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本研究旨在构建由细菌人工染色体(Bacteria artificial chromosome,BAC)携带的单纯疱疹I型病毒质粒及携带绿色荧光蛋白(Green fluorescent protein,GFP)的重组型BAC-HSV-1感染性子代病毒。构建了携带HSV-1同源臂的质粒C223-UL43左臂-UL47右臂。将该质粒线性化后与HSV-1基因组共转染至Vero细胞,通过真核细胞内同源重组产生了含有GFP报告基因的BAC-HSV-1重组病毒,噬斑纯化筛选出阳性重组病毒,并再次感染Vero细胞,Hirt法提取BAC-HSV-1环形基因组并将其电穿孔入DH10B感受态细胞,由PCR和酶切法鉴定BAC-HSV-1质粒。为研究BAC-HSV-1子代病毒的生物学特性,将实验组和对照组细胞分别给予BAC-HSV-1质粒和HSV-1基因组DNA,收取病变细胞的上清液,以MOI=0.1再次感染Vero细胞,半数组织培养感染剂量(50% tissue culture infective dose,TCID50)法测定两组的病毒滴度。PCR和酶切法分别鉴定BAC-HSV-1,结果示BAC-HSV-1构建成功。TCID50法测定实验组和对照组病毒滴度,经统计学分析两组病毒滴度间差异无统计学意义(P>0.05)。本研究成功地构建了真核细胞和原核细胞间穿梭的HSV-1-BAC重组病毒/质粒。
The purpose of this study was to construct the herpes simplex I virus plasmid carried by Bacteria artificial chromosome (BAC) and the recombinant BAC-HSV-1 infectious progeny virus carrying green fluorescent protein (GFP). The C223-UL43 left arm-UL47 right arm carrying the HSV-1 homology arm was constructed. The plasmid was linearized and co-transfected with HSV-1 genome into Vero cells. BAC-HSV-1 recombinant virus containing GFP reporter gene was generated by homologous recombination in eukaryotic cells. The positive recombinant virus was screened by plaque purification. Vero cells were infected again. The BAC-HSV-1 circular genome was extracted by Hirt method and electroporated into DH10B competent cells. The BAC-HSV-1 plasmid was identified by PCR and restriction enzyme digestion. In order to study the biological characteristics of BAC-HSV-1 progeny virus, BAC-HSV-1 plasmid and HSV-1 genomic DNA were respectively administered to the experimental group and the control group, and the supernatant of the diseased cells was collected at MOI = 0.1 Vero cells were infected and the virus titers of both groups were determined by the method of TCID50 (50% tissue culture infective dose). BAC-HSV-1 was identified by PCR and restriction enzyme digestion, respectively. The results showed that BAC-HSV-1 was successfully constructed. TCID50 method was used to determine the titer of virus in experimental group and control group. There was no significant difference in virus titer between the two groups by statistical analysis (P> 0.05). In this study, HSV-1-BAC recombinant virus / plasmid was successfully constructed to shuttle between eukaryotic cells and prokaryotic cells.