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1.
颗粒化重组戊型肝炎病毒衣壳蛋白及其抗原性与免疫原性 总被引:18,自引:0,他引:18
过去的研究发现大肠杆菌表达的戊型肝炎病毒(HEV)衣壳蛋白ORF2的aa394-606片段NE2可以形成同源多聚体,并具有良好的免疫保护性,但纯化后的免疫原性较弱。这里表达了3个NE2蛋白的N端延伸突变体,发现对应于ORF2 aa368_606的重组蛋白HEV239在体外可以形成颗粒性抗原。HEV 239抗原颗粒与戊肝患者血清反应性良好,对中和性单克隆抗体8C11的反应性与NE2抗原相当,而对另一中和性单克隆抗体8H3的反应性较NE2抗原有显著提高,表明HEV 239抗原颗粒具有比NE2更好的抗原性。纯化后的HEV 239抗原颗粒直径约为15~30nm。铝佐剂吸附的HEV 239免疫Balb/c小鼠的半数有效剂量(ED50)在0.08~0.25μg之间,而同样以铝佐剂吸附的NE2抗原60μg剂量免疫的抗体阳转率仅25%,表明HEV 239抗原颗粒具有更好的免疫原性。 相似文献
2.
重组蛋白NE2包含了戊型肝炎病毒(HEV)衣壳蛋白(pORF2)的aa394~606片段.在NE2上已鉴定出了2个HEV中和表位,并获得了3个识别中和表位的单克隆抗体(MAb)8C11、13D8和8H3.这3个MAb间的交叉阻断ELISA实验发现,8C11和13D8可以彼此完全阻断,8H3对8C11和13D8均不能阻断,而8C11非但不能阻断8H3,反而显著增强了8H3与抗原的结合.用生物传感器进行的抗体与抗原结合的动力学分析也证实了这一现象.这些结果提示,在NE2上8H3表位区域受到抗原上某些结构的掩盖,而8C11与NE2的结合引起了抗原空间结构的改变,导致了掩盖8H3表位的结构的去除和8H3表位的充分暴露.免疫捕获RT-PCR发现,8C11同样可以显著增强8H3对天然HEV病毒的捕获能力,提示这种结合诱导的衣壳蛋白空间构象改变在天然HEV病毒颗粒上同样存在. 相似文献
3.
大肠杆菌表达的戊型肝炎病毒ORF2片段的聚合现象研究 总被引:29,自引:4,他引:25
在大肠杆菌中表达了戊型肝炎病毒(HEV)ORF2的a.a.394~a.a.604片段,得到的重组蛋白NE2在SDSPAGE中主要以可被尿素解聚的二聚体形式存在,二聚体对病人血清的反应性明显强于单体;质谱分析表明NE2可形成从二聚体到至少六聚体的多种聚体;动态光散射测定表明平均分子半径约4nm,相当于四聚体,但分散度较大,提示为多种大小不一的聚合体的混合物。这些证据表明NE2蛋白可形成以同源二聚体为基本单位的多种聚合体形式,其中以二聚体间的结合最为紧密,并且以二聚体为基础可进一步装配出多种更高级结构,从而具有作为HEV疫苗及诊断试剂抗原的良好前景。
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4.
迄今所发现的唯一的戊型肝炎病毒(HEV)中和表位定位于开放读码框架2(ORF2)编码蛋白的第578和第607氨基酸(aa)之间的区域。将对应此区域的基因片段通过一段柔性的甘氨酸铰链与乙型肝炎病毒(HBV)表面抗原(HBsAg)基因的3′端相连,构建成HBV/HEV融合基因。该融合基因在毕赤酵母细胞内的表达产物物为分子量约29kDa的融合蛋白,具有组装成嵌合病毒样颗粒(VLP)的能力。此嵌合VLP具有与HBsAgVLP相似的特性且保留了天然HBV/HEV双重抗原性。对此嵌合VLP特性的初步研究提示其可能具有HBV/HEV双价重组疫苗的潜在应用前景。 相似文献
5.
