人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

Study on immunogenicity of DNA vaccine encoding human cytomegalovirus gB and pp150 fusion gene

目的 构建人巨细胞病毒（human cytomegalovirus,HCMV）糖蛋白（gB）和胞膜蛋白（pp150）的单基因及融合基因的真核表达载体并比较其免疫学活性,为研制HCMV核酸疫苗奠定实验基础.方法 PCR扩增HCMV gB及pp150并将其连接成融合基因,分别克隆至peDNA3.1（＋）真核表达载体;制备纳米粒DNA疫苗并免疫BALB/e小鼠,检测其体液免疫与细胞免疫应答.结果 成功制备了peDNA3.1（＋）-gB、peDNA3.1（＋）-pp150和peDNA3.1（＋）-gB-pp150纳米核酸疫苗;gB和gB-pp150核酸疫苗均能刺激小鼠产生较强的特异性抗体（P〈0.01）;pp150和gB-pp150核酸疫苗免疫组的小鼠脾细胞能产生较多的IFN-γ和较高的刺激指数.结论 HCMV gB单基因核酸疫苗能诱导较强的体液免疫应答,pp150单基因核酸疫苗则能诱导较强的细胞免疫应答;而gB-pp150的融合基因疫苗能诱导较强的体液免疫和细胞免疫应答.

Objective To provide experimental evidence for development of human cytomegalovirus （HCMV） nucleic acid vaccine,HCMV surface protein（gB）,membrane protein（pplSO）,and gB-pp150 fused gene eukaryotie expression vector were constructed.Methods gB and pp150 genes were amplified and fused into gB-pp150,then were cloned into pcDNA 3.1（＋） to obtain recombinant expression plasmids pcDNA 3.1（＋）-gB,pcDNA 3.1（＋）-pp150 and pcDNA 3.1（＋）-gB-pp150,which were encapsulated with chitosan.Mouse were vaccinated and the humoral and eell immune response were determined by ELISA,specific proliferative response of plenie lymphocytes.Results The gB,pp150 and gB-pp150 fusion gene eukaryotie expression vector were successfully constructed.The antibodies A value induced by peDNA3.1（＋）-gB or peDNA3.1（＋）-gB-pp150 were much higher than that of peDNA3.1（＋）（P〈0.01）.The IFN-γ levels induced by pcDNA3.1（＋）-pp150 and peDNA3.1（＋）-gB-pp150 were significantly higher than that of pcDNA3.1（＋）.There are significant diferenee between the stimulating indexes of pcDNA3.1（＋）-pp150 or peDNA3.1（＋）-gB-pp150 immunized and normal mice.Conclusion The DNA vaccine pcDNA3.1（＋）-gB can induce significant humeral immunity response.and pcDNA3.1 （＋）-pp150 can induce high cellular immune response,whereas pcDNA3.1（＋）-gB-pp150 can induce both humeral and cellar immune responses in BALB/c mice.