目的：构建1型单纯疱疹病毒(HSV-1)糖蛋白B(gB)和糖蛋白D(gD)重组抗原表位核酸疫苗，并进一步研究该疫苗在动物模型中的免疫保护作用及组织表达。

方法：筛选HSV-1的gB、gD表位基因进行串联，构建重组蛋白编码基因X，并转入原核表达载体PET28(a)，利用重组蛋白制备单克隆抗体，免疫新西兰大白兔心脏采血分离血清检测纯化蛋白的免疫原性及血清中的多克隆抗体并获取血清抗体效价。构建真核表达载体pcDNA3.1-X，并采取肌肉注射的方式对4-6 wk BALB/c雌性小鼠进行3针免疫程序，ELISA实验法检测血清中抗体及免疫相关的细胞因子，免疫组化检测小鼠眼球、三叉神经节和脑组织中蛋白X的表达。

结果：重组蛋白X免疫大兔后，在兔血清中检测到目的多克隆抗体，血清抗体的效价为1：3200。小鼠免疫真核重组质粒pcDNA3.1-X后，血清中检测到了实验组的HSV-1 IgM抗体含量为12.13±0.85 ng/L，显著高于载体对照组(0.49±0.44 ng/L)(t=21.07，P<0.001)。实验组细胞因子IL-2、IL-4、IL-10、IFN-γ的含量分别为11.63±0.60、22.65±1.47、85.75±14.12、114.90±6.39 ng/L，均显著高于载体对照组和空白对照组(均P<0.05)。免疫组织化学染色法表明实验组眼球、三叉神经节和脑组织中均发现目的蛋白X的表达。

结论：本研究成功构建了HSV-1 gB和gD串联表位核酸疫苗，能够诱导机体产生较强的免疫应答，并能在眼球、三叉神经节和脑组织中稳定表达，具有较强的免疫原性，为HSV-1重组抗原表位串联疫苗的研制提供了基础数据。

METHODS: The HSV-1 gB and gD epitope genes were selected and tandem assembled to construct the recombinant protein-coding gene X, which was transducted into the prokaryotic expression vector pET28(a). The recombinant protein was synthesized and utilized to generate monoclonal antibodies, which were subsequently used to immunize New Zealand white rabbits. The immunogenicity of the purified protein and the presence of polyclonal antibodies in the serum were tested through separating serum from cardiac blood, and the serum antibody titers were determined. The pcDNA3.1-X was successfully constructed as a eukaryotic expression vector and immunized the female BALB/c mice aged 4 to 6 wk via intramuscular injection. Serum antibodies and immune-related cytokines were quantified using enzyme-linked immunosorbent assay(ELISA). The expression of the X protein in the ocular, trigeminal ganglion, and brain tissues of the mice was assessed.

RESULTS: The target polyclonal antibody was identified with a serum antibody titer of 1:3200 in the rabbit serum after immunized by recombinant protein X. Upon immunizing mice with the eukaryotic recombinant plasmid pcDNA3.1-X, the concentration of HSV-1 serum IgM antibodies of the experimental group was 12.13±0.85 ng/L, which was significantly higher than that of the vector control group(0.49±0.44 ng/L; t=21.07, P<0.001). The concentrations of cytokines interleukin IL-2, IL-4, IL-10, and IFN-γ in the experimental group were 11.63±0.60, 22.65±1.47, 85.75±14.12, and 114.90±6.39 ng/L, respectively, all of which were significantly higher than those in the vector control group and the blank control group(all P<0.05). Immunohistochemical staining revealed the presence of target protein X in the eyeball, trigeminal ganglion, and brain tissue.

CONCLUSION: The HSV-1 gB and gD tandem epitope nucleotides vaccine pcDNA3.1-X was successfully constructed, which activates a remarkable immune response and is stably expressed in the eyeball, trigeminal ganglion, and brain tissue. This study provides a foundation for further research of an HSV-1 recombinant antigen epitope tandem vaccine.