【作者】 刘合宾；

【导师】 刘克洲； 朱智勇；

【作者基本信息】 浙江大学 ， 内科学， 2004， 博士

【摘要】 汉坦病毒(Hantavirus)是引起人肾综合征出血热(Hemorrhagic fever with renal syndrome，HFRS)和人汉坦病毒性肺炎综合征(Hantavirus pulmonary syndrome，HPS)的病原体，在世界各大洲均有流行。汉坦病毒感染及其引起的相关疾病已成为严重的世界性公共卫生问题之一。 汉坦病毒基因组为分三节段的单股负链RNA，即大(L)、中(M)、小(S)三个基因片段，依次编码病毒的RNA聚合酶L蛋白，外膜蛋白G1、G2以及核蛋白(NP)。由S基因编码的核蛋白是汉坦病毒重要的结构蛋白成份，对调节病毒的复制，刺激宿主保护性细胞免疫反应以及调控细胞凋亡信号路径等方面有重要的作用。 汉坦病毒Z10和Z37株是两个分离自不同宿主及不同血清型的重要中国疫苗株，其中Z10株分离自出血热病人血清，血清学分型为汉滩型病毒(Hantaan，HTNV)；Z37株分离自家鼠，其血清学分型为汉城型病毒(Seoul，SEOV)。具有血凝滴度高和诱导中和性抗体能力强的特点。本论文主要研究了汉坦病毒疫苗株Z10和Z37核蛋白体内、体外表达，核蛋白与基因组末端反向重复序列相互作用以及核蛋白对鼠源性巨噬细胞生长的影响。 核蛋白相对保守，常作为汉坦病毒分子进化亲缘性分析的依据。为了解汉坦病毒疫苗株Z10与Z37基因分型及与其他毒株在进化上的亲缘性，克隆并序列测定了Z10和Z37核蛋白全基因，二者核蛋白在核苷酸水平和氨基酸水平的同源性分别为75.1％和80.6％；进化树分析表明Z10株与HTNV代表株76／118以脚红久~弓理多弓护脱耀_户学企讼戈茄萝及中国H丁NV分离株Ag有一定的遗传差异，在进化上偏向于HTNV型，但却是介于HTNV型与SEOV型之间的毒株:而237株不仅与SEOV型中国分离株R22，甚至与SEOV型英国分离株皿461都有很大的亲缘性。该结果说明不同地域来源的SEOV型毒株之间有较大的保守性，而HTNV型毒株因地理区域的不同变异性较大。 核蛋白能不仅能刺激宿主保护性细胞免疫，而且可聚合形成的病毒样颗粒 (vLP)，在作为亚单位肤类疫苗目标蛋白以及分型诊断抗原具有很大的应用潜力。为了获得纯化的可溶性重组核蛋白，构建了210和237株核蛋白原核表达载体并在大肠杆菌中表达，我们发现在较低的温度(18“C)和较低浓度IPTG(100mM)条件下诱导表达过夜可提高可溶性核蛋白的产量。然后以液相色谱分别经Ni一NAT亲和柱和HIPreP 16厂10 DEAE离子交换柱二步纯化获得高纯度的重组核蛋白，产量约为0.2 mg每升菌液。并对纯化的210和237株核蛋白分别以胰蛋白酶不完全消化后用\\飞stem blot进行区分和鉴定。本研究建立的表达纯化条件对大规模生产纯化的核蛋白具有很好的参考价值。 核蛋白在细胞内不同区域的分布，表达以及存在方式不仅对病毒本身的复制水平而且对被感染的宿主细胞基因调控有重要的影响，为了解内源性核蛋白的表达及细胞内的分布情况，构建了汉坦病毒210株核蛋白基因与鼠干细胞反转录病毒(入ISCv)重组载体，通过磷酸钙转染法导入产病毒的包装细胞系BOSC23中产生完整的重组MscV一FlagNP病毒。然后以重组病毒直接感染NIH 3T3细胞，利用P盯。mycin对感染细胞进行连续压力筛选，获得了转核蛋白抗性细胞。分别用Southem blot和PCR方法可检测到核蛋白基因在抗性细胞染色体中完整地整合，并且用westem blot在抗性细胞中可检测到核蛋白稳定的表达。进一步以Flag单克隆抗体介导的免疫荧光染色联合共聚焦激光扫描荧光显微镜分析了内源性Flag融合核蛋白在抗性细胞内分布，发现核蛋白主要分布于胞浆及胞核周围区，并且部分核蛋白可聚集形成胞浆包涵体。核蛋白主要分布于胞浆及胞核周围区，并且部分核蛋白可聚集形成胞浆包涵体。 汉坦病毒基因组的一个重要特点是每个基因片段两个末端都具有一段长为18个核普酸的高度保守的反向重复序列，互补可形成双链发夹结构。为了研究葫乏乙久弓势治鳌二笋炭，厚士学夕落忿戈蔺要该双链结构在核蛋白包装病毒基因组RNA中的作用与功能，以T4 DNA激酶同位素标记一对人工合成互补的18个核营酸反向重复序列，制备双链探针，以模拟基因片段末端反向重复序列形成的双链发夹结构。然后以电泳移动阻滞实验(E MSA)分析了210和237株纯化重组核蛋白在非变性胶中与反向重复序列双链探针相互作用的可能性，发现重组210和237核蛋白在体外非变性胶中均能特异结合基因组末端反向重复序列形成的双链探针，该结果提示基因片段末端反向重复序列形成的双链发夹结构可能是保证核蛋白正确包装病毒基因组重要信号序列和作用位点。 在汉坦病毒感染过程中，巨噬细胞是除上皮细胞外最早被感染的细胞。为了解核蛋白在汉坦病毒感染巨噬细胞时的作用，刺激或细胞内大量表达对鼠源性巨噬细胞系分析了210和237核蛋白细胞外J774的影响。和237大肠杆菌表达的重组的核蛋白与鼠源性巨噬细胞系发现以Zmg/l的210J774共培养可引起明显的生长抑制以及细胞出现脱落，破碎等病理变化;而在转染有210核蛋白真核瞬时表达载体的巨噬细胞中虽然用Westem blot能检测到核蛋白大量表达，但并?更多还原