戊型肝炎病毒(HEV)根据易感宿主的差别可以分为两大类:一类只分离自人的H(Human)类,包括HEV-1和HEV-2;一类为人畜共患的Z(Zoonosis)类,包括HEV-3和HEV-4。本研究通过比较这两类HEV的ORF2aa368~606区段,发现存在4个类保守的差异位点,均位于HEV的主要中和表位区域aa459~606,分别是aa483、aa492、aa497和aa599;对这四个位点进行定点替换突变,以一组能够捕获HEV-1和/或HEV-4的单克隆抗体比较各种突变体的免疫反应性,结果表明仅aa497的差异造成了这两类HEV中和表位构象的部分差异,提示aa497及其相关的病毒表面结构差异在H类和Z类HEV宿主选择中可能扮演重要角色。 相似文献
6.
c-Abl是非受体酪氨酸激酶,它在细胞内被一些基因毒性的、氧化的及其它形式的压力所激活。目前研究证明:应用标记的c-Abl发现其在细胞内可以相互形成同源二聚体,并且一分子c-Abl的N末端区域与相应的另一分子的C末端相互作用形成二聚体。实验进一步表明: cAbl SH3 结构域结合到另一c-Abl 分子富含脯氨酸的C-末端约958-982氨基酸区域。如果去除c-Abl 富含脯氨酸的结构域,就会阻止二聚体的形成。这些结果首先证实了c-Abl在细胞内可以相互形成同源二聚体,并暗示着二聚体的形成可能影响着c-Abl活性的调节。 相似文献
7.
戊型肝炎病毒细胞吸附模型的建立及病毒吸附区域初步研究 总被引:7,自引:3,他引:7
E.coli中表达的HEV衣壳蛋白片段P239(aa368~606)形成的类病毒颗粒与戊肝患者恢复期血清及中和单抗具有良好的反应性,较好地模拟了天然HEV病毒颗粒的表面空间结构。利用P239吸附HepG2细胞的模型来模拟HEV对宿主细胞的吸附,多株中和单抗对吸附的阻断验证了吸附的特异性。P239与多株传代细胞系的吸附结果则表明了这种特异性吸附的细胞选择性。对阻断P239吸附的线性单抗进行定位,初步确定了P239与细胞相互作用的区域:ORF2上的aa423~443很可能和病毒上的细胞膜受体结合部位非常靠近,或可能直接参与构成了病毒与细胞特异性识别的表位。此本研究为进一步研究HEV与宿主细胞的相互作用提供一定的线索。 相似文献
8.
肝炎病毒的感染常常导致人体肝脏代谢能力变化,研究表明甲型肝炎病毒(Hepatitis A Virus,HAV)、乙型肝炎病毒(Hepatitis B Virus,HBV)和丙型肝炎病毒(Hepatitis C Virus,HCV)的感染都会影响肝细胞对药物的代谢能力。在本研究中,通过酵母双杂交系统在人肝cDNA文库中筛选与戊型肝炎病毒重组衣壳蛋白E2结合的蛋白,发现了与细胞色素P450 2A6(CYP2A6)部分片段高度同源的蛋白序列。通过pull-down、免疫共沉淀以及特异性底物催化反应评价等方法进一步证实了CYP2A6与重组衣壳蛋白E2、p239间的相互作用,并发现与p239结合可以降低CYP2A6对其特异性底物香豆素的催化能力。上述结果提示CYP2A6可能在戊肝病毒入侵细胞后的细胞病变过程中起作用。 相似文献
9.
李青 《微生物学免疫学进展》2010,38(4):70-74
戊型肝炎(Hepatitis E,HE)是由戊型肝炎病毒(Hepatitis E Virus,HEV)引起的一种非甲非乙型病毒性肝炎。HE主要在亚非拉等发展中国家流行,但在欧美等发达国家也有散发病例。HEV是一种非包膜单股正链RNA病毒,具有四种基因型,仅有一种血清型,所以有望能够研究出对HEV所有基因型均有保护性的疫苗。因HEV尚无合适的细胞培养系,目前,戊肝疫苗研究开发主要集中于基因工程疫苗。戊肝病毒开放阅读框2(ORF2)含有重要的中和抗原表位,目前已有ORF2许多片段在不同表达系统中成功地进行了表达,如原核、昆虫、酵母、植物细胞等表达系统,且表达产物均具有一定的免疫原性。虽然戊肝疫苗的研究已开展多年并取得了很大进展,迄今全球仍无戊肝疫苗被批准上市。但有两种戊肝候选疫苗现已进入Ⅲ期临床试验阶段,有望成为预防戊肝的有效手段。 相似文献
10.
