【作者】 王林；

【导师】 林建银； 林旭；

【作者基本信息】 福建医科大学 ， 病原生物学， 2009， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus, HBV)是一种部分双链环状嗜肝DNA病毒,宿主范围狭窄,主要是灵长类中的人类和大猩猩。HBV在人类可引起急、慢性乙型肝炎、肝纤维化、并与原发性肝细胞癌的发生发展关系密切。α-干扰素(Interferon-α, IFN-α)仍是目前最常用的治疗乙型肝炎病毒感染的药物。它通过建立细胞内抗病毒状态和免疫调节作用抑制并清除病毒。但临床发现约2/3的患者表现为对α-干扰素治疗无反应,即没有出现HbeAg血清转换和血清HBV DNA水平降低,除了宿主因素外还与病毒本身抗干扰素作用有关。目前比较明确的是HBV DNA聚合酶的抗干扰素作用。Foster等发现,表达HBV DNA聚合酶末端蛋白(该“末端蛋白”事实上还包括部分Spacer区,详见本研究第一部分)可显著抑制细胞对α-干扰素反应,并认为是由于TP表达阻断α-干扰素通路引起,但其抗IFN-α作用的功能区域迄今尚不清楚。中国地区HBV感染主要为B基因型及C基因型,临床资料显示,B基因型HBV感染对干扰素治疗的反应好于C基因型,我们通过对大样本HBV DNA聚合酶区氨基酸序列分析后发现,B、C基因型HBV DNA聚合酶在TP区及Spacer区分别存在14及44个氨基酸差异,目前尚不清楚这些氨基酸差异是否与B、C基因型HBV感染对干扰素治疗反应差异有关。另TP位于DNA聚合酶的氨基端,通常情况下只能以DNA聚合酶的形式出现,并不存在单独游离的TP。但随着扩增HBV全基因组DNA方法的建立,在乙肝患者中分离到多种HBV基因组剪接变异体、缺失突变体。其中在2309nt以后发生剪接和缺失的HBV亚基因组DNA能编码TP相关蛋白(HBV DNA聚合酶的编码区起始于2309 nt)。对于这些剪接和缺失的HBV亚基因组DNA及其编码的各种TP相关蛋白的抗干扰素作用还未有过系统研究。为此,本研究内容包括三部分:㈠乙型肝炎病毒TP+Spacer抗干扰素作用功能区域分析以HBV全基因组DNA重组质粒FMU013为模板,PCR扩增获得DNA聚合酶TP+Spacer区各功能区基因片段,克隆到真核表达载体pcDNA3.1/HisC中,构建各功能区缺失突变体,与干扰素反应报告质粒p6-16CAT共转染Huh7肝细胞,检测干扰素处理后CAT表达改变,确定抗IFN-α反应的功能区域。㈡比较B、C基因型TP相关蛋白抗干扰素作用的强弱收集干扰素治疗有反应、无反应慢性乙型肝炎患者治疗前血清,抽提血清DNA,以限制性片段长度多态性方法确定基因型。PCR获得各B、C基因型标本TP+Spacer区N端257aa基因片段,克隆到真核表达载体pcDNA3.1/HisC中。各重组表达载体与干扰素反应报告质粒p6-16CAT共转染Huh7肝细胞,检测干扰素处理后CAT表达改变,比较B、C基因型TP+Spacer区抗干扰素作用差别㈢乙型肝炎病毒剪接变异缺失突变体编码的TP相关蛋白抗干扰素作用收集中、重度慢性乙型肝炎患者血清,抽提血清DNA,PCR扩增HBV基因组DNA克隆到pUC18载体中并测序。与标准株HBV序列进行相似性比较,分析其结构,找出符合本研究的所有可能存在的剪接变异类型(在2309nt以后发生剪接)和各种缺失突变体(在2309nt以后发生缺失),克隆各剪接变异体和缺失突变所编码的TP相关蛋白基因到真核表达载体pcDNA3.1/HisC中,各TP相关蛋白基因重组载体与干扰素反应报告质粒p6-16CAT共转染Huh7肝细胞,检测干扰素处理后CAT表达改变,确定各TP相关蛋白抗干扰素作用并分析其发挥抗干扰素作用的功能区域。本研究确定了:TP相关蛋白抗干扰素作用的功能区域与Spacer区密切相关,且其发挥作用需要完整的TP区;B基因型HBV感染对干扰素治疗的反应性显著好于C基因型HBV感染,但这种差异与HBV TP+Spacer区抗干扰素作用无关。分离获得了8种剪接变异体、5种缺失突变体,证实其编码的两种TP相关蛋白具有抗干扰素作用,TP+Spacer是其发挥作用的功能区。更多还原

