Construction Of Nucleic Acid Vaccine With Neisseria Gonorrhoeae Surface Protein A Fused With B Subunit Of Escherichia Coli Heat-labile Enterotoxin And Study On Its Immunocompetence

Objectives:Amplify the NspA gene and LTB gene, fuse LTB and NspA with a linker by recombination PCR, and insert them into pcDNA3.1(-) to construct the recombination eukaryotic expression vectors; encapsulate the constructed recombination eukaryotic plasmids with chitosan to be used as vaccine; detect the level of humoral and cellular immunity induced by nucleic acid vaccines in BALB/c mice immunized through vaginal. These results will provide foundation for the development of Neisseria gonorrhoeae DNA vaccine.Methods:The NspA gene and LTB gene were amplified from Neisseria gonorrhoeae WHO-A strain and Escherichia coli H 44815 strain. The NspA gene and LTB gene were fused with a linker by recombination PCR. The PCR products were purified and cloned into pcDNA3.1(-) vertor. After cleavage and sequencing amalysis, the NspA gene and the LTB-NspA gene were cloned into pcDNA3.1(-) in the correct orientation. The recombination eukaryotic plasmids were extracted and purified from E.coli JM109 by scale and were encapsulated wih chitosan for vaccination.On the other side,the recombination prokaryotic expression vectors(pET-30a/NspA,pET-30a/LTB,pET-30a/LTB-NspA)were constructed and induced to express in E.coli BL21; the 4 weeks old female BALB/c mice were immunized with pcDNA3.1(-)/NspA and pcDNA3.1(-)/LTB-NspA encapsulated wih chitosan. The expression of NspA and LTB in vaginal mucosal tissue of immunized mice were detected by immunoflourescence analysis. The level of anti-gonorrhoeae NspA sIgA antibody in vaginal fluid and IgG antibody in the immunized mice sreum were detected by indirect ELISA. The level of IFN-γinduced in supernatant of T lymphocytes culture from immunized mice was detected by ELISA. The proliferation of splenocytes was determined by MTT colormetry.Results:(1) The recombinant eukaryotic expression vetors pcDNA3.1(-)/NspA and pcDNA3.1(-)/LTB-NspA were successfully constructed by sequencing analysis, the chitoan encapsulated vaccines were uptaked, the NspA gene and LTB gene were expressed in vaginal mucosal tissue of immunized mice by immunoflourescence assay.(2) The anti-gonorrhoeae NspA sIgA antibody in vaginal fluid and IgG antibody in sreum were induced in the vaccinated mice; and the level of these antibodies increased with the weeks, and the level of anti-gonorrhoeae NspA sIgA antibody induced in the mouse group vaccinated with pcDNA3.1(-)/LTB-NspA was significantly higher than the mouse group vaccinated with pcDNA3.1(-)/NspA (P<0.05).(3) The cytokine IFN-γinduced in supernatant of T lymphocytes culture from immunized mice was significantly increased. The level of IFN-γin the mice immunized with pcDNA3.1(-)/NspA was reached 135.86±13.97pg/mL(P<0.01), and the value in the mice immunized with pcDNA3.1(-)/LTB-NspA was 140.18±20.54pg/mL(P<0.01). But none significant difference was tested between the mice group immunized with pcDNA3.1(-)/LTB-NspA and the mice group immunized with pcDNA3.1(-)/NspA (P>0.05).(4) The proliferation response of spleen cells of mice vaccinated with pcDNA3.1(-)/NspA group(SI=1.518±0.010) and vaccinated with pcDNA3.1(-) /LTB-NspA group(SI=1.573±0.012) was significantly higher than those of mice immunized with pcDNA3.1(-)(SI=1.134±0.007) (P<0.01). But there was no significant difference between the mouse group vaccinated with pcDNA3.1(-)/ LTB-NspA and the mouse group vaccinated with pcDNA3.1(-)/NspA(P>0.05).Conclusions:(1) The recombinant eukaryotic expression vetors pcDNA3.1(-)/NspA and pcDNA3.1(-)/LTB-NspA were successfully constructed. And used the chitoan encapsulated these recombinant plasmids as vaccines, the vaccines were uptaked, the NspA gene and LTB gene were also expressed in vaginal mucosal tissue of immunized mice.(2) The recombination prokaryotic expression vectors(pET-30a/NspA, pET-30a/LTB, pET-30a/LTB-NspA)were constructed correctly and the recombination protein of NspA and LTB-NspA were successfully induced to express in E.coli BL21.(3) Humoral and cellular immunity could be induced by the nucleic acid vaccine of pcDNA3.1(-)/NspA and pcDNA3.1(-)/LTB-NspA in vaccinated BALB/c mice. With the molecular aduvant,the vaccine of pcDNA3.1(-)/LTB-NspA could significantly increase sIgA antibody in the vaccinated mice vaginal fluid, but it could not significantly improve cellular immunity and increase specific IgG antibody in vaccinated mice serum.