Construction, expression of Neisseria gonorrhoeae porB and Escherichia coli ltB fusion gene and analysis of its Immunocompetence

Abstract:

Abstract: [Objective] To construct prokaryotic fusion gene expression vector pET-30a/ltB-porB, express the recombinant fusion protein LTB-PorB and analyze the immunocompetence of the recombinant fusion protein in female BALB/c mice through intranasally immunization. [Methods] B subunit of Escherichia coli heat-labile enterotoxin (LTB) and Neisseria gonorrhoeae Porin B (PorB)fusion gene, LTB gene and PorB gene were cloned into prokaryotic vector pET-30a. The recombinants were identified by Polymerase Chain Reaction(PCR), enzyme digestion and DNA sequencing, and then expressed efficiently in Escherichia coli BL21 in the form of inclusion bodies. The renatured recombinant proteins had antigenicity, which was confirmed by Western blot. Female BALB/c mice were inoculated with renatured recombinant proteins without endotoxin through intranasally immunization at the days 0, 14, 28. Next, humoral immunoresponse and cellullar immunologic response were detected in female BALB/c mice by enzyme linked immunosorbent assay(ELISA) and methyl thiazolyl tetrazolium(MTT) colorimetric assay. [Results] The level of PorB specific sIgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in LTB-PorB group, A450 of sIgA in LTB-PorB group was 0.66 at the day 42, which was significantly higher than controls (P<0.01), and the titer was up to 1:1280. A450 of serum IgG in LTB-PorB group was 0.60 at the day 28, which was significantly higher than the LTB and the Solution Buffer controls (P<0.01), and the titer was up to 1:2560. However, the IgG between LTB-PorB group and PorB control(A450:0.57)had no significant difference (P>0.05). Stimulation index of the splenic lymphocyte in LTB-PorB group was significantly higher than the LTB and the Solution Buffer controls (P<0.05). But the level of IFN-γ induced by splenic lymphocyte between LTB-PorB group and controls had no significant difference (P>0.05). [Conclusion] The recombinant fusion protein LTB-PorB could induce high level of humoral immunoresponse and slightly cellullar immunologic response in female BALB/c mice through intranasally immunization. For the first time to our knowledge, the mucosal adjuvant LTB could assist PorB to induce high level of mucosal immune response in the genital tract mucosa of mice.

Key words:Keywords: Neisseria gonorrhoeae; Porin B; B subunit of Escherichia coli heat-labile enterotoxin; gene fusion; prokaryotic expression; Immunocompetence