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目的构建单纯疱疹病毒Ⅰ型(herpes simplex virus I,HSV-I)SM44株截短糖蛋白B基因序列,为研制联合基因疫苗奠定基础,用于角膜炎的防治。方法利用聚合酶链反应(polymerase chain response,PCR)技术从感染HSV-I SM44株的vero细胞中扩增出HSV-gBt编码基因,经双酶切鉴定后将目的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和测序鉴定。结果对pcDNA3-gBt双酶切后,电泳可见目的基因(1.5kb)和线性质粒pcDNA3(5.4kb)两条带;测序结果表明,克隆基因插入方向正确,与基因库中登录的HSV-1F株gB基因序列比较,同源性达99.5%。结论经证实成功地构建了重组真核表达质粒pcDNA3-gBt,为进一步研究其免疫学效应及构建病毒糖蛋白联合疫苗并最终用于单疱病毒角膜炎的预防和治疗奠定理论基础
Objective To construct the sequence of truncated glycoprotein B gene of herpes simplex virus I (HSV-I) SM44 and lay the foundation for the development of combined gene vaccine for the prevention and treatment of keratitis. Methods HSV-gBt gene was amplified from vero cells infected with HSV-I SM44 by polymerase chain reaction (PCR) technique. After double enzyme digestion, the target gene was inserted into eukaryotic expression plasmid pcDNA3 The recombinant eukaryotic expression plasmid pcDNA3-gBt was constructed and verified by restriction analysis and sequencing. Results After double digestion with pcDNA3-gBt, two bands of target gene (1.5kb) and linear plasmid pcDNA3 (5.4kb) were obtained by electrophoresis. Sequencing results showed that the cloned gene was correctly inserted and interacted with HSV-1F strain gB gene sequence comparison, homology 99.5%. Conclusion The recombinant eukaryotic expression plasmid pcDNA3-gBt has been successfully constructed, which lays a theoretical foundation for further study of its immunological effects and construction of a virus glycoprotein conjugate vaccine for the prevention and treatment of herpes simplex virus keratitis