Outer Membrane Protein NMB0315 From Neisseria Meningitidis Serogroup B As A Novel Vaccine Candidate

[Objective]To explore the roles of prokaryotic recombinant outer membrane protein NMB0315 in inducing specific humoral and cellular immune response in mice, the bactericidal activity of the immune serum in vitro and the immunoprotection of the NMB0315 vaccine against N.meningitidis serogroup B, so as to evaluate preliminarily the possibility of the recombinant NMB0315 as a novel vaccine candidate.[Methods]The whole NMB0315 gene was amplified by PCR from the standard strain MC58 genomic DNA, cloned into a plasmid p ET-30 a, identified by sequencing analysis and double enzyme digestion. The vector p ET-30a/NMB0315 was transformed into E.coli BL21 to express recombinant protein. The protein fractions were purified through his-tag/Ni-NTA resin, identified by Western-blot and RP-HPLC. Anti-NMB0315 sera were obtained by intraperitoneally inoculating BALB/c mice with the purified recombinant protein. The indirect ELISA method was used to detect the levels of specific Ig G, Ig G1, Ig G2 a, Ig G2 b and Ig G3 in serum, specific s Ig A in genital tract, IL-4 and IFN-γ in culture supernatant of the spleen lymphocytes from the immuned mice. The CCK8 assay was used to detect the stimulation index(SI) related with the proliferation of spleen lymphocyte. The immune protective effect was investigated with the recombinant protein vaccine and the bactericidal titer of the immune serum was determined by serum bactericidal assay(SBA) in vitro.[Results]The prokaryotic recombinant vetor p ET-30a/NMB0315 was successfully constructed, the r NMB0315 proteins were expressed in supernatant with soluble state. The specific humoral and cellular immune responses were effectively induced by the r NMB0315 in the inoculated mice. In the FA+r NMB0315 group, the levels of serum Ig G, Ig G1, Ig G2 a, Ig G3, Ig G2 b and genital tract s Ig A were significantly higher than in controls(P<0.001), the titers of the antibody at the week 6 were 1:150,000, 1:85,000, 1:60,000, 1:35,000, 1:30,000 and 1:30,000 respectively. At the week 2, 4, 6, the serum Ig G2a/Ig G1 ratios were 0.673,0.810 and 0.685 respectively in the FA+r NMB0315 group, all ratios were less than 1. At the week 6,the stimulation index(1.56±0.056) of the splenic lymphocyte was significantly higher than those of the PBS(0.146±0.032)and FA(0.31±0.044)groups(P<0.001), the levels of IL-4(210.86pg/m L) and INF-γ(157.59pg/m L) in culture supernatant of the spleen lymphocytes were significantly higher than the PBS and FA groups(P<0.001). The bactericidal titer of the FA+r NMB0315 group reached 1:128 following three immunizations, the protection rate of the vaccine group was 90% against the N. meningitidis strain MC58.[Conclusion] 1. The r NMB0315 could effectively induce specific humoral immunity and cellular immunity in BALB/c mice, especially for Th2 humoral immunological response and mucosal immune response. 2. The r NMB0315 showed effective immunoprotection against N. meningitides serogroup B, the immune serum had strong bactericidal activity in vitro.