title: Packaging, amplification, and appraisal of the recombinant tumor-selective type I herpes simplex virus carrying GALV.fus gene.
abstract: The aim of the study was to successfully construct three plasmids, which include the GALV.fus gene plasmid regulated by the herpes simplex virus type 1 (HSV-1) late expression gene-UL38 promoter and induced by HSV-1 (HSV-UL38P-GALV.fus), the cytomegalovirus promoter without tumor specificity (CMVP) GALV.fus plasmid (HSV-CMVP-GALV.fus), and the control plasmid in which the GALV.fus gene fragment was replaced by the enhanced green fluorescent protein (EGFP) gene fragment (HSV-CMVP-EGFP). The three constructed plasmids were all packaged and named as Synco-2, Synco-1, and Baco-1. The plasmids were amplified in coliform bacterium and transfected into Vero cells using lipofectamine. These recombinant HSV-1 were amplified in Vero cells and purified by conventional methods of cesium chloride, TCID50 method is used to measure virus titers. The total RNA was then extracted from the HepG2 cells transfected by Synco-1 and Synco-2, and the expression of GALV.fus mRNA was detected by RT-PCR. The three recombinant HSV-1 vectors were propagated in Vero cells and purified by cesium chloride density gradient centrifugation, titrated by TCID50 method, and packaged. The titers of Baco-1, Synco-1, and Synco-2 were 3 × 10(10), 1 × 10(11), and 4 × 10(10) pfu/ml. The GALV.fus gene was identified in the infected HepG2 cells by RT-PCR method.
authors.affiliation: Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China, drzhubing@tom.com.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Bing
authors.forename: Yang
authors.initials: Y
authors.lastname: Jianru
authors.forename: Jiang
authors.initials: J
authors.lastname: Yuequan
authors.forename: Chen
authors.initials: C
authors.lastname: Shifeng
authors.forename: Fu
authors.initials: F
authors.lastname: Xinping
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy; D056945:Hep G2 Cells; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics*; D008175:Lung Neoplasms / Q000235:genetics / Q000628:therapy; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D034702:RNA-Binding Protein FUS / Q000235:genetics*; D014162:Transfection; D014709:Vero Cells; D019065:Virus Assembly
chemical_list: D004274:DNA, Recombinant; D034702:RNA-Binding Protein FUS; D012313:RNA