccine titer was determined by double-layer agar plate method, and the recombinant P6-M13 phage morphology was observed under transmission electron microscope. Ninety 5-week-old BALB/c female mice were randomly divided into PBS group, M13 phage group and recombinant P6-M13 phage vaccine group, 30 for each, and intraperitoneally injected with PBS(500 μL/mouse), M13 phage[1 × 10~(12)pfu/(500 μL·mouse)]and recombinant P6-M13 phage vaccine[1 × 10~(12)pfu/(500 μL·mouse)]on the 0, 14th and28th day, separately. Two weeks after the last immunization, the levels of specific IgG in serum and IFNγ, IL-2, IL-4, IL-5and IL-17A in spleen lymphocyte culture supernatant were detected by ELISA, and the proliferation of spleen lymphocytes was analyzed by CCK-8. Three weeks after the last immunization, the mice were challenged with 1. 5 × 108cfu/mL NTHi through the nasal cavity. After one week of challenge, the pathological changes of nasal mucosa and lung tissue were observed by HE staining. Four weeks after the last immunization, the heart, liver, spleen, lung and kidney of mice were weighed, the organ coefficients were calculated, and histopathological sections were prepared for pathological observation.Results The recombinant P6-M13 phage could correctly express P6-M13 PⅢ fusion protein with a titer of 5. 5 × 10~(14) pfu/mL,and the recombinant P6-M13 phage with regular morphology was observed under microscope. Compared with M13 phage group and PBS group, the level of serum specific antibody IgG in mice of recombinant P6-M13 phage vaccine group was significantly higher(F = 71. 489, P < 0. 05); the levels of IFNγ, IL-2, IL-4 and IL-5 secreted by mouse spleen cells decreased significantly(F = 8. 315, 16. 986, 39. 204 and 6. 291, respectively, each P < 0. 05), while there was no significant difference in IL-17 level among the three groups(F = 0. 863, P > 0. 05); the spleen cell stimulation index increased significantly(F =22. 952, P < 0. 05). After challenge, the nasal mucosa and lung tissue structures of mice in PBS group and M13 phage group were seriously damaged, and inflammatory cells increased, while in the recombinant P6-M13 phage vaccine group, the structures of nasal mucosa and lung tissue were normal with few inflammatory cells. There was no significant difference in the organ coefficients of heart, liver, spleen, lung and kidney of mice in each group(F = 1. 012, 1. 642, 0. 300, 2. 079, and 0. 405, respectively,each P > 0. 05), and no pathological changes were found in the general color morphology and pathological sections of the main organs. Conclusion The constructed recombinant P6-M13 phage vaccine targeting NTHi outer membrane protein P6 can induce effective humoral and cellular immunity in mice with certain immune protection ability and good safety.
