丙型肝炎病毒与宿主细胞蛋白的相互作用及其意义

蛋白－蛋白的结合是病毒与宿主细胞相互作用的分析基础。随着酵母双杂交系统等研究蛋白－蛋白结合新技术的广泛应用,人们对病毒复制的本质及致病机制有了更新的认识,本文综述了丙型肝炎病毒（ＨＣＶ）核心蛋白Ｃ及非结构蛋白５Ａ（ＮＳ５Ａ）与宿主蛋白相互作用的进展,并讨论了它们在ＨＣＶ致病中的作用。     

SARS冠状病毒N蛋白和CYP4F3的相互作用

目的：研究SARS冠状病毒（SARS-CoV）N蛋白与CYP4F3的相互作用。方法：应用免疫共沉淀、GST—pull down、BIACORE实验验证SARS—CoVN蛋白与CYP4F3的相互作用。结果：SARS—CoVN蛋白与CYP4F3能够相互作用,BIA-CORE实验证实CYP4F3与SARS—CoVN蛋白的亲和常数为Ko=4．3×10^-11。结论：SARS—CoVN蛋白与CYP4F3在细胞内、外均能形成复合物,这为进一步探讨SARS—CoVN蛋白在SARS致病机理中的作用奠定了基础。     

SARS-CoV N蛋白抗原表位的筛选和鉴定

利用噬菌体展示的线性12肽库从马抗SARS-CoV IgG筛选SARS-CoV的抗原表位.经生物淘洗富集的噬菌体克隆被测序.获得两个共有序列DXXDP和TXTLL.它们分别与SARS-CoV N蛋白341-345和392-396位氨基酸序列高度同源.含共有序列的克隆在ELISA竞争抑制试验中与SARS-CoV N蛋白竞争结合马抗SARS-CoVIgG.将两个共有序列肽通过基因重组技术成功展示到大肠杆菌鞭毛,获得重组菌F1和F2.用重组菌F1和F2免疫接种试验Balb/c小鼠产生的血清均能与SARS-CoVN蛋白特异结合.说明DXXDP和TXTLL是SARS-CoVN蛋白的两个连续抗原表位.     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoVN蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Westernblotting和间接免疫荧光方法分别检测了4株抗SARS-CoVN蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoVN蛋白对上述4种单克隆抗体的识别表位进行了初步定位。结果显示:(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoVN蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoVN蛋白的aa30-60,1-1C2针对的抗原表位则位于SARS-CoVN蛋白的aa170-184。这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料。     

SARS-CoV N蛋白与病毒mRNA相互作用的初步研究

SARS冠状病毒(SARS-CoV)是一种新型的冠状病毒,其基因组大小约为30,000 nt,为单股正链RNA.病毒基因中的1-72个核甘酸为前导序列.核衣壳(Nucleocapsid,N)蛋白是冠状病毒的主要结构蛋白,它在病毒基因转录,翻译以及病毒颗粒包装中起重要作用.在本研究中,我们通过PCR的方法从SARS-CoV cDNA中克隆N基因,将基因克隆到大肠杆菌表达载体中,经表达纯化获得大量重组蛋白,通过亲和层析和凝胶过滤层析获得高纯度的N蛋白.同时构建前导RNA的转录模板,经体外转录得到地高辛标记的RNA.使用Northwestern分析技术,我们证实纯化的N蛋白在体外可以与RNA发生特异性的结合.N蛋白与病毒RNA的结合特性及其在病毒生活周期中的所起作用的初步研究,为下一步设计出有效的阻断病毒周期从而达到抗病毒目的的药物或疫苗奠定了基础.     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoV N蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Western blotting和间接免疫荧光方法分别检测了4株抗SARS-CoV N蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoV N蛋白对上述4种单克隆抗体的识别表位进行了初步定位.结果显示(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoV N蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoV N蛋白的aa 30-60,1-1C2针对的抗原表位则位于SARS-CoV N蛋白的aa 170-184.这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料.     

