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我们先前已报道了构建成一种能在ＨＳＶｔｓＫ株辅助下进行复制和包装，并表达β－半乳糖苷酶基因ｌａｃＺ的ＨＳＶ－１扩增子质粒ｐＨＳＬ，以及它的应用〔１〕。该质粒中依次含有ＨＳＶ－１复制起点ｏｒｉＳ序列及ＩＥ６８启动子、ｌａｃＺ基因、ＳＶ４０ｐｏｌｙＡ、ＨＳＶ－１包装信号‘ａ’序列和大肠杆菌质粒骨架。然而，该质粒中的报告基因ｌａｃＺ无法用简单的酶切方法卸载下来，继而装入目的基因。本研究在此基础上，新构建了一系列质粒型单纯疱疹病毒载体。首先构建了ＨＳＶ－１扩增子载体质粒ｐＨＳＶＩＥ６８，在ＩＥ６８启动子的下游带有可供外源基因插入的ＨｉｎｄⅢ、ＳａｌＩ、ＸｂａＩ、ＢａｍＨＩ等克隆位点，其后没有ｐｏｌｙＡ加尾信号。为检验ｐＨＳＶＩＥ６８载体的有效性，将报告基因ｌａｃＺ－ＳＶ４０ｐｏｌｙＡ片段通过ＨｉｎｄⅢ和ＢａｍＨＩ位点插入该载体中，构建成重组扩增子质粒ｐＨＳＶ－ｌａｃＺ１。将该质粒转入ＢＨＫ细胞，并辅以ＨＳＶ－１ｔｓＫ株病毒感染，３１℃培养４８～７２小时后收获的细胞上清感染新鲜的ＢＨＫ细胞，用Ｘ－ｇａｌ液染色可见有大量细胞蓝染，提示所构建的ｐＨＳＶＩＥ６８质粒能有效地工作。在此基础上，我们又构建了含有ＳＶ４０ｐｌｏｙ?
We have previously reported the construction of HSV-1 amplicon plasmid pHSL, which can be replicated and packaged with the aid of the HSVtsK strain and expressed the lacZ gene of β-galactosidase, and its use [1]. The plasmid contains in sequence oriS sequence of HSV-1 origin of replication and IE68 promoter, lacZ gene, SV40 polyA, HSV-1 packaging signal ’a’ sequence and Escherichia coli plasmid backbone. However, the reporter gene lacZ in this plasmid can not be unloaded by a simple digestion method, and then the target gene is loaded. In this study, based on this, a new series of plasmid herpes simplex virus vectors were constructed. Firstly, HSV-1 amplicon vector plasmid pHSVIE68 was constructed and cloned into HindⅢ, SalI, XbaI, BamHI and other sites downstream of IE68 promoter for insertion of exogenous gene, followed by no polyA tailing signal. To test the effectiveness of pHSVIE68 vector, the reporter gene lacZ-SV40polyA fragment was inserted into the vector through HindIII and BamHI sites to construct recombinant amplicon plasmid pHSV-lacZ1. The plasmid was transfected into BHK cells supplemented with HSV-1tsK virus infection, and the freshly harvested BHK cells were harvested after culturing at 31 ° C for 48-72 hours. The cells were stained with X-gal to show a large amount of cell blue stain, Suggesting that the constructed pHSVIE68 plasmid can work effectively. On this basis, we have built with SV40ploy?