【作者】 黄锋涛； 崔晓娴； 曹胜波； 熊传喜； 王敏； 王卫民； 吴兵； 刘学芹；

【Author】 Feng-Tao Huang~1,Xiao-Xian Cui~2,Sheng-Bo Cao~2,Chuan-Xi Xiong~1,Ming Wang~1 Wei-Ming Wang~1 Bin Wu~1 Xue-qin Liu~(1*) (1.College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China. 2.College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China.)

【机构】 华中农业大学水产学院； 华中农业大学动物医学院；

【摘要】 研究已证实杆状病毒可以进入各种脊椎动物细胞,包括哺乳动物细胞,禽类细胞,以及鱼类细胞。由于杆状病毒能高效转导哺乳动物细胞,在疫苗研究,基因治疗,以及基因功能研究方面都有一定应用,现已成为一种新型的基因导入载体。然而研究发现,杆状病毒转导鱼类细胞系效率低下,这就阻碍了杆状病毒在鱼类细胞中的应用。本研究构建了重组杆状病毒Bac-CMV-EGFP,以CMV为启动子,EGFP为报告基因。用重组杆状病毒转导了6种常规的鱼类细胞系,利用流式细胞仪检测了重组杆状病毒对鱼类细胞的转导效率。研究发现,重组杆状病毒可以高效转导鱼类细胞,特别是鲤鱼上皮瘤细胞(EPC),胖头鲤肌肉细胞(FHM),并且报告基因在细胞中能够持续存在很长时间。本研究结果,为杆状病毒载体在鱼类细胞中的应用奠定了基础。更多还原

【Abstract】 The baculovirus Autographa californica multiple nucleopolyhedrovirus(AcMNPV),a type member of the family Baculoviridae,is a rod-shaped nucleocapsid virus with a large genomic DNA (approximately 134 kb).It has been employed widely to overexpress recombinant proteins in insect cells for many years.However,in 1995,Hofmann et al.reported,for the first time,that recombinant baculovirus carrying mammalian expression cassette with cytomegalovirus promoter,was able to transduce hepatic cell and drive expression of reporter gene.Subsequently,a large number of variety of vertebrate cells have been transduced by baculovirus successfully,not only including mammalian cells but also cells of avian and piscine origin.Baculovirus has emerged to be an excellent gene delivery vector in recent years.It has been suggested that baculovirus can be used as a useful vector for studying gene functions and developing vaccines and gene therapy strategies.Studies on transduction of mammalian cells have shown baculovirus can be an effective vector for gene delivery.However,in previous studies,it has been found the gene delivery efficiency was very low in differentiated fish cells.In this study,we constructed a recombinant baculovirus containing cytomegalovirus immediate-early(CMV-IE) promoter,and used enhanced green fluorescent protein(EGFP) gene as the reporter gene.The transduction efficiency of baculovirus was measured by flow cytometry and the persistence of EGFP was monitored by inverted fluorescence microscope.Our findings were sharply different with previous studies.Firstly,we demonstrated baculovirus can mediate highly efficient gene delivery into differentiated fish cells.Moreover,we found growth medium is better than PBS as the surrounding solution to enhance the transduction efficiency in some fish cells. Finally,the transgene in fish cells can persist for a long time,and the expression of transgene can reach the highest level several days later after transduction.The results suggest baculovirus can also be an excellent gene delivery vector used in fish cells.更多还原