ABSTRACT
Objective To construct the Neisseria surface protein A (NspA) DNA vaccine of Neisseria gonorrhoeae and evaluate the humoral and cellular immune responses induced by this vaccine in mice model.Methods The recombinant expression vector pcDNA3.1 (+)/NspA was constructed by inserting NspA gene into the eukaryotic expression vector pcDNA3.1 (+) and confirmed by poly merase chain reaction (PCR),restriction enzymes HindⅢ,XbaⅠand DNA sequencing.NspA mR- NA in transfected RAW264.7 cells and NspA protein expression in transfected COS-7 cells were de- tected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical stai- ning,respectively.Forty-five male BALB/c mice were immunized with pcDNA3.1 (+)/NspA recom binant plasmid.The level of serum anti-Neisseria gonorrhoeae antibody of the immunized mice was detected by tube agglutination test,and the level of interieron (IFN)-?was assayed by enzyme-linked immunosorbent assay (ELISA).The proliferation of splenocytes was determined by methyl thiazolyl tetrazolium (MTT) colormetry.The NspA gene in BALB/c mice was identified by PCR with the total DNA extracted from quadriceps femoris in immunized sites.Results Restriction enzymes digestion a- nalysis and DNA sequencing results revealed that the pcDNA3.1 (+)/NspA had been constructed successfully.NspA gene had been transcripted and expressed in mammalian cells.The peak titer of specific antibody was 1:640 in pcDNA3.1(+)/NspA immunized group and there was no specific an- tibody detected in both pcDNA3.1 (+) immunized group and PBS group.The IFN-?level in pcD NA3.1 (+) immunized group was (23.79?11.85)pg/mL and that in pcDNA3.1 (+)/NspA immu- nized group was(169.71?30.52)pg/mL (P