【Abstract】 Hepatitis B virus (HBV) is the member of the Hepadnaviridae family, with a narrow host range which are human or gorillas, infection of HBV resulted in a broad spectrum of clinical manifestations, including asymptomatic carrier, acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Interferon-α(IFN-α) is one of the most commonly used drugs for the treatment of hepatitis B, it can inhibit and ultimately clear the HBV by immunomodulation and establishing intracellular antiviral state. However, it was found that approximately two thirds of IFN-αtreated patients showed IFN-αnon-responseness, i.e., no loss of HBeAg and reduction of serum HBV DNA lever. In addition to the host factors, it is also related to the anti-IFN-αeffects of virus itself. At present,the anti-IFN-αeffects of the HBV DNA polymerase was recognized specificly. Foster et al demonstrated that terminal protein (TP) of HBV DNA polymerase could dramatically inhibit the cellular response to IFN-αby blocking the IFN pathway, yet the related functional region of TP was remained largely obscure.The genotype B and C HBV are the predominant genotype in china. Several studies have shown that patients with genotype B have a higher probability of hepatitis B e antigen (HBeAg) seroconversion and the reduce of virus titer than patients with genotype C during IFN-αtreatment. Our analysis of a large sample of HBV DNA polymerase amino acid sequences ascertains that there are separately 14 and 44 diversity in TP and Spacer regionsre spectively.Whether these diversity were corresponded with the different responds to IFN-αtreatment in patients with genotype B and patients with genotype C was still uncertainty now.In fact , TP,which is located in the N-terminal of HBV DNA polymerase , can only exist in the form of pol,but, a novel method for efficient amplification of whole hepatitis B virus genome maked it possible to isolate a variety of splice variants and deletion mutants from serum of chronic hepatitis patients. these HBV subgenomic DNA which generated by Splicing and deletion beyond 2309nt from the HBV complete genomic DNA can encode TP Associated proteins. It is presently lack of the systematic study about the anti-IFN-αeffects of these TP Associated protein. To address all these issues, our study includes three parts1. Analysis of the functional region for anti-IFN-αeffects by TP+Spacer of HBV DNA polymeraseThe fragments of the serial deletants of TP+Spacer were amplificated from FMU013 which a recombinated vector of HBV complete genome and cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring deleted TP+Spacer,then, treated with IFN-α.The intracellular CAT expression were calculated to evaluate the anti- IFN-αeffects.2. Comparison of the anti-IFN-αeffects of genotype B and C TP+Spacer regionIsolated DNA from IFN pre-treatment serum samples which from chronic hepatitis patients including responsers and non-responsers. Genotypes of all samples were determined by restriction fragment length polymorphism。The Tp257 fragments of all samples were amplificated from isolated DNA and cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring Tp257, then, treated with IFN-α.The intracellular CAT expression were calculated to comparison of the anti-IFN-αeffects of genotype B and C TP associated protein.3. The anti-IFN-αeffects of the Tp associated protein encoded by HBV splice variants and deletion mutantsIsolated DNA from serum samples of chronic hepatitis patients, complete HBV genomes were amplificated from isolated serum DNA and cloned into pUC18 vector and sequenced. Identified all the possible existent Splice variants and deletion mutants which spliced and deleted beyond 2309nt. The PCR fragment of the Tp Associated proteins encoded by these HBV subgenomic DNA were cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring the fragment of the Tp Associated proteins , then, treated with IFN-α.The intracellular CAT expression were calculated to determine the anti-IFN-αeffects of the Tp associated protein encoded by HBV splice variants and deletion mutants . Our study demonstrated that functional region for anti-IFN-αeffects of the Tp associated protein was closely related to the Spacer region, and the complete TP indispensable for this effect. Although,the differences of amino acids sequences from Tp + Spacer region between genotype B and C were significant, it showed that they were not attributed to the different anti-IFN-αeffects. Eight kinds of splice variants and five kinds of deletion mutants were obtained,and two kinds of TP associated proteins with anti-IFN-αeffects were confirmed.更多还原