粘膜佐剂LTB优化的抗淋病ltB-nspA融合基因疫苗鼻饲免疫效果研究

Immunocompetence of a DNA vaccine of Neisseria gonorrhoeae surface protein A fused with B subunit of Escherichia coli heat-labile enterotoxin

目的:用真核重组质粒pcDNA3.1(-)/ltB-nspA鼻饲免疫小鼠,探索粘膜佐剂LTB辅佐NspA所诱发的特异性体液免疫应答和细胞免疫应答水平。方法:对真核重组质粒行PCR及双酶切鉴定后大量制备,经鼻饲途径免疫雌性BALB/c小鼠,间接ELISA法检测血清中NspA特异性IgG抗体水平及小鼠生殖道灌洗液中NspA特异性sIgA抗体水平;MTT法检测脾淋巴细胞增殖水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清IFN-γ含量。结果:融合基因pcDNA3.1(-)/ltB-nspA组小鼠生殖道灌洗液中sIgA(A450:0.316±0.045)明显高于pcDNA3.1(-)/nspA单基因组(P<0.05)和其它对照组(P<0.01);小鼠血清中IgG(A450:0.643±0.156)水平、脾淋巴细胞刺激指数(SI:1.65±0.32)和脾淋巴细胞诱生的IFN-γ(160.56±25.67pg/ml)水平均明显高于pcDNA3.1(-)/ltB、空质粒pcDNA3.1(-)和PBS对照组(P<0.01)。结论:pcDNA3.1(-)/ltB-nspA融合基因疫苗经鼻饲免疫,能够诱导小鼠产生较强的特异性体液免疫应答和细胞免疫应答;粘膜佐剂LTB可辅佐NspA诱导小鼠产生更高水平的生殖道粘膜免疫。

Objective：To detect NspA-specific humoral immunological response and cellular immunological response in BALB/c mice which were induced by a DNA vaccine pcDNA3.1（-）/ltB-nspA through intranasal immunization.Methods：Recombinant eukaryotic expression plasmids pcDNA3.1（-）/ltB,pcDNA3.1（-）/nspA,pcDNA3.1（-）/ltB-nspA were identified by Polymerase Chain Reaction（PCR）and enzyme digestion.Female BALB/c mice were inoculated with the recombinant eukaryotic expression plasmids through intranasally immunization at the weeks 0,2 and 4.Then humoral immunological response and the cellular immunological response were detected in female BALB/c mice by enzyme linked immunosorbent assay（ELISA）and methyl thiazolyl tetrazolium（MTT）colorimetric assay.Results：The level of NspA-specific sIgA（A450：0.316±0.045）in genital tract in the group pcDNA3.1（-）/ltB-nspA was showed significantly higher than in the group pcDNA3.1（-）/nspA and other controls.The levels of serum IgG（A450：0.643±0.156）,stimulation index（SI：1.65±0.32）of the splenic lymphocytes and IFN-γ（160.56±25.67 pg/ml）induced by splenic lymphocytes in the group pcDNA3.1（-）/ltB-nspA were significantly higher than in the pcDNA3.1（-）/ltB,pcDNA3.1（-）and PBS controls（P〈0.01）,but had no significant difference compared with pcDNA3.1（-）/nspA group（P〉0.05）.Conclusion：Humoral immunological response（especially for mucosal immunological response）and cellular immunological response could be effectively induced by the DNA vaccine of pcDNA3.1（-）/ltB-nspA in mice through intranasally immunization,and the mucosal adjuvant LTB could assist NspA to induce high level of mucosal immune response in the genital tract mucosa of mice.