携带GALV.fus基因的重组单纯疱疹病毒的组装、扩增及鉴定

Packaging,amplification and identification of recombinant tumor-selective type I herpes simplex virus vector containing GALV.fus gene

目的:包装已成功构建的受Ⅰ型单纯疱疹病毒(HerpessimplexvirusⅠ,HSV-Ⅰ)晚期表达基因-UL38启动子调控的、嗜肿瘤单纯疱疹病毒介导的,长臂猿白血病病毒致融性外膜糖蛋白(Gibbon ape leukemia virus membrance fusion glycopratein,GALV.fus)基因质粒(HSV-UL38P-GALV.fus),无肿瘤特异性巨细胞病毒启动子(Cytomegalovirus promoter,CMVP)GALV.fus质粒(HSV-CMVP-GALV.fus),GALV.fus基因片段被增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因片段所取代的治疗对照质粒(HSV-CMVP-EGFP),分别命名为Synco-2、Synco-1、Baco-1。方法:扩增已成功构建的HSV-UL38P-GALV.fus、HSV-CMVP-GALV.fus、HSV-CMVP-EGFP质粒,然后分别在Vero细胞用脂质体转染、包装成重组嗜肿瘤单纯疱疹病毒。重组单纯疱疹病毒的扩增及纯化采用Vero细胞及氯化铯纯化常规方法,病毒滴度测定采用TCID50法。Synco-1、Synco-2病毒感染肝癌细胞株HepG2,分别提取受染肝癌细胞HepG2的总RNA,以RT-PCR的方法鉴定GALV.fus基因的表达。结果:成功包装了3种重组单纯疱疹病毒Baco-1、Synco-1和Synco-2,按需要在Vero细胞内扩增后以氯化铯密度梯度离心法进行纯化,TCID50法测得病毒滴度分别为3×1010 pfu/ml1,×1011 pfu/ml和4×1010 pfu/ml。受染肝癌细胞株HepG2中以RT-PCR法检测到GALV.fus基因的表达。结论:成功包装、扩增及纯化3种重组单纯疱疹病毒,受染肝癌细胞株HepG2中以RT-PCR法检测到GALV.fus基因的表达。

Objective：Package of constructed recombinant tumor-selective type I Herpes simplex virus vector plasmid containing UL38 and CMV promoter and GALV.fus gene （HSV-UL38P-GALV.fus、HSV-CMVP-GALV.fus）,GALV.fus gene was replaced by EGFP in control plasmid （HSV-CMVP-EGFP）. The three recombinant viruses were named Synco-2、Synco-1 and Baco-1 respectively。Methods： At first the three constructed plasmids were amplificated in coliform bacterium and transfected into Vero cells by using lipofectamine respectively. Then,these three recombinant type I Herpes simplex viruses were propagated in Vero cells and purified by cesium chloride density purification,titrated by TCID50 method. In the end,Total RNA was extracted from the HepG2 cells which were transfected by Synco-1 and Synco-2 and GALV.fus mRNA was detected by RT-PCR. Results：The three recombinant HSV-1 vectors were packaged successfully,which were propagated in Vero cells and purified by cesium chloride density purification,titrated by TCID50 method. The titre of Baco-1、Synco-1 and Synco-2 were 3×10 10 pfu/ml,1×10 11 pfu/ml and 4×10 10 pfu/ml respectively.GALV.fus gene were identified in the infected HepG2 cells. Conclusion：We packed,amplificated and purified Recombinant HSV-I virus successfully and the expression of GALV.fus gene was identified in the infected HepG2 cells by RT-PCR.