关键词: 淋病奈瑟球菌, 细菌菌苗, MetQ蛋白, 鼻腔免疫, 免疫保护

Abstract: 【Abstract】 Objective To prokaryotically express the Neisseria gonorrhoeae MetQ protein in Escherichia coli （E.coli）, and to evaluate the immunoprotective effect of MetQ protein against Neisseria gonorrhoeae by intranasal immunization in female BALB/c mice. Methods A pCold TF-metQ-E.coli BL21 expression strain was constructed by gene recombination technology, and MetQ protein was expressed and purified. Twelve female BALB/c mice aged 6 - 8 weeks were randomly and equally divided into two groups （6 mice in each group）: MetQ group received nasal drips of a mixture of MetQ protein and cholera toxin B subunit （CTB） adjuvant at a ratio of 1∶1 （10 μg per mouse）; phosphate-buffered saline （PBS） group received nasal drips of a mixture of PBS and CTB adjuvant at a ratio of 1∶1 （10 μg per mouse）; all mice were immunized once every 2 weeks for a total of 3 sessions. Enzyme-linked immunosorbent assay （ELISA） was performed to detect antibody titers in serum samples and vaginal secretions from 3 mice in the MetQ group after the last immunization, Western blot analysis to determine the protein expression of MetQ in Neisseria gonorrhoeae strains FA1090 and F19, as well as 6 randomly selected clinical isolates of Neisseria gonorrhoeae, and indirect ELISA to analyze the binding ability of MetQ antiserum to whole bacterial antigens. The bactericidal activity of MetQ antiserum against Neisseria gonorrhoeae and its inhibitory effect on the adhesion of Neisseria gonorrhoeae to ME-180 cells were evaluated. Measurement data were expressed as mean ± standard deviation, and t test was used for comparisons between different treatment groups. Results The signal peptide-removed MetQ protein was successfully expressed. After intranasal immunization, specific IgG, IgG1, IgG2a, and IgA antibodies were detected in the mouse serum samples in the MetQ group, with the antibody titers being 1 × 107, 1 × 105, 2 × 104, and 1 × 102 respectively, and specific IgG and IgA antibodies were detected in the mouse vaginal secretions in the MetQ group, with the antibody titers being 1 × 103 and 1 × 102 respectively. Western blot analysis showed that MetQ protein was expressed in all 8 Neisseria gonorrhoeae strains. Indirect ELISA showed that the binding ability of MetQ antiserum to whole bacterial antigens of Neisseria gonorrhoeae was 20. Serum bactericidal activity test revealed that the effective bactericidal titer of MetQ antiserum could reach 1∶320 with the survival rate of Neisseria gonorrhoeae being 46.33% ± 6.67% （t = 8.05, P = 0.001, compared with the serum-free control group ［100%］）, and when the MetQ antiserum was 1∶160 and 1∶640 diluted, the survival rates of Neisseria gonorrhoeae were 22.01% ± 6.80% （t = 11.47, P < 0.001） and 84.55% ± 6.99% （t = 2.21, P > 0.05） respectively. Adhesion inhibition assay showed that MetQ antiserum diluted at 1∶20, 1∶40 and 1∶80 could significantly inhibit the adhesion of Neisseria gonorrhoeae to ME-180 cells with the adhesion rates being 17.25%, 28.23% and 54.51% respectively （t = 48.44, 31.88, 7.29, P < 0.001, < 0.001, = 0.002, respectively, compared with the serum-free control group ［100%］）. Conclusions MetQ protein was expressed in different Neisseria gonorrhoeae strains, and high-potency specific antibodies could be detected in the serum samples and vaginal secretions from mice after intranasal immunization. The MetQ antiserum had a high affinity with Neisseria gonorrhoeae, and exerted favorable bactericidal activity and adhesion-inhibiting effect, indicating that it is a potential candidate vaccine protein against Neisseria gonorrhoeae infection.

Key words: Neisseria gonorrhoeae, Bacterial vaccines, MetQ protein, Intranasal immunity, Immune protection