【作者】 宋建华；

【导师】 陈新文；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2006， 博士

【摘要】 杆状病毒是一类专一性感染节肢动物的DNA病毒,作为生物杀虫剂广泛应用于害虫防治。随着分子水平的基础研究迅猛发展,杆状病毒作为一种新型的基因治疗载体受到人们的高度重视。我们的研究拓宽了杆状病毒敏感细胞范围,同时,通过杆状病毒在哺乳动物细胞中介导功能基因的研究,进一步证明杆状病毒是一种优良的基因治疗载体。本文还通过杆状病毒抗凋亡基因p35在植物中广谱抗病反应的研究,揭示杆状病毒p35基因的较高应用价值。我们的研究进一步深化了杆状病毒及其基因的研究,为扩展其应用奠定了基础。第一章以综述的形式描述了杆状病毒的病毒学特点,同时介绍了杆状病毒的应用研究和杆状病毒广谱抗凋亡基因p35的研究现状。第二章阐明了重组杆状病毒Ac-CMV-eGFP(含有CMV启动子控制绿色荧光蛋白的重组AcMNPV)作为基因转移载体在脊椎动物鸡和鸭细胞中的转导能力,结果分析表明Ac-CMV-eGFP能高效转导鸡胚和鸭胚细胞,且在鸭胚细胞中转导能力强于鸡胚细胞。进一步的分析表明AcMNPV能转导来自鸡的不同组织如肝、肺和肾细胞,其中对肺细胞的转导率最高,达49.8%,其次为肾细胞(44%)和肝细胞(43%)。去乙酰化酶的抑制剂丁酸,能促进沉默基因的表达,显著提高目的基因在鸡、鸭细胞中的表达水平。另外,杆状病毒的转导对禽原代细胞没有毒性。初步的结果显示杆状病毒通过受体介导的膜融合机制进入禽类细胞,与杆状病毒感染和转导哺乳动物细胞的机制相似。该结果进一步拓宽了杆状病毒的应用范围。杆状病毒由于其独特的生物学特性显示了作为基因治疗载体的潜力和优势。第三章研究了杆状病毒载体介导的p35基因抗哺乳动物细胞凋亡。研究发现重组杆状病毒Ac-CMV-GFP和Ac-CMV-p35均能转导人胚肾细胞293细胞系,介导更多还原

【Abstract】 The Baculoviridae is a diverse family of arthropod-specific viruses with a double-stranded genomic DNA of which most members infect Lepidoptera. It has been widely utilized for the expression of recombinant proteins and as a biopesticide. With the development of biology, baculovirus became a new gene therapy vector. we have initiated experiments to determine whether baculovirus vectors are capable of delivering transgene to avian. One of our aims of this thesis is to analyze the efficiency of baculoviruses transducing into avian cells, to illuminate the response of avian cells stimulated by baculoviruses and the mode of entry of baculovirus into avian. We also expressed baculoviral p35 gene in kidney cells mediated by baculovirual vector and checked the suppression of apoptosis. We are very interested if expression of the baculovirus P35 protein in plant could enhance the broad-spectrum resistance against diseases. Our studies broadened the range of susceptible cells of baculovirus system and developed the studies of baculovirus as well as genes, which made a soild foundation for futher application research.In Chapter one, an overview outlines the character of baculovirus, major on the application of baculoviruse and the broad-spectrum anti-apoptotic gene p35.Baculovirus AcMNPV has shown the ability of high efficiently transducing into a variety of mammalian cell types. Chicken and duck cells were tested for their ability to be transduced by Ac-CMV-eGFP, a recombinant baculovirus capable of expressing an eGFP reporter gene under the control of the CMV promoter. Our results (Chapter 2) showed that chicken and duck cells were transducible, as demonstrated by flow cytometry assay, and the transduction efficiency of duck cells was higher than that of chicken cells. Addition of histone deacetylase inhibitor sodium butyrate enhanced the transduction of baculovirus into both chicken and duck cells. In addition,更多还原