单纯疱疹病毒Ⅰ型基因治疗载体的快速构建

背景：单纯疱疹病毒Ⅰ型载体因具有独特的优点目前被广泛应用,但其构建尚缺乏一种快速有效的方法。目的：利用Cre/Loxp高效重组系统构建单纯疱疹病毒Ⅰ型载体。方法：分离单纯疱疹病毒HSV-1,将含Cre重组酶的c66-SV40-cre质粒转染Vero细胞,构建一株带有Loxp位点的重组HSV-1框架载体HSVLoxp。构建穿梭载体pShuttle-SV40-Cre-Loxp-IRES及重组单纯疱疹病毒Ⅰ型载体HSV-GDNF,用HAT培养基筛选出阳性毒株后用GDNF引物做PCR鉴定,扩增培养后测定滴度。结果与结论：成功构建pHV-TK-GFP质粒,并在Vero细胞内发生重组,分离出缺失了Us3基因的重组病毒HSVtk-Loxp-GFP01。成功构建HSV-1框架载体HSVLoxp及穿梭载体pShuttle-SV40-Cre-Loxp-IRES,成功获得GDNF基因,并将其转移到了HSV-1基因组上,成功构建了表达GDNF的单纯疱疹病毒HSV-1载体,测定其滴度约为2.25×106IU/mL。

Rapid construction of herpes simplex virus type I vector for gene therapy

BACKGROUND：Herpes simplex virus typeⅠ（HSV-1） vector has been currently widely used due to its unique advantages,but a rapid and effect method is needed to construct this vector.OBJECTIVE：To develop a rapid method using Cre/loxp site specific recombination system to construct HSV-1 vector.METHODS：HSV-1 was isolated and c66-SV40-cre plasmid containing Cre recombinase was used to transfect Vero cells.Then HSV-1 HSVLoxp was constructed.Shuttle vector pShuttle-SV40-Cre-Loxp-IRES and HSV-I vector HSV-GDNF were constructed.The positive strains screened by HAT culture medium were identified by PCR taking GDNF as primer.After amplification,titer was determined.RESULTS AND CONCLUSION：A strain of HSV-1 was isolated and pHV-TK-GFP was constructed successfully.Recombinant virus HSVtk-Loxp-GFP01 without Us3 gene was isolated.HSV-1 HSVLoxp and shuttle vector pShuttle-SV40-Cre-Loxp-IRES were successfully constructed.GDNF gene was successfully obtained and transferred into HSV-1 vector.Thus,HSV-1 vector expressing GDNF was successfully constructed and the titer was 2.25×106 IU/mL.