Immunocompetence Of Nucleic Acid Vaccines Of Neisseria Gonorrhoeae Surface Protein A Fused With B Subunit Of Escherichia Coli Heat-labile Enterotoxin

Objective:NspA-special humoral immune response(especially mucosal immune response) and cellular immune response were produced by immunizing BALB/c mice through intranasally with the recombinated eukaryotic expression vectors (pcDNA3.1(-)/nspA,pcDNA3.1(-)/ltB,pcDNA3.1(-)/ltB-nspA) to evaluate the immulonogic effect.Methods:Eukaryotic expression vectors pcDNA3.1(-)/nspA,pcDNA3.1(-)/ltB, pcDNA3.1(-)/ltB-nspA were identified by PCR and Digestion with BamHⅠand HandⅢ. Recombinated eukaryotic expression vectors (pcDNA3.1(-)/nspA,pcDNA3.1(-)/ltB,pcDNA3.1(-)/ltB-nspA) were multiplicity prepared and purified. 4-week-old female BALB/c mice were immunized respectively with pcDNA3.1(-)/nspA, pcDNA3.1(-)/ltB-nspA, pcDNA3.1(-)/ltB, pcDNA3.1(-) and PBS through intranasally. 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 Stimulation index by MTT, and TNF-γlevels were tested in cultured supernatant. The expression of NspA,LTB-NspA,LTB in intranasal mucosal tissue of the immunized mice were detected by immunohistochemistry analysis.Results:Anti-gonorrhoeae NspA SIgA antibody in vaginal secretions was detected in pcDNA3.1(-)/nspA and pcDNA3.1(-)/ltB-nspA groups, and that of pcDNA3.1(-)/ltB-nspA was more than pcDNA3.1(-) /nspA and controls. The level of anti-gonorrhoeae NspA IgG antibody in serum of the pcDNA3.1(-)/nspA and pcDNA3.1(-)/ltB-nspA groups showed significant higher than controls, but that of pcDNA3.1(-) /ltB-nspA was no more than pcDNA3.1(-)/nspA. Stimulation index of the spleen cells and TNF-γlevels in cultured supernatant in pcDNA3.1(-)/nspA and pcDNA3.1(-)/ltB-nspA groups were higher than controls. The expression of NspA, LTB-NspA, LTB in intranasal mucosal tissue of the immunized mice were detected by immunohistochemistry.Conclusions:1. Humoral immune response(especially mucosal immune response) and cellular immune response could be induced by the nucleic acid vaccine of pcDNA3.1(-)/nspA and pcDNA3.1(-)/ltB-nspA in vaccinated BALB/c mice.2. The eukaryotic expression vectors(pcDNA3.1(-)/nspA,pcDNA3.1(-)/ltB,pcDNA3.1(-)/ltB-nspA) were expressed in intranasal mucosal tissue of the immunized mice.3. SIgA antibody in the vaginal fluid of mice vaccinated through intranasally was significantly increased in the vaccine of pcDNA3.1(-)/ltB-nspA with the mucous aduvant LTB .