单纯疱疹病毒Ⅰ型糖蛋白B基因疫苗的构建及细胞免疫学研究  

The construction and cellular immunological study of herpes simplex virus type Ⅰ glycoprotein B DNA vaccine

作　　者：李光源[1] 贺冰[1] 冯非[1] 孟祥俊[1] 宋忆淑[2] 王晓祺[3] 

机构地区：[1]吉林大学第一医院眼科,长春130021 [2]东北师范大学遗传与细胞研究所,长春130021 [3]东北师范大学中心研究室,长春130021

出　　处：《中国免疫学杂志》2005年第7期535-539,共5页Chinese Journal of Immunology

基　　金：吉林大学新技术新疗法课题(200104);长春市科技发展(03170509)基金资助

摘　　要：目的:构建、制备单纯疱疹病毒Ⅰ型(HSVⅠ)糖蛋白BDNA疫苗,检测其诱导机体产生的细胞免疫应答。方法:PCR扩增编码HSVⅠgB去除N端部分信号肽序列(39bp)的基因片段(2673bp),定向插入pcDNA3载体中,构建pcDNA3gB基因疫苗并对其进行PCR、酶切及测序鉴定。于BALBc小鼠注射免疫3次,观察小鼠CD4+、CD8+T细胞亚群的变化,CFSEPI双标记的流式细胞计数法检测CTL活性。结果:双酶切分析结果为目的基因(2.7kb)和线性质粒pcDNA3(5.4kb);PCR扩增结果为特异产物(2.7kb);测序结果与GenBankgB基因序列同源性达99.5%;CTL活性增强;BALBc小鼠脾CD4+T细胞增加。结论:经鉴定证实了HSVⅠgB基因疫苗的构建;HSVⅠgB基因疫苗可以诱导较强的细胞免疫应答,应用于预防HSVⅠ的感染具有良好的前景。Objective:To construct the DNA vaccine against herpes simplex virus type Ⅰ glycoprotein B(HSV-Ⅰ,gB) and evaluate its effects on cell-mediated immunity.Methods:The gB encoding sequence(2 673 bp) with the removal of signal peptide(39 bp) was amplified by polymerase chain reaction(PCR),and then was directionally cloned into the vector pcDNA3,the recombinant vector pcDNA3-gB was confirmed by the restricted 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 and the influence of cell-mediated immunity represented by CTL was assayed through CFSE/PI flow cytometry.Results:Double-enzyme digestion showed two bands which are HSV-Ⅰ gB gene(2.7 kb) and linear pcDNA3(5.4 kb);PCR analysis shows the specific band(2.7 kb);sequence analysis showed the rate of homology was 99.5% compared with GenBank.The mice immunized with pcDNA3-gB induced high levels of CTL;the amount of CD4+T cell increased.Conclusion:The construction of the HSV-Ⅰ DNA vaccine is identified;it is powerful to induce cell-mediated immunity and is promising for the treatment of HSV-Ⅰ infection.

关 键 词：单纯疱疹病毒 DNA疫苗 糖蛋白B 流式细胞术 CFSE 

分 类 号：R392[医药卫生—免疫学]