乙型肝炎病毒（hepatitis B virus, HBV）感染是全球性的公共衛(wèi)生問(wèn)題,全世界有超過(guò)3.6億的慢性感染者。進(jìn)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(fù)制、轉(zhuǎn)錄機(jī)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(guò)將HBV DNA重組質(zhì)粒轉(zhuǎn)染到肝癌細(xì)胞系（如HepG2和Huh7細(xì)胞）來(lái)進(jìn)行的。在體外可復(fù)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(fù)制重組體比較困難。文獻(xiàn)報(bào)道,多數(shù)HBV體外重組體都需通過(guò)多步復(fù)雜的酶切連接過(guò)程對(duì)實(shí)驗(yàn)室標(biāo)準(zhǔn)病毒株進(jìn)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(shí)驗(yàn)研究。本研究利用一種新的策略構(gòu)建了兩株來(lái)源于臨床樣本的HBV全基因組1.3倍體重組質(zhì)粒,并通過(guò)實(shí)驗(yàn)證實(shí)了它們可體外復(fù)制,同時(shí)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(jìn)行實(shí)驗(yàn)證實(shí)其在體外可以復(fù)制,可用于HBV復(fù)制、轉(zhuǎn)錄調(diào)節(jié)機(jī)制的研究和抗病毒藥物的篩選。方法:1.選擇... 

【文章來(lái)源】：重慶醫(yī)科大學(xué)重慶市

【文章頁(yè)數(shù)】：75 頁(yè)

【學(xué)位級(jí)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(jìn)行后期 PCR 反應(yīng)，得到相應(yīng)擴(kuò)增產(chǎn)物，說(shuō)明病毒 DNA 提取成功。2.3 HBV0.4 倍體和 0.9 倍體的 PCR 片段擴(kuò)增結(jié)果以 2 個(gè)病人血清中提取的 HBV 為模板，采用事先合成好的對(duì)應(yīng)引物分別擴(kuò)增相應(yīng)片段獲得 HBV0.4 倍體和 HBV0.9 倍體。取 2μl 擴(kuò)增產(chǎn)物進(jìn)行 1%瓊脂糖凝膠電泳，通過(guò)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(duì)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(xiàn)】：
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(zhǔn)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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