含Ⅰ型单纯疱疹病毒包装信号序列片段的分离及扩增子质粒双表达载体的构建

Isolation of HSV-1 a sequence involved in the cleavage-packaging of HSV virus and construction of HSV-1 amplicon vector

利用基因工程技术分离出含有Ⅰ型单纯疱疹病毒（HSV－1）包装信号序列和HSV－1基因组复制起点序列的片段，表达外源基因所必需的启动子序列以及其下游作为报告基因的lacZ基因片段，构建成质粒型HSV－1载体pHSVL。用脂质体介导的方法可将该质粒转染入经辅助病毒HSV－1tsK株超感染的Vero细胞，并将其包装成HSV－1假型病毒颗粒，可如天然病毒一样再感染传代细胞和神经细胞并在其中进行基因表达。在pHSVL的基础上，我们还构建了一种可同时表达两种外源基因的质粒型疱疹病毒载体pHLCL，即在pHSVL中插入第二个转录单位：HCMVIE启动子和其下游的荧光素酶基因（lucgene），由此获得的pHLCL病毒接种传代细胞证明它可在其中同时表达两种报告基因．本研究成功地构建了两种质粒型HSV－1载体，其独特的基因转移方式使之在神经生理、病理及神经系统疾病的基因治疗的实验研究中都具有广阔的应用前景。

Fragment containing HSV-1 a sequence has been isolated by subcloning procedures using 3' end DIG-ddUTP labelled DNA probes. With it we developed an HSV-1 amplicon vector pHSVL which contains an HSV-1 ori,HSV-1 IE68 promotor and E. coli lacZ gene. Employing the replication functions of the helper virus HSV-1 tsK, the amplicons are wide tropic vectors, capable of entry, replication and gene expression in both Vero cells and neurons. We inserted HCMV IE promoter and firefly luciferase gene into the pHSVL and name it pHLCL. The pHLCL virus,which was obtained by the same way as pHSVL,proved to be able to introduce reporter genes into immotalized cells and neurons, and express them simultaneously. Both vectors are expected to be usefull in transferring genes into postmitotic cells (including neurons),and in the study of neurobiology, neuropathology and gene therapy of the diseases of the nervous system.