病毒-宿主蛋白相互作用组学研究进展

病毒和宿主之间的蛋白相互作用贯穿其整个生命周期.对病毒-宿主蛋白相互作用组的研究不仅可以阐明病毒的感染过程和机体的防御机制,而且还可以揭示潜在的抗病毒治疗靶点.本文回顾了病毒-宿主蛋白相互作用组学常用的研究方法,并探讨了每种方法的优点及局限性.     

SARS-CoVN蛋白抗原表位的筛选和鉴定

利用噬菌体展示的线性12肽库从马抗SARS-CoVIgG筛选SARS-CoV的抗原表位。经生物淘洗富集的噬菌体克隆被测序。获得两个共有序列:DXXDP和TXTLL。它们分别与SARS-CoVN蛋白341-345和392-396位氨基酸序列高度同源。含共有序列的克隆在ELISA竞争抑制试验中与SARS-CoVN蛋白竞争结合马抗SARS-CoVIgG。将两个共有序列肽通过基因重组技术成功展示到大肠杆菌鞭毛,获得重组菌F1和F2。用重组菌F1和F2免疫接种试验Balb/c小鼠产生的血清均能与SARS-CoVN蛋白特异结合。说明DXXDP和TXTLL是SARS-CoVN蛋白的两个连续抗原表位。     

SARS冠状病毒S蛋白在昆虫细胞中的表达和纯化

导致严重急性呼吸综合征(sevcre acute rcspiratory syndrome,SARS)的元凶是一种新型的冠状病毒(SARS coronavirus,SARS-CoV)。SARS-CoV感染入侵宿主细胞关键的一环是病毒自身的棘突蛋白(spike protein,S-protein)与细胞受体的相互作用,故而S蛋白己成为SARS研究的主要热点。     

SARS冠状病毒核衣壳蛋白抑制胞质分裂和细胞增值

目的:SARS冠状病毒(SARS-CoV)的核衣壳蛋白(N蛋白)能够结合病毒RNA形成螺旋状的核衣壳。在3种不同的细胞系中分别表达SARS-CoVN蛋白,研究它对转染细胞生长的影响。方法:将重组质粒pEGFP-N和pEGFP(作为对照)分别转染人胚肾HEK293细胞、成纤维细胞3T3、人宫颈癌HeLa细胞,通过激光共聚焦显微镜、荧光显微镜观察SARS-CoVN蛋白在细胞内的定位以及细胞的生长变化。结果:SARS-CoVN蛋白能定位于细胞质,并不像其他冠状病毒N蛋白那样能够定位到细胞核。同时发现SARS-CoVN蛋白能够诱导形成多核细胞,多核细胞的百分率可达33.9%。结论:SARS-CoVN蛋白抑制胞质分裂,延缓细胞生长。     

严重急性呼吸综合征(SARS)冠状病毒核蛋白的鉴定与分析

利用蛋白质组学技术,对纯化的严重急性呼吸综合征(SARS)冠状病毒颗粒所含核蛋白进行初步分离与鉴定。质谱分析结果最终表明,SARS冠状病毒核蛋白的分子量位于47kD与52kD之间,所获得的SARS冠状病毒核蛋白的质谱分析数据覆盖了所预测病毒核蛋白氨基酸序列的87％,且符合率为100％。从而首次从蛋白质水平对SARS冠状病毒核蛋白的氨基酸序列进行了证实。     

