Expression, Purification Of Neisseria Gonorrhoeae Surface Protein A Recombinant Protein And Studies On Its Immunocompetence

Objective:pET-30a/nspA, pET-30a/ltB and pET-30a/ltB-nspA recombinant protein were expressed in E.coli BL21, and then purified , NspA-special humoral immune response(especially mucosal immune response) and cellular immune response were produced in BALB/c mice immunized in intranasally with the recombinant proteins NspA, LTB-NspA to evaluate the immulonogic effect.Methods:pET-30a/nspA, pET-30a/ltB and pET-30a/ltB-nspA prokaryotic recombinant plasmid were extracted, and then identificated by PCR and double digestion. The recombinated plasmids were transformated into E.coli BL21 to express proteins. The recombinant proteins were then puried using Ni+-NAT Magnetic Agarose Beads and renatured by Urea concentration gradient. Antigenicity of recombinant proteins was analyzed by Western-blot. Rabits were immunized respectively with recombinant proteins LTB-NspA, NspA, LTB and Freund's adjuvant, serum were collected and antibody titers of anti-NspA were detected by indirect ELISA. 4-week-old female BALB/c mice were immunized respectively with NspA, LTB-NspA, LTB and solution buffer through intranasally after multiplicity prepared. All groups were immunized three times at 2w intervals. Serum and vaginal secretions were collected on weeks 0, 2, 4 and 6. Serum were obtained from tail vein blood samples and stored at -20℃.Vaginal secretions were obtained by pipetting 0.05 ml PBS in and out of the genital duct twice in each mouse. The level of anti-gonorrhoeae NspA SIgA antibody in vaginal secretions and IgG antibody in serum of the immunized mice were assayed by indirect ELISA. The spleenic cells obtained from the immunized mice were tested its proliferative ratio by MTT and TNF-γand IL-4 levels were tested in cultured supernatant.Results:Prokaryotic expression vectors pET30a/nspA,pET30a/ltB,pET30a/ltB-nspA induced by IPTG were expressed as 6-His recombinant proteins in EcoliBL21, three recombinant proteins had good antigenicity and immunogenicity by Western-blot. and then rabbits were immunized respectively. 4 weeks old female BALB/c mice was immunized with NspA, LTB-NspA, LTB and Solution Buffer through intranasally. The ELISA results showed the anti-gonorrhoeae NspA SIgA antibody in vaginal secretions were detected in test groups, and that of LTB-NspA was more than NspA and control. The level of anti-gonorrhoeae NspA IgG antibody in serum of the test groups showed significant higher than control, but that of LTB-NspA was no more than NspA. Proliferative ratio of the spleenic cells and TNF-γ, IL-4 levels in cultured supernatant in test groups were higher than control.Conclusions:(1) pET-30a/nspA, pET-30a/ltB and pET-30a / ltB-nspA were successfully expressed in E.coli BL21;(2) Humoral and cellular immunity were induced in mice immunized with recombinant protein by nasal feeding, and LTB-NspA and NspA-induced IgG,IL-4,IFN-γlevels and spleen lymphocyte proliferation stimulation index in the experimental group are significantly higher than control;(3) With mucosal adjuvants LTB-NspA mucosa induced by specific mucosal SIgA levels were significantly higher than those of without adjuvant NspA; but both induced IgG,IL-4,IFN-γlevels and lymphocyte proliferation stimulation index difference There was no significant。...