单纯疱疹病毒1型截短糖蛋白B基因疫苗的构建及其诱导小鼠细胞免疫应答

目的:构建、制备单纯疱疹病毒1型截短糖蛋白B DNA疫苗，检测其诱导机体产生的细胞免疫应答效果。方法:利用PCR技术从HSV-1 SM44毒株基因组中扩增出编码HSV-1 gB14～507氨基酸序列的一段基因。定向插入真核表达质粒pcDNA₃载体中，构建出重组真核表达质粒pcDNA₃-gBt，并对其进行酶切分析、PCR鉴定及测序鉴定。于BALB/c鼠注射免疫3次，抗体分析CD4⁺、CD8⁺T细胞亚群的变化，羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)/碘化丙碇(PI)双标记的流式细胞计数法检测CTL活性。结果:PCR及测序鉴定结果显示，插入的克隆基因与GenBank中HSV-1F株gB基因序列一致，证实了HSV-1 gBt核酸疫苗的构建；pcDNA₃-gBt免疫组BALB/c鼠的CD4⁺T细胞数较空质粒（pcDNA₃）对照组和生理盐水对照组增加，CTL活性也较对照组明显增强，但是pcDNA₃-gBt免疫组BALB/c鼠的CD8⁺T细胞、CD4⁺/ CD8⁺T细胞比值与对照组比较差异均无显著性（P＞0.05）。结论:HSV-1 gBt核酸疫苗诱导较强的细胞免疫应答。

Construction and cellular immune response of herpes simplex virus type 1 truncated glycoprotein B DNA vaccine in mice

Objective To construct an eukaryotic expression plasmid containing the truncated gene encoding the part of herpes simplex virus type 1 glycoprotein B(HSV-1,gB) and evaluate its effects on cell-mediated immunity.Methods The part encoding sequence of the glycoprotein B 14-507 amino acid was amplified from HSV1 SM44 DNA genome by polymerase chain reaction(PCR),and then was directionally cloned into eukaryotic expression vector pcDNA3,the recombinant vector pcDNA3gBt was confirmed by the restrictived endonuclease analysis,PCR and sequence analysis.It was employed to evaluate immune response of the mice inoculated triply with the DNA vaccine.The transformation of CD4 /CD8 T lymphocyte was detected by means of antibody and the influence of cell-mediated immunity represented by CTL was assayed through CFSE/PI flow cytometry.Results The construction of the HSV-1 gBt DNA vaccine was identified;cytotoxic activity was strengthened;the amount of CD4 T cell from BALB/c mice immuned with pcDNA3-gBt was higher than mice immuned with plasmid pcDNA3 and saline,the tendacy was the same for CTL,but the amount of CD4 T cell and CD4 /CD8 from BALB/c mice immuned with pcDNA3-gBt had no obvious difference with mice immuned with plasmid pcDNA3 and saline.Conclusion The HSV-1 gBt DNA vaccine is powerful to induce cell-mediated immunity.