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题名: 中国对虾白斑综合症杆状病毒的研究-增殖模型,精细结构,核酸与结构蛋白性质,检测技术,同源性比较
作者: 黄灿华
答辩日期: 2000
导师: 吴清江 ; 陈棣华
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 对虾白斑综合症杆状病毒 ; 病毒体内增殖模型 ; 精细结构 ; 核酸与蛋白特性 ; 检测技术 ; 同源性比较
摘要: 对虾白斑综合症杆状病毒(WSSV)是对我国及世界上其它国家水产养殖业造成巨大经济损失的一种对虾病毒。WSSV在海洋甲壳纲动物中有较广泛的宿主范围,然而对淡水甲壳纲动物的侵染活性人们还了解不多。本研究在阐明WSSV对常见淡水甲壳纲动物的侵染活性的同时,成功地建立了WSSV淡水克氏螯虾体内增殖模型,并通过电镜技术、核酸探针斑点杂交与原位杂交等手段证实所建立的增殖模型的可靠性。在完整WSSV病毒粒子纯化技术上取得重要研究进展。采用改良的密度梯度离心介质建立了一套成熟的WSSV纯化方案。许多研究结果如病毒精细结构的阐明、完整病毒结构多肽的揭示、病毒基因组酶切图谱分析等,填补或完善了国内外相关领域的研究空白,为深入探讨病毒基因组结构和功能,确立该病毒的分类地位提供了重要参数。利用pUCl8质粒部份克隆了中国对虾白斑综合症杆状病毒WSSV基因组,其中两个插入片段L46、M13分别经地高辛标记制备成核酸探针,建立了斑点杂交与原位杂交等检测与诊断技术,对我国沿海地区病虾样品进行了跟踪检测。同时利用原位杂交技术动态研究了对虾白斑综合症杆状病毒在虾体内的感染与发生过程。斑点杂交与原位杂交检测诊断技术的建立为对虾病害的检测诊断提供了准确、直观的手段。利用WSSV兔抗血清建立的快速免疫金银染色(IGSS)法将适用于对虾白斑综合症杆状病毒的现场诊断与检测。比较我国沿海不同海域收集对虾白斑综合症杆状病毒(WSSV)三个分离株基因组酶切多态(RFLP)以及病毒结构蛋白图谱,结果完全一致,证实造成我国从南至北对虾爆发性流行病的WSSV为同一种病毒病原。中国对虾白斑综合症杆状病毒与报道的日本对虾杆状病毒(RV-PJ=PRDV)、斑节对虾白斑综合症杆状病毒(WSBV=PmNOBIII)部份基因组核酸序列比较,同源率分别为97%与100%,结果提示亚洲及太平洋地区对虾白斑综合症杆状病毒可能为同一种病毒或同种病毒的不同株系。研究养虾水体中盐度、溶氧量、及pH值等主要理化指标的变化与对虾爆发性流行病之间的关系。以低剂量病毒感染中国对虾,人工改变海水盐度、溶氧量、pH值等理化指标,发现其变化与对虾发病死亡之间存在明显的诱发关系。结果对阐明对虾爆发病毒性流行病成因及指导虾农科学养虾具有重要参考价值。除了上述研究结果外,本研究还概述了迄今所发现的对虾病毒种类、可能的分类地位,及一些重要的对虾病毒研究的新进展。同时从虾池生态环境与对虾病害关系,对虾病毒对水产养殖业及野生对虾资源的潜在冲击,对虾病毒性疾病的防治措施等方面进行了分析与展望。
英文摘要: White Spot Syndrome Virus (WSSV), a penaeid shrimp virus, causes signifi-cant economic loss in both the Chinese and global aquaculture industry. WSSV hasa wide host range in the saltwater crustaceans, but little is known about the suscep-tibility of freshwater crustaceans to WSSV. In this study, an experimental modelwas developed and established with freshwater crayfish Cambarus clarkii to prolif-erate White Spot Syndrome Virus (WSSV) in vivo. The success and reliability of this model was confirmed by transmission electron microscopy (TEM), dot - blotand hybridization in situ etc. It is a very important progress that a new procedure was developed using a re-formative gradient medium and by ultra- centrifugation for the isolation and purifi-cation of intact WSSV viral particles from the infected penaeid shrimp or crayfish.Some results from this current study such as the fine structure of WSSV, restriction enzyme patterns of the WSSV genome, and characteristic polypeptides features, provide new and additional scientific information in this field as well as important parameters for further research of structures and functions of WSSV genome and proteins, evolution and phylogeny of WSSV, and potential approaches for the pre-vention and control of WSSV viral diseases worldwide.WSSV genome DNA was partially cloned into pUC18 plasmid. Two fragments from L46 and M13 clones were selectively labeled by Dig-ll-dUTP and used as molecular probes in two detection methods, dot-blot and hybridization in situ, for the investigation and early diagnosis of viral diseases in shrimp samples obtained from different regions of China. Meanwhile, WSSV infection process in penaeid shrimp was also dynamically studied with hybridization in situ. Both dot - blot and hybridization in situ can provide nicer and intuitionistic results for the early diagno-sis and detection of WSSV. Immunogold silver - enhancing staining (IGSS) detec-tion method was also set up with WSSV polyclonal antiserum prepared by immuniz-ing the rabbit with intact WSSV. This method can easily be applied for on-site di-agnosis of WSSV.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12620
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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中国对虾白斑综合症杆状病毒的研究-增殖模型,精细结构,核酸与结构蛋白性质,检测技术,同源性比较.pdf(9068KB)----暂不开放-- 联系获取全文

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中国对虾白斑综合症杆状病毒的研究-增殖模型,精细结构,核酸与结构蛋白性质,检测技术,同源性比较.黄灿华[d].中国科学院水生生物研究所,2000.20-25
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