HBV全基因组1.3倍体构建新策略及初步应用
发布时间：2021-07-10 02:25
乙型肝炎病毒（hepatitis B virus, HBV）感染是全球性的公共卫生问题,全世界有超过3.6亿的慢性感染者。进行病毒体外研究是HBV研究的重要方面,但目前缺乏有效的HBV体外感染模型,这极大地限制了HBV复制、转录机制和抗病毒等方面的研究。现有的多数与HBV相关的体外研究都是通过将HBV DNA重组质粒转染到肝癌细胞系（如HepG2和Huh7细胞）来进行的。在体外可复制的HBV DNA重组体至少要包含1.1倍以上的HBV基因组,由于HBV基因组的特殊结构,构建体外可复制重组体比较困难。文献报道,多数HBV体外重组体都需通过多步复杂的酶切连接过程对实验室标准病毒株进行片段置换而形成的,这些方法受制于HBV高度异质性,因此通用性较差,难以用于多样本的临床实验研究。本研究利用一种新的策略构建了两株来源于临床样本的HBV全基因组1.3倍体重组质粒,并通过实验证实了它们可体外复制,同时探讨该策略用于HBV耐药研究的可行性。目的:建立一种构建HBV全基因组1.3倍体的新策略,利用该策略进行实验证实其在体外可以复制,可用于HBV复制、转录调节机制的研究和抗病毒药物的筛选。方法:1.选择...
【文章来源】：重庆医科大学重庆市
【文章页数】：75 页
【学位级别】：硕士
【部分图文】：
HBV成熟颗粒rcDNA结构示意图
图 1-2: HBV1.3 倍体构建策略Figure2: Strategy for 1.3 HBV genome length units construction.The red pairs of primers (minus strand as the template) were used to amplify fragment1073-1798 from HBV genome and the green pairs (plus strand as the template) for1654-3215-803. The two fragments obtained have an overlapped sequnce, i.e. 1654-1798, whichwould be used to anneal these two fragments and extend them to get a template for anadditional PCR to get the fragment 1037-3215-803. By a similar method, the fragment 598-2067can be produced. Then, the fragments 1073-3215-803 and 598-2067 were liagted after SpeⅠdigestion.13
2.2 血清提取病毒 DNA 结果以病人血清提取病毒 DNA 为模板进行后期 PCR 反应，得到相应扩增产物，说明病毒 DNA 提取成功。2.3 HBV0.4 倍体和 0.9 倍体的 PCR 片段扩增结果以 2 个病人血清中提取的 HBV 为模板，采用事先合成好的对应引物分别扩增相应片段获得 HBV0.4 倍体和 HBV0.9 倍体。取 2μl 扩增产物进行 1%琼脂糖凝胶电泳，通过电泳图显示，得到约 1.5kbp 的 HBV0.4 倍体和 3.0kbp 的 HBV0.9 倍体图 1-3 各型 HBV 序列比对结果Fig.1-3 Alignment of 8 types HBV sequences831 complete HBV DNA sequences were extracted from GenBank and categorized intogenotypes. The sequences of various genotypes were resulted from Clustal X analysis withthreshold frequency of 95%. As shown in the figure, the SpeⅠsite is fairly conserved among the sequences analyzed.
【参考文献】：
期刊论文
[1]Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients[J]. Yin-Ping Lu, Tao Guo, Ji-Hua Dong, Jian-Fang Zhu, Zhao Liu, Department of Virology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China Yin-Ping Lu, Bao-Ju Wang, Dong-Liang Yang, Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China Meng-Ji Lu, Institute of Virology, Duisburg-Essen University, Essen 45122, Germany. World Journal of Gastroenterology. 2008(22)
[2]Quantifying anti-HBV effect of targeted ribonuclease by real-time fluorescent PCR[J]. Jun Liu Ying-Hui Li Jin Ding Wei-Dong Gong Cai-Fang Xue Ya Zhao Yu-Xiao Huang Department of Etiology,Fourth Military Medical University,Xi’an 710032,Shaanxi Province,China. World Journal of Gastroenterology. 2004(19)
[3]慢性乙型肝炎患者病毒准种特性的初步研究[J]. 兰林,王宇明,黄燕萍. 中华肝脏病杂志. 2003(04)
本文编号：3274991
【文章来源】：重庆医科大学重庆市
【文章页数】：75 页
【学位级别】：硕士
【部分图文】：
HBV成熟颗粒rcDNA结构示意图
图 1-2: HBV1.3 倍体构建策略Figure2: Strategy for 1.3 HBV genome length units construction.The red pairs of primers (minus strand as the template) were used to amplify fragment1073-1798 from HBV genome and the green pairs (plus strand as the template) for1654-3215-803. The two fragments obtained have an overlapped sequnce, i.e. 1654-1798, whichwould be used to anneal these two fragments and extend them to get a template for anadditional PCR to get the fragment 1037-3215-803. By a similar method, the fragment 598-2067can be produced. Then, the fragments 1073-3215-803 and 598-2067 were liagted after SpeⅠdigestion.13
2.2 血清提取病毒 DNA 结果以病人血清提取病毒 DNA 为模板进行后期 PCR 反应，得到相应扩增产物，说明病毒 DNA 提取成功。2.3 HBV0.4 倍体和 0.9 倍体的 PCR 片段扩增结果以 2 个病人血清中提取的 HBV 为模板，采用事先合成好的对应引物分别扩增相应片段获得 HBV0.4 倍体和 HBV0.9 倍体。取 2μl 扩增产物进行 1%琼脂糖凝胶电泳，通过电泳图显示，得到约 1.5kbp 的 HBV0.4 倍体和 3.0kbp 的 HBV0.9 倍体图 1-3 各型 HBV 序列比对结果Fig.1-3 Alignment of 8 types HBV sequences831 complete HBV DNA sequences were extracted from GenBank and categorized intogenotypes. The sequences of various genotypes were resulted from Clustal X analysis withthreshold frequency of 95%. As shown in the figure, the SpeⅠsite is fairly conserved among the sequences analyzed.
【参考文献】：
期刊论文
[1]Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients[J]. Yin-Ping Lu, Tao Guo, Ji-Hua Dong, Jian-Fang Zhu, Zhao Liu, Department of Virology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China Yin-Ping Lu, Bao-Ju Wang, Dong-Liang Yang, Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China Meng-Ji Lu, Institute of Virology, Duisburg-Essen University, Essen 45122, Germany. World Journal of Gastroenterology. 2008(22)
[2]Quantifying anti-HBV effect of targeted ribonuclease by real-time fluorescent PCR[J]. Jun Liu Ying-Hui Li Jin Ding Wei-Dong Gong Cai-Fang Xue Ya Zhao Yu-Xiao Huang Department of Etiology,Fourth Military Medical University,Xi’an 710032,Shaanxi Province,China. World Journal of Gastroenterology. 2004(19)
[3]慢性乙型肝炎患者病毒准种特性的初步研究[J]. 兰林,王宇明,黄燕萍. 中华肝脏病杂志. 2003(04)
本文编号：3274991
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