通过 Western blot、体外捕获PCR、ELISA阻断实验及合成的多肽库等方法,对23株抗戊型肝炎病毒(HEV)单克隆抗体(单抗)识别HEV ORF2表位的作用进行系统研究.结果显示,7株线性单抗识别表位都位于ORF2aa408~458之间,16株构象型单抗识别表位都定位于ORF2 aa459~606之间,大部分构象型单抗识别表位都在天然病毒表面.对这些单抗识别表位系统地了解将为HEV疫苗、诊断、病毒受体和病毒感染机制等方面的研究提供重要工具. 相似文献
11.
Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies 总被引:13,自引:0,他引:13
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Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection. 相似文献
12.
Min Zhao Xiao-Jing Li Zi-Min Tang Fan Yang Si-Ling Wang Wei Cai Ke Zhang Ning-Shao Xia Zi-Zheng Zheng 《The Journal of biological chemistry》2015,290(32):19910-19922
The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process. 相似文献
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14.
猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣bingen毒株Erns糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位~ 35 6位或 348位~ 35 0位、384位~ 386及 32 2位~ 32 3位、380位~ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异 相似文献
15.
Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus 总被引:3,自引:0,他引:3
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Li TC Takeda N Miyamura T Matsuura Y Wang JC Engvall H Hammar L Xing L Cheng RH 《Journal of virology》2005,79(20):12999-13006
Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions. 相似文献
16.
The sequence of the 3' 4508 nucleotides (nt) of the genomic RNA of the Therien strain of rubella virus (RV) was determined for cDNA clones. The sequence contains a 3189-nt open reading frame (ORF) which codes for the structural proteins C, E2 and E1. C is predicted to have a length of 300 amino acids (aa). The N-terminal half of the C protein is highly basic and hydrophilic in nature, and is putatively the region of the protein which interacts with the virion RNA. At the C terminus of the C protein is a stretch of 20 hydrophobic aa which also serves as the signal sequence for E2, indicating that the cleavage of C from the polyprotein precursor may be catalyzed by signalase in the lumen of the endoplasmic reticulum. E2 is 282 aa in length and contains four potential N-linked glycosylation sites and a putative transmembrane domain near its C terminus. The sequence of E1 has been previously described [Frey et al., Virology 154 (1986) 228-232]. No homology could be detected between the amino acid sequence of the RV structural proteins and the amino acid sequence of the alphavirus structural proteins. From the position of a region of 30 nt in the RV genomic sequence which exhibited significant homology with the sequence in the alphavirus genome at which subgenomic RNA synthesis is initiated, the RV subgenomic RNA is predicted to be 3346 nt in length and the nontranslated region from the 5' end of the subgenomic RNA to the structural protein ORF is predicted to be 98 nt. In a different translation frame beginning at the 5' end of the RV nt sequence reported here is a 1407 nt ORF which is the C terminal region of the nonstructural protein ORF. This ORF overlaps the structural protein ORF by 149 nt. A low level of homology could be detected between the predicted amino acid sequence of the C-terminus of the RV nonstructural protein ORF and the replicase proteins of several positive RNA viruses of animals and plants, including nsp4 of the alphaviruses, the protein encoded by the C-terminal region of the alphavirus nonstructural ORF. However, the overall homology between RV and the alphaviruses in this region of the genome was only 18%, indicating that these two genera of the Togavirus family are only distantly related. Intriguingly, there is a 2844-nt ORF present in the negative polarity orientation of the RV sequence which could encode a 928-aa polyprotein. 相似文献
17.
Xing L Wang JC Li TC Yasutomi Y Lara J Khudyakov Y Schofield D Emerson SU Purcell RH Takeda N Miyamura T Cheng RH 《Journal of virology》2011,85(2):1117-1124
Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T=1 virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T=1 HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope. 相似文献