摘要目的:研究内部核糖体进入位点(internal ribosome entry site，IRES)序列连接"抗原-佐剂"的单纯疱疹病毒2型(herpes simplex virus type 2, HSV-2)DNA疫苗对免疫小鼠的保护作用。方法:利用IRES重组构建HSV-2包膜糖蛋白D(glycoprotein D，gD)和CCL28佐剂双表达DNA疫苗pgD-IRES-CCL28和pCCL28-IRES-gD，经测序验证，肌肉注射免疫BALB/c小鼠2次后，定期收集小鼠的血清和阴道灌洗液样品，分离免疫后的脾细胞、肠系膜淋巴结细胞和直肠黏膜组织。通过终点ELISA法检测免疫后小鼠的抗原特异性抗体滴度，采用体外中和试验检测免疫后及攻毒后血清和阴道灌洗液中的中和抗体滴度，采用脾细胞和肠系膜淋巴结细胞趋化试验和直肠组织的免疫组织化学染色检测组织和器官内的CCL28响应性免疫细胞变化。对免疫后小鼠进行阴道攻毒试验，采用荧光定量PCR、称量体重及观察疾病严重程度的方法评估各组小鼠抵抗病毒感染的能力。通过与1∶1混合的单独表达gD和CCL28的DNA疫苗(pgD+pCCL28)的免疫保护效果比较，分析IRES双表达DNA疫苗诱导的小鼠体液免疫和细胞免疫应答水平，以及免疫保护效果。结果:pCCL28-IRES-gD免疫小鼠后可产生与pgD+pCCL28组相似的血清IgG滴度、阴道灌洗液IgA抗体滴度，以及较高的抗体中和能力、直肠黏膜IgA+浆细胞和黏膜组织内CCL28响应性免疫细胞，攻毒后血清中和抗体出现较晚，但小鼠未出现体重减轻、疾病症状和死亡。但pgD+pcDNA3.1和pgD-IRES-CCL28对小鼠的免疫保护无效。结论:pCCL28-IRES-gD双表达DNA疫苗可诱导小鼠产生较强的黏膜免疫应答，有较强的免疫保护作用。

abstractsObjective:To study the protective effects of bicistronic DNA vaccines carrying herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and adjuvant CCL28 sequences that were connected by internal ribosome entry site (IRES) sequence in mouse model.Methods:The recombinant DNA vaccines, pgD-IRES-CCL28 and pCCL28-IRES-gD, encoding HSV-2 gD and adjuvant CCL28 were constructed with IRES sequence. After verified by sequencing, they were intramuscularly injected twice into BALB/c mice. Serum samples and vaginal lavage fluids were collected regularly. Splenocytes, mesenteric lymph node cells and rectal mucosa tissues were separated and collected. The titers of antigen-specific antibodies in immunized mice were analyzed with end-point ELISA. In vitro neutralization assay was used to measure neutralizing antibody titers in serum and vaginal lavage fluid after vaccination and virus challenge. CCL28-responsive immune cells in splenocytes, mesenteric lymph node cells and rectal tissues were detected by chemotaxis experiment and immunohistochemical staining. The protective effects of the bicistronic DNA vaccines were evaluated by fluorescent quantitative PCR, weighing and disease severity assessment. Humoral and cellular immune responses induced by the bicistronic DNA vaccines and their efficacy in immunoprotection were analyzed by comparing with pgD+ pCCL28 group. Results:IgG titers in serum samples and IgA antibody titers in vaginal lavage fluids of mice immunized with pCCL28-IRES-gD were similar to those in pgD+ pCCL28 group. The neutralizing ability of antibodies, the number of rectal mucosal IgA+ plasma cells and CCL28-responsive immune cells in mucosal tissues were increased in pCCL28-IRES-gD group. Serum neutralizing antibodies were not produced immediately in the mice challenged with HSV-2, but no weight loss, disease symptoms or death was observed. However, pgD+ pcDNA3.1 and pgD-IRES-CCL28 were ineffective against HSV-2 infection in mice.Conclusions:The recombinant bicistronic DNA vaccine of pCCL28-IRES-gD could induce stronger mucosal immune response in mice and provide better protective effects.