Construction Of Nucleic Acid Vaccine With Neisseria Gonorrhoeae Surface Protein A And Study On Its Immunocompetence In Mice

Objectives:The NspA gene was amplified by PCR according to the sequence of Neisseria gonorrhoeae WHO-A putative NspA gene (AY157539, Genbank). The eukaryotic expression vector of NspA was constructed by molecular biological methods.BALB/c mice were immunized with pcDNA3.1(+)/NspA by intramuscular. The levels of humoral and cellular immunity induced by pcDNA3.1(+)/NspA in BALB/c mice were detected. These results will provide foundation for the development of Neisseria gonorrhoeae DNA vaccine.Methods:A pair of primers were synthesized after being designed by PRIMER5.0 software aaccording to the sequence of Neisseria gonorrhoeae WHO-A putative NspA gene. The NspA gene was amplified by PCR.The PCR products were purified and cloned into pUCm-T vertor. After cleavage and sequencing, the NspA gene was subcloned to pcDNA3.1(+) in the correct orientation. Then the constructed plasmid was transfected into RAW264.7 and COS-7 cells by liposome-mediated gene transfer method. The level of NspA mRNA in transfected RAW264.7 cells was assayed by RT-PCR and NspA expressed in transfected COS-7 cells was detected by immunocytochemistry. 6 weeks old BALB/c mice were immunized with pcDNA3.1(+)/NspA.The level of anti-gonorrhoeae NspA antibody in the immunized mice serum was detected by tube agglutination test. The level of IFN-γ was detected by ELISA. The proliferation of splenocytes was determined by MTT colormetry. The existence of NspA gene in BALB/c mice was indentified by PCR.