单纯疱疹病毒I型扩增子系列载体的构建

THE CONSTRUCTION OF A SERIES OF DEFECTIVE HERPES SIMPLEX VIRUS VECTORS

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 a Herpes simplex virus type 1(HSV-1) amplicon plasmid pHSL containing a HSV-1 package signal sequence, an origin for viral replication and a β-galactosidase gene under the control of IE68 promoter. The plasmid could replicate and be packaged into HSV-1 virons, as well as express lacZ gene in the presence of HSV-1 tsK infection. However, the reporter lacZ gene in pHSL can not be simply cut off by restrictive endonucleases and then substituted by target genes. In this study, we constructed a series of HSV-1 amplicon plasmids based on the previous work. First, the pHSVIE68 was constructed. Downstream of the IE68 promoter were the multiple cloning sites including HindⅢ, SalI, XbaI and BamHI for inserting exogenous genes. In order to test the feasibility of the construct, lacZ gene was inserted into pHSV-1 between HindⅢ and BamHI sites, resulting in recombinant plasmid pHSV-lacZ1. BHK cells infected by HSV-1 tsK strain were transfected with pHSV-lacZ1 DNA and incubated at 31℃. 48-72 hours later, the supernatant of the cells was harvested and used to infect BHK cells. With X-gal staining, a large number of cells were stained blue, suggesting that the constructed pHSVIE68 could work effectively. Because there is no polyA sequence included in pHSVIE68, another vector pHSVIE68pA, a HSV-1 amplicon plasmid for general use, was constructed. In addition, we constructed a bicistronic HSV-1 amplicon plasmid aimed at expressing exogenous gene as well as lacZ gene simultaneously. These vectors could be used conveniently to carry exogenous genes in defective HSV-1 vector system studies.