SARS冠状病毒核衣壳蛋白在酵母中的表达与活性检测

用PCR扩增SARS冠状病毒N蛋白全长cDNA,克隆到酵母表达载体pPIC3.5K,构建pPIC3.5K-SCoVN酵母表达质粒。表达质粒线性化后电转化到毕赤酵母GS115中,经G418-RDB, MM/MD平板与PCR扩增筛选获得His⁺ Mut⁺ 重组菌株。比较研究了不同的培养基、溶解氧以及甲醇浓度对菌株生长与重组蛋白表达的影响。结果表明：FBS培养基最适宜重组菌的生长与表达,溶氧对菌体的生长与表达有显著的影响,甲醇诱导最佳终浓度为1％（V/V）,SDS-PAGE分析重组蛋白的表达量,发现重组N 蛋白表达量占细胞总蛋白的6％,每升培养基可以生产410mg重组N蛋白,生物量达45OD600。Western　blotting结果表明,重组N 蛋白对鼠源单克隆抗体以及SARS病人恢复期血清具有较强的特异性反应。对摇瓶培养条件进行了发酵罐放大实验,结果生物量达到348OD600,表达量达到 2.5g/L,分别为摇瓶表达的7.7倍和6.1倍,为SARS早期血清学诊断研究以及为N蛋白在病毒复制以及致病机理的研究奠定了一定的基础。     

SARS病毒核蛋白基因的克隆及其表达研究

从一例输入性传染性非典型性肺炎病人血清中提取病毒RNA,通过RT—PCR方法扩增出SARS病毒核蛋白基因片段,克隆入质粒载体pUCm—T后,进行核苷酸序列的测定及分析,与已公布的SARS病毒基因序列进行比较,证实为SARS冠状病毒核蛋白基因。为了解该病毒核蛋白的抗原特性,将核蛋白基因插入表达载体,构建重组质粒pET28a—SN,转导大肠杆菌BL21(DE3)后,加IPTG诱导表达。产物经SDS—PAGE电泳分析,表达出相对分子量约为50kDa的蛋白,占整个菌体的45％左右。Westem—blot分析表明,表达产物仅与SARS阳性病人血清起反应,而与正常血清不起反应。间接ELISA免疫检测,抗原滴度达1：12500。表明表达的核蛋白为SARS特异性抗原,这为SARS病毒的诊断试剂的研制提供了方便而安全的抗原来源。     

SARS病毒核蛋白基因的克隆及其表达研究

从一例输入性传染性非典型性肺炎病人血清中提取病毒RNA,通过RT-PCR方法扩增出SARS病毒核蛋白基因片段,克隆入质粒载体pUCm-T后,进行核苷酸序列的测定及分析,与已公布的SARS病毒基因序列进行比较,证实为SARS冠状病毒核蛋白基因.为了解该病毒核蛋白的抗原特性,将核蛋白基因插入表达载体,构建重组质粒pET28a-SN,转导大肠杆菌BL21(DE3)后,加IPTG诱导表达.产物经SDS-PAGE电泳分析,表达出相对分子量约为50kDa的蛋白,占整个菌体的45%左右.Western-blot分析表明,表达产物仅与SARS阳性病人血清起反应,而与正常血清不起反应.间接ELISA免疫检测,抗原滴度达112500.表明表达的核蛋白为SARS特异性抗原,这为SARS病毒的诊断试剂的研制提供了方便而安全的抗原来源.     

鼻咽癌下调新基因NOR1相互作用蛋白的筛选和鉴定

NOR1基因是新的鼻咽癌相关基因,该基因在鼻咽癌细胞系HNE1和鼻咽癌组织中表达下调．在鼻咽癌细胞HNE1中恢复NOR1基因表达抑制了鼻咽癌细胞的生长和增殖能力．为了探讨NOR1基因的生物学功能,以NOR1基因为诱饵运用酵母双杂交技术在人胎脑文库中筛选其交互作用蛋白,挑选阳性克隆,进行DNA序列分析和同源检索,阳性克隆编码7个不同的蛋白质,其中一个阳性克隆编码线粒体ATP合成酶亚基OSCP蛋白．瞬时转染pCMV-myc-NOR1质粒进入鼻咽癌5-8F细胞,通过密度梯度离心法分离线粒体蛋白,Western blot检测表明myc-NOR1蛋白分布于线粒体与胞浆．免疫荧光检测表明在鼻咽正常上皮细胞NP69中内源性NOR1蛋白与线粒体存在明显共定位．随后采用特异性酵母双杂交、免疫荧光共定位、免疫共沉淀技术证实了NOR1与OSCP在线粒体内存在交互作用．提示,NOR1是一个新的线粒体蛋白,可能通过结合OSCP蛋白调控细胞能量代谢,为深入探讨其功能提供了重要线索．     

