乙型肝炎病毒（hepatitis B virus, HBV）感染是全球性的公共衛(wèi)生問題,全世界有超過3.6億的慢性感染者。進行病毒體外研究是HBV研究的重要方面,但目前缺乏有效的HBV體外感染模型,這極大地限制了HBV復制、轉(zhuǎn)錄機制和抗病毒等方面的研究�，F(xiàn)有的多數(shù)與HBV相關(guān)的體外研究都是通過將HBV DNA重組質(zhì)粒轉(zhuǎn)染到肝癌細胞系（如HepG2和Huh7細胞）來進行的。在體外可復制的HBV DNA重組體至少要包含1.1倍以上的HBV基因組,由于HBV基因組的特殊結(jié)構(gòu),構(gòu)建體外可復制重組體比較困難。文獻報道,多數(shù)HBV體外重組體都需通過多步復雜的酶切連接過程對實驗室標準病毒株進行片段置換而形成的,這些方法受制于HBV高度異質(zhì)性,因此通用性較差,難以用于多樣本的臨床實驗研究。本研究利用一種新的策略構(gòu)建了兩株來源于臨床樣本的HBV全基因組1.3倍體重組質(zhì)粒,并通過實驗證實了它們可體外復制,同時探討該策略用于HBV耐藥研究的可行性。目的:建立一種構(gòu)建HBV全基因組1.3倍體的新策略,利用該策略進行實驗證實其在體外可以復制,可用于HBV復制、轉(zhuǎn)錄調(diào)節(jié)機制的研究和抗病毒藥物的篩選。方法:1.選擇... 

【文章來源】：重慶醫(yī)科大學重慶市

【文章頁數(shù)】：75 頁

【學位級別】：碩士

【部分圖文】：

HBV成熟顆粒rcDNA結(jié)構(gòu)示意圖

圖 1-2: HBV1.3 倍體構(gòu)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 結(jié)果以病人血清提取病毒 DNA 為模板進行后期 PCR 反應，得到相應擴增產(chǎn)物，說明病毒 DNA 提取成功。2.3 HBV0.4 倍體和 0.9 倍體的 PCR 片段擴增結(jié)果以 2 個病人血清中提取的 HBV 為模板，采用事先合成好的對應引物分別擴增相應片段獲得 HBV0.4 倍體和 HBV0.9 倍體。取 2μl 擴增產(chǎn)物進行 1%瓊脂糖凝膠電泳，通過電泳圖顯示，得到約 1.5kbp 的 HBV0.4 倍體和 3.0kbp 的 HBV0.9 倍體圖 1-3 各型 HBV 序列比對結(jié)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.

【參考文獻】：
期刊論文
[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準種特性的初步研究[J]. 蘭林,王宇明,黃燕萍.  中華肝臟病雜志. 2003(04)



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