B群脑膜炎奈瑟菌外膜蛋白NMB0315核酸疫苗的免疫活性和免疫保护作用初步研究

Immunocompetence and Immunoprotection of a DNA vaccine NMB0315 against N. meningitidis serogroup B in mice

目的:构建B群脑膜炎奈瑟菌pc DNA3.1(+)/NMB0315真核表达重组质粒,检测其诱导小鼠产生的特异性体液免疫和细胞免疫应答水平、免疫血清体外杀菌效果及免疫保护作用,初步探讨NMB0315核酸疫苗的免疫活性和免疫保护效果,为研制一种新的预防B群流脑的核酸疫苗提供实验依据。方法:以B群脑膜炎奈瑟菌标准株MC58基因组DNA为模板设计引物,PCR扩增NMB0315基因,克隆至pc DNA3.1(+)真核表达载体中,经Bam HⅠ和XhoⅠ双酶切及测序鉴定重组质粒。构建成功的pc DNA3.1(+)/NMB0315重组质粒转染COS-7细胞和RAW264.7细胞,免疫细胞化学染色法和Western blot法检测重组蛋白在真核细胞中的表达。大量提取重组质粒,肌注免疫BALB/c小鼠,检测小鼠体内抗NMB0315的特异性体液免疫和细胞免疫水平;测定免疫血清体外杀菌滴度,观察核酸疫苗对实验小鼠的免疫保护效果。结果:构建的核酸疫苗pc DNA3.1(+)/NMB0315能够在真核细胞中有效转录和表达;免疫小鼠后能诱导产生特异性体液免疫应答和细胞免疫应答。在疫苗免疫后的2、4、6周,pc DNA3.1(+)/NMB0315组和pc DNA3.1(+)/NMB0315+Cp G组血清总Ig G及Ig G1、Ig G2a、Ig G2b和Ig G3亚类抗体水平呈递增趋势,均明显高于PBS、pc DNA3.1(+)、Cp G对照组(P<0.001);小鼠血清Ig G2a/Ig G1比值均小于1;生殖道灌洗液中特异性s Ig A水平均明显高于PBS、pc DNA3.1(+)、Cp G对照组(P<0.01)。免疫第6周,脾淋巴细胞刺激指数(SI)均明显高于对照组(P<0.01,P<0.01,P<0.05)。pc DNA3.1(+)/NMB0315+Cp G、pc DNA3.1(+)/NMB0315疫苗组免疫血清补体介导的体外杀菌效价分别为1∶128和1∶64;在致死量B群脑膜炎奈瑟菌MC58攻击后72 h存活率分别为70%和65%,对照组[PBS,pc DNA3.1(+),Cp G]72 h存活率为0。结论:NMB0315核酸疫苗能够诱导小鼠产生较高水平的体液免疫(包括黏膜免疫)和细胞免疫应答;免疫血清补体介导的体外杀菌活性较高;NMB0315核酸疫苗对感染B群脑膜炎奈瑟菌小鼠具有较强的免疫保护作用。

Objective： To construct NMB0315 eukaryotic expression recombinant vector,detect specific humoral and cellular immune response induced by the recombint DNA vaccine intramuscularly in female BALB / c mice,evaluate the immunocompetence and immunoprotection of the vaccine,so as to provide experimental basis for the development of a novel nucleic acid vaccine against N. meningitidis serogroup B. Methods： The whole NMB0315 gene was amplified by PCR from the standard strains MC58 genomic DNA,cloned into a plasmid pc DNA3. 1（ ＋）,identified by double digestion of the recombinant plasmid with restriction enzymes and sequencing. The recombinant vector pc DNA3. 1（ ＋） / NMB0315 was transfected into eukaryotic COS-7 cells and RAW264. 7 cells,the NMB0315 protein was detected by immunocytochemical method and Western blot respectively. The levels of specific humoral and cellular immune response were detected after inoculating in female BALB / c mice intramuscularly with the recombinant plasmid. The immune protective effect was investigated with the DNA vaccine and the bactericidal titer of the immune serum was deter mined by serum bactericidal assay（ SBA） in vitro. Results： The recombinant pc DNA3. 1（ ＋） / NMB0315 was effectively transcripted and expressed in eukaryotic cells and the specific humoral and cellular immune responses were induced in the inoculated mice. In the recombinant pc DNA3. 1（ ＋） / NMB0315 group,the levels of serum Ig G,Ig G1,Ig G2 a,Ig G2 b and Ig G3 and genital tract s Ig A were significantly higher than in controls（ P 〈0. 001）. The stimulation index in the culture supernatant of the spleen lymphocytes of the vaccine group was higher than that of the control group（ P 〈0. 05）. The ratios of serum Ig G2 a / Ig G1 in the DNA vaccine group were less than 1. The bactericidal titer of the NMB0315 ＋ Cp G group reached 1∶ 128 following three immunizations,the protection rate of the vaccine group was 70%against the N. meningitidis strain MC58. Conclusion： The NMB0315 nucleic acid vaccine could induce higher levels of humoral immunity and cellular immunity and showed effective protection against N. meningitidis serogroup B,the immune serum had strong bactericidal activity in vitro.