一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先，将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN，其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1，但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明，删除IR区的重组HSV-1保留了复制能力，能够携载并表达外源基因，建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.

The Development of a Herpes Simplex Virus Type 1 Vector System

The herpes simplex virus type 1 (HSV-1) was used to produce oncolytic viruses. In this study, a new type vector system of HSV-1 was developed based from bacterial artificial chromosome technology. Both internal inverted repeat sequences (IR) of HSV-1 were cloned into pKO5. A shuttle plasmid pKO5/BN was constructed and transfected to E.coli with pHSV-BAC by electroporation. We also obtained the recombinant pHSVΔIR BAC without the IR. Recombinant HSVΔIR (MH1001) virus was used to infect pHSVΔIR-BAC transfected Vero cells. To produce HSVΔIR/EGFP (MH1002) recombinant virus expressing EGFP, pHSVΔIR BAC-containing E. coli and pKO5/BN was used. The proliferation of Vero cells infected with MH1001 and MH1002 was slightly attenuated than wild-type HSV-1 infection, but was insignificant in P values. Western blotting analysis showed decreased expression levels of early gene ICP0, ICP4, ICP8, ICP22 and ICP27 in infected cells. Immunofluorescence and confocal microscopy showed that both the recombinant and wild-type viruses resided in the cytoplasm. The results showed that recombinant HSV-1 without IR region could retain the ability to replicate and express foreign genes.