肺炎嗜衣原体MOMP和人IL-2融合基因DNA疫苗的免疫原性研究

Immunogenicity of the DNA vaccine with monogene and fusion gene of the major outer membrane protein in Chlamydia pneumoniae and human IL-2

目的构建肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)主要外膜蛋白(MOMP)单基因及momp和人IL-2融合基因的真核表达载体并比较其免疫反应性,为研制Cpn核酸疫苗提供理论和实验依据。方法扩增Cpn momp及人IL-2基因并将二者融合,将momp和momp-IL-2分别克隆至pcDNA3.1(+)真核表达载体;制备纳米粒DNA疫苗免疫BALB/c小鼠,免疫荧光组化法检测重组质粒在小鼠组织内的稳定表达;ELISA法检测小鼠血清中的特异性抗体和小鼠脾细胞培养上清中IFN-γ水平;MTT法测定脾淋巴细胞特异性增殖反应。结果成功制备了pcDNA3.1(+)-momp和pcD-NA3.1(+)-momp-IL-2的纳米核酸疫苗;Cpn momp单基因和momp-IL-2融合基因核酸疫苗均能刺激小鼠产生特异性抗体,且pcDNA3.1(+)-momp-IL-2能诱导小鼠产生更高效价的特异性抗体(P<0.05);pcDNA3.1(+)-momp-IL-2诱导小鼠脾细胞产生IFN-γ的量及对特异抗原的刺激指数明显高于pcDNA3.1(+)-momp。结论Cpn momp单基因核酸疫苗和momp-IL-2融合基因核酸疫苗均能刺激小鼠产生较强的体液免疫和细胞免疫应答;pcDNA3.1(+)-momp-IL-2的免疫效果强于pcDNA3.1(+)-momp。

To provide the theoretical and experimental basis for the development of nucleic acid vaccine, the eukaryotic expression vector containing the major outer membrane protein （MOMP） of Chlamydia pneumoniae monogene or its fusion gene with human IL-2 gene was constructed and their immunogenieities were compared. These genes were amplified , fused by recombinant PCR and then cloned into eukaryotic expression vector pcDNA3. 1 （＋） to obtain the recombinant plasmid pcD- NA3.1 （-） momp and pcDNA3. 1 （＋）-momp-IL-2. BALB/c mice were vaccinated intramuscularly with these two kinds of plasmid combined plasmids respectively. The humoral immune responses of mice after vaccination were determined by ELISA assay and the cell-mediated response was evaluated by the specific proliferative response and the level of IFN-γ of splenic lymphocytes. It was demonstrated that the eukaryotic expression vectors containing the momp gene or the momp-IL-2 fusion gene were successfully constructed and they could elicit production of specific antibodies in the vaccinated mice. The antibody titer e licited by plasmid peDNA3, 1（＋）-momp-IL-2 was much higher than that of peDNA3, 1（＋）-momp （P〈0.05）. In addition, the stimulation index （SI） and the IFNγ level also showed the similar pattern as antibody titer （P〈0.05）. It is evident that both the pcDNA3.1 （＋）-momp and ptasmid peDNA3.1 （＋） can induce strong humoral and cell-mediated immune responses in mice inoculated with these DNA vaccines and the immunogenicity induced by the former were significantly higher than that of the latter.