【Abstract】 Hantaviruses are the major causative agents of human hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which have been becoming one of global serious issues for human public health. Hantavirus possesses a tripartite, single-strand RNA genome, with segments designated as small (S), medium (M) and large (L) encoding the viral nucleocapsid protein (NP), envelope glycoproteins (Gl and G2) and RNA polymerase L protein, respectively. The viral NP is involved in the virus replication, stimulation of the protective cell immune response and regulation of apoptosis pathway.Hantaviruses Z10 and Z37 are two important Chinese vaccine strains, which were isolated from the serum of a HFRS patient and apodemus agrarius, and serologically grouped into HTNV and SEOV type, respectively. To study the evolutionary relationship of these two Chinese vaccine strains to other recognized hantaviruses, the cDNAs encoding complete NPs of Z10 and Z37 were cloned and subsequently sequenced. Phylogenetic analysis of the NP sequences at both nucleotide and deduced amino acid levels revealed that Z37 strain is closely related to SEO virus Chinese strain R22 and AH09 and even UK strain IR461, whereas Z10 strain is genetically distinct from the HTNV Korean strain 76-118 and Chinese isolateA9, forming a sub-lineage between HTNV and SEOV serotype. This data suggest that HTNV serotype with geographic differences is more variable than SEOV.To obtain soluble and native NPs, the sequences encoding NPs of Z10 and Z37 were expressed in E.coli at 18 with 100 mM IPTG overnight and the His- tagged recombinant NPs were then purified with Ni-NAT affinity chromatography and DEAE ion exchange chromatography by using a FPLC system. The purified recombinant NPs of Z10 and Z37 were distinguished by trypsin incomplete digestion, followed by western blot using anti-His monoclonal antibody.A unique structure feature of the viral genome is a highly conserved about 18-nucleotide inverted repeat sequence is present at the terminals of each gene segment in different types of Hantaviruses, which can form a special hairpin structure when it got complemented. However, the function of the hairpin structure has not been clearly known. Electrophoresis mobility shift assay (EMSA) showed that both NPs of Z10 and Z37 hantaviruses bound specifically to the double strand DNA probe possessing the 18- nucleotide inverted repeat sequences in vitro, suggesting the inverted repeat sequence at the ends of each gene segment could be an important target site for nucleocapcid protein, and therefore plays an important role during Hantavirus packaging the viral apparatus into a complete viral particle.To investigate the intracellular NP expression and translocation, the NP coding sequence of hantavirus Z10 was introduced into NEH 3T3 cells through a retrovirus MSCV system. The integration of NP gene in the selected cell clones was confirmed by PCR and southern blot, and the expression of NP was detected by western blot. IFA in combination with laser scanning confocal fluorescent microscope shows the NP predominantly locates and partially polymerizes into inclusion bodies in the cytoplasm and membrane region of nucleus in selected cells.Macrophages are one of the target cells of Hantavirus at the early stage of infection. To explore the possible roles of NP during hantavirus infection in macrophages, the effects of NP on a mouse macrophage lineage J774 were studied. Itwas found that over-expression of Z10 NP had very little effects on J774 cells compared to the control cells, whereas there was an obvious inhibitory and cytopathic effects on macrophages J774 by stimulating with E.coli produced recombinant NPs of both Z10 and Z37 at a concentration of 2 mg/ml. These data indicate that hantavirus NP has cytopathic effects on macrophages as a extracellular stimulating signal.更多还原