SARS冠状病毒中的核衣壳蛋白

严重急性呼吸综合征(SARS)的元凶是一种新冠状病毒,研究病毒结构蛋白的功能有助于了解病毒的感染、复制和包装等生理过程。其中核衣壳蛋白是SARS冠状病毒中含量最丰富和最保守的结构蛋白,自身聚合后包被病毒RNA基因组形成螺旋状核壳体是SARS冠状病毒成熟的关键步骤;核衣壳蛋白能与病毒或宿主细胞中多种蛋白质相互作用,还能影响宿主细胞的多个通路。因此核衣壳蛋白是一个重要的多功能蛋白质,参与了病毒感染、复制和病毒包装等过程。     

Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.The assembly of coronaviruses is driven principally by homotypic and heterotypic interactions between the two most abundant virion proteins, the membrane protein (M) and the nucleocapsid protein (N) (14, 32). The M protein is a triple-spanning transmembrane protein residing in the virion envelope, which is derived from the cellular budding site, the endoplasmic reticulum-Golgi intermediate compartment. More than half of the M molecule, its carboxy-terminal endodomain, is situated in the interior of the virion, where it contacts the nucleocapsid (46, 50). Also found in the virion envelope is the spike protein (S), which, although crucial for viral infectivity, is not an essential participant in assembly. The other canonical component of the coronavirus envelope is the small envelope protein (E), the function of which is enigmatic. Some evidence suggests that the E protein does not make sequence-specific contacts with other viral proteins (27) but instead functions by modifying the budding compartment, perhaps as an ion channel (56, 57). Alternatively, or additionally, E could act in a chaperone-like fashion to facilitate homotypic interactions between M protein monomers or oligomers (4).The N protein is the only protein constituent of the helically symmetric nucleocapsid, which is located in the interior of the virion. Coronavirus N proteins are largely basic phosphoproteins that share a moderate degree of homology across all three of the phylogenetic groups within the family (29). Some time ago, we proposed a model that pictured the N protein as comprising three domains separated by two spacers (A and B) (40). This arrangement was originally inferred from a sequence comparison of the N genes of multiple strains of the prototypical group 2 coronavirus, mouse hepatitis virus (MHV), and its validity seemed to be reinforced by numerous sequences that later became available. Part of this model, the delineation of spacer B and the acidic, carboxy-terminal domain 3, has been well supported by subsequent work (22, 25, 41, 42). However, a wealth of recent, detailed structural studies of bacterially expressed domains of the N proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) and of infectious bronchitis virus (IBV) has much more precisely mapped boundaries within the remainder of the N molecule (8, 16, 21, 23, 47, 51, 60). The latter studies have shown that the N protein contains two independently folding domains, designated the N-terminal domain (NTD) and the C-terminal domain (CTD). It should be pointed out that this nomenclature can be misleading: the NTD does not contain the amino terminus of the protein, and the CTD does not contain the carboxy terminus of the protein. Specifically, the CTD does not include spacer B and domain 3. The NTD and the CTD are separated by an intervening serine- and arginine-rich region; this region was previously noted to resemble the SR domains of splicing factors (42), and it has recently been shown to be intrinsically disordered (6, 7).In the assembled virion, the three known partners of the N protein are the M protein, the genomic RNA, and other copies of the N protein itself. We have sought to develop genetic and molecular biological methods that will begin to elucidate the varied ways in which the N molecule interacts during MHV infection. We previously found that the fusion of N protein domain 3 to a heterologous marker, green fluorescent protein (GFP), results in incorporation of GFP into virions (22). In the present study, we similarly fused each of the individual domains of N to GFP, and we thereby uncovered two strong modes of N protein-N protein interaction that likely contribute to